i THE IMPACT OF DIABETES MELLITUS IN PREGNANCY ON MATERNAL HEALTH OUTCOMES Veronique Nicolaou Student number 9702987E A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy. Supervisors Professor Naomi Levitt Professor Shane Norris Johannesburg, South Africa 2021 ii Declaration This dissertation is submitted for the degree of Doctor of Philosophy at the University of Witwatersrand. The research described herein was conducted under the supervision of Professor Shane Norris in the Department of Paediatrics, University of Witwatersrand and Professor Naomi Levitt in the Department of Endocrinology, University of Cape Town, between period January 2018 and December 2020. This work to the best of my knowledge is original, except where acknowledgements and references are made to previous work. Neither this, nor any substantially similar dissertation has been or is being submitted for any other degree or diploma or other qualification at any other University. Signature 12 th day of November 2021 iii Dedication “Every great dream begins with a dreamer. Always remember, you have within you the strength, the patience and the passion to reach for the stars to change the world”. Harriet Tubman Professor Huddle, without you I may have never realized this dream! To my dearest husband Stefano and two beautiful girls, Michela and Sofia who were behind me all the way. It is with thanks to you all that I achieved this dream. iv Acknowledgements I am deeply indebted to Professor Shane Norris for a priceless opportunity to pursue my PhD under his supervision. I appreciate his vast knowledge and skills, guidance, and input in building a critical intellectual argument, hypothesis generation and writing the reports from research proposal, ethics application, publications, and the whole thesis. He provided not only the platform, but also brought on board international expertise in my area of interest. A special thanks go to Professor Naomi Levitt for co-supervising my work all the way from Cape Town. She was always ready to hop on a plane to meet up and discuss some critical issues surrounding the project and was always readily available to assist and advise were necessary. I thank you for your expertise and time devoted in preparing my academic arguments and execution of hypotheses. Furthermore, I need to thank various other experts and colleagues, for contributing in various ways. Professor Huddle, from the outset of my career in Internal Medicine and Endocrinology, you have been a phenomenal mentor and colleague. Your intimate knowledge you have always shared with me during my years of training as a clinician and more recently the support you have given me during my PhD path has been invaluable. To a newly acquired colleague and friend who set out on her PhD journey alongside me, Larske Soepnel, thank you for being a pillar of strength as we tackled the many challenges together with the blissful moments in achieving our goals throughout this process. I wish you all the best with your PhD. Professor Kerstin Klipstein-Grobusch, the mentoring and guidance you provided throughout this journey was a welcome addition to my research. Your keen knowledge and ongoing support were deeply appreciated. To the research team at the DPHRU, a special word of thanks to the research nurses and assistants, especially Sr Gladys who selflessly supported this project despite the numerous challenges. Without you this would not have been possible. Thank you for your hard work and dedication. I am eternally grateful. To the laboratory staff, Elisa Masiakwala and Yusuf Guman- thank you for all your hard work in the laboratory and for seeing this project through from start to end. Your meticulous work ethic and dedication to the study is so appreciated. To Wihan Taljaard, your intimate knowledge and kind manner was a tremendous help with setting up our REDCap data capturing platform. Thank you! To Glory Chidumwa, your generous contribution in assisting me with understanding statistics and applying it correctly to the thesis is commendable and I am grateful to you for having provided me with this valuable tool. v The women and their babies in my study- thank you for your contribution to Science and for helping me better understand the consequences of gestational diabetes in our country. I hope that through this work we can assist in improving health care platforms to encourage better screening and awareness around screening for non-communicable diseases following a diabetic pregnancy together with interrupting the vicious cycle of intergenerational disease. Lastly, I am so grateful to my husband Stefano and two daughters, Michela and Sofia who were a constant inspiration on my journey. vi Thesis Material and Contributions Together with my supervisors, I contributed to the conceptualisation of the new data collection for this thesis. I contributed to the development of the study protocol, which included designing the data collection templates, formulating the questionnaire, the standard operating procedures and training staff where necessary. I was actively involved in performing all data collection which included interviewing participants, performing anthropometric measurements, blood venesection, performance of an oral glucose tolerance test and ultrasonography. In addition, I contributed to the project management, quality control and data management, as well as statistical analysis of the results presented. Funding and Awards This study was made possible through the support of the SAMRC/Wits Developmental Pathways for Health Research Unit. The support of the SAMRC/Wits Developmental Pathways for Health Research Unit, towards this research is hereby acknowledged. Opinions expressed and conclusions arrived at are those of the author and not necessarily attributed to the Centre of Excellence in Human Development. vii Presentations and publications Presentations Scientific meetings 1. Characteristics of women with diabetes in pregnancy in Soweto and the associated maternal and neonatal outcomes Conference: Research day University of Witwatersrand Date: August 2018 Place: University of the Witwatersrand, South Africa. 2. The cardiometabolic outcomes in a cohort of women exposed to hyperglycaemia first detected in pregnancy at 3-6 years post-partum in an urban South African setting. Conference: DOHaD around the world seminar series Date:7 th April 2021 Conference proceedings 1. Maternal and neonatal outcomes following a diabetic pregnancy within the context of HIV. Conference: International Congress of Endocrinology (ICE) Date: December 2018 Place: Cape Town, South Africa. 2. The cardiometabolic outcomes in a cohort of women exposed to hyperglycaemia first detected in pregnancy at 3-6 years post-partum in an urban South African setting. Conference: ENDO 2021(virtual) Date: March 20-23rd 2021 3. The metabolic outcomes following hyperglycaemia first detected in pregnancy in Soweto, South Africa Conference: SEMDSA (South Africa) Date: 25-28 th March 2021 Awarded prize for best oral presentation in the “Diabetes” category. viii Publications The research findings from this PhD were written up as three separate manuscripts all submitted to peer-reviewed journals. Two manuscripts were accepted for publication and published by the respective journals and the last manuscript has been submitted and is currently under review. The signed co-author agreement for the use of each publication within this thesis can be found in Appendix A. 1. Nicolaou V, Soepnel L, Huddle KRH, Klipstein-Grobusch, Levitt N & Norris, S.A. (2019) Maternal and neonatal outcomes following a diabetic pregnancy within the context of HIV. International Journal of Obstetrics and Gynecology, 147: 1-9. DOI: 10.1002/ijgo.12956. (Appendix I) 2. Nicolaou V, Soepnel L, Huddle KRH, Levitt N, Klipstein-Grobusch & Norris, S.A. (2020) Maternal and neonatal outcomes following the introduction of oral hypoglycaemic agents for gestational diabetes were comparable to insulin monotherapy within two historic cohorts. South African Medical Journal, 110: 154-158. doi:10.7196/SAMJ. 2020.v110i2.14024.(Appendix J) 3. Nicolaou V, Soepnel L, Huddle KRH, Klipstein-Grobusch, Levitt N & Norris, S.A The cardiometabolic outcomes in a cohort of women exposed to hyperglycaemia first detected in pregnancy at 3-6 years post-partum in an urban South African setting, submitted to PLOS ONE Journal on 31 st July 2021-provisionally accepted subject to minor revisions. (Appendix K) PhD student’s contribution to each publication: The student was responsible for the overall study design and the conceptualisation of each sub- study, as well as project coordination that involved overseeing data collection, designing of standard operating procedures, and training of staff. Data cleaning, statistical analyses and interpretation of results were performed by the student as was the writing and revision of each manuscript. Co-authors provided methodological advice, some statistical assistance, and critically reviewed the manuscripts. ix Abstract Background Hyperglycaemia in pregnancy, encompassing all types of diabetes, is on the rise with an alarming global prevalence of 17%, of which 84% of these being accounted for by hyperglycaemia first detected in pregnancy (HFDP). Alongside the well-established adverse short-term outcomes of poorly managed or untreated disease, HFDP has a significant impact on the future health of both mother and offspring, playing a crucial role in the global diabetes epidemic. Until recently, HFDP, for which the highest prevalence occurs in low to middle income countries (LMIC), played a minor role in the shadow of more obvious determinants of maternal and fetal morbidity in these low-resource settings. More recently it has been recognised as an important public health issue given its immediate burden on maternal health services and the transgenerational and population level impact of the far-reaching ramifications for mother and child alike. Though our knowledge surrounding the burden, determinants, and immediate consequences of HFDP in South Africa (SA) is growing, the appreciation of its long-term consequences is poorly explored. Understanding and appreciating the adverse health consequences of HFDP creates an informed platform for developing health interventions for a condition which is aggressively fuelling the non-communicable diseases (NCD) burden. Objective: Our study seeks to investigate the cardiometabolic outcomes in an urban cohort of black SA women with a history of HFDP, 3-6 years following their delivery, compared to a group of women non-exposed to HFDP on the backdrop of a high human immunodeficiency virus (HIV) burden. Additionally, we explore the safety and efficacy of exposure to oral hypoglycaemic agents (OHAs) on maternal and neonatal outcomes. Design and Methods: An initial retrospective review of all women identified with diabetes in pregnancy, including type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM) and HFDP at the Gestational Endocrine Clinic at Chris Hani Baragwanath Academic Hospital (CHBAH) for the period 2012 through to 2018 was performed in order to investigate the immediate maternal and neonatal outcomes as well as to explore the impact of HIV infection and exposure to OHAs on these outcomes. Twin pregnancies and more than one pregnancy for the same individual x during the time frame were excluded. Secondly, a prospective follow up study of women identified with prior HFDP between 2014 and 2017 were recruited for a cross-sectional analysis between March and November 2019 (3-6 years following their index pregnancy) and compared with women time-matched post-partum, who tested negative for HFDP (non-HFDP group) using the same diagnostic test and criteria. Two hundred and four participants were enrolled of which 103 were exposed and 101 non-exposed to HFDP. All participants were subject to a detailed questionnaire as well as various measurements including anthropometric parameters, blood pressure, the performance of an oral glucose tolerance test (OGTT), fasting serum creatinine, insulin, lipids, and glycated haemoglobin (HbA1C), urine analysis, body composition analysis via dual-energy x-ray (DXA) and ultrasonography of the carotid arteries to determine intima media thickness (cIMT) and the presence of plaque. For the retrospective review, descriptive statistics were used for comparing various maternal and neonatal outcomes by diabetes groups and for statistically significant outcomes, logistic regression analysis was performed using backwards selection following univariate analysis. For the prospective evaluation of HFDP exposed vs. non-exposed HFDP participants, unadjusted and multivariate adjusted odds ratio were estimated for the outcomes T2DM, metabolic syndrome (MetS) and 10-year cardiovascular risk calculated using Framingham risk score (FRS) from logistic regression models. Further multivariate models were designed to explore the potential association of historical maternal factors with the outcomes including fat mass index (FMI), T2DM, and cIMT using logistic or linear regressions. Results: Of the initial 1071 diabetic pregnancies retrospectively identified, majority of the women had pregestational diabetes (57%), with the remainder having HFDP (43%). Within the HFDP group, 51% had “overt” GDM (DIP) vs. 49% “true” gestational diabetes (GDM). Despite good glycaemic control across all the groups (as measured by HbA1C at term), adverse maternal and neonatal outcomes were higher than in the background population with initial maternal HbA1C and body mass index (BMI) being predictors of poorer neonatal outcomes. Overall HIV prevalence within the group was 24%, lowest in those with HFDP (17.4%) and accounted for significantly higher perinatal mortality (PNM) in HIV-infected 9.4% than non-HIV-infected pregnancies 1.8%, p<0.001. The exposure to either oral hypoglycaemic agents (metformin and glibenclamide) versus insulin on immediate maternal and neonatal outcomes in two cohorts of women diagnosed with HFDP, was found to be safe and effective and was only significant for xi an association between glibenclamide and macrosomia. In the prospective, cross-sectional analysis of 103 women exposed to HFDP (identified using 75g OGTT, adopting IADPSG criteria) versus 101 women not exposed to HFDP, higher rates of progression to all three outcomes were noted with 44.6% of those exposed progressing to T2DM (adjusted risk of 10.5(95%CI 3.7-29.5)), 40.8% having MetS (adjusted risk 6.3(2.2-18.1)) and higher cardiovascular risk as identified by FRS (adjusted risk 4.3(1.6-11.5)), however cIMT did not remain significant after adjusting for confounders. Though HFDP exposure was a significant predictor for T2DM, MetS and cardiovascular risk, certain maternal factors present either pre- pregnancy, during the index pregnancy and/or post-partum were significantly associated with the outcomes. HIV, however, was not found to play a role in progression to any of these outcomes. Body composition was significantly altered in women exposed to HFDP versus those non-exposed with significantly higher FMI (13.9 vs. 12.3, p=0.008) and visceral fat indices between the groups and this remained significant after adjusting for confounders. Conclusion: The growing epidemic of obesity, T2DM, and cardiovascular diseases (CVD), particularly amongst LMIC, poses a significant public health burden in addition to the persistent infectious disease scourge. Our study confirmed the significant immediate adverse maternal and neonatal outcomes in women with diabetes in pregnancy and provided reassuring data on the safety and efficacy of OHAs in pregnancy when compared with the gold standard of insulin. Additionally, we identified a group of young black women at high risk of cardiometabolic diseases in the short-term following HFDP exposure. Appreciating these risks on the background of the growing prevalence of HFDP assists in providing evidence to incorporate universal screening programmes for detecting HFDP, expansion of specialized antenatal services for these women in addition to the post-partum interventions including targeted screening, counselling, and lifestyle and/or therapeutic interventions in order to preserve future maternal health and interrupt the intergenerational cycle of chronic diseases. Future prospective studies are needed to explore the best timing and impact of these interventions in curtailing these adverse outcomes and improving cardiometabolic health in this vulnerable group of women. xii Preface “The future belongs to those who believe in their dreams”. Eleanor Roosevelt Pregnancy offers the clinician a unique opportunity to meaningfully affect the outcome of two individuals. Additionally, the post-partum period offers a further opportunity to influence future health for both mother and child alike. With this knowledge, my passion and interest in the field of diabetes in pregnancy, was first sparked when Professor Huddle, Head of Clinical Medicine at the Chris Hani Baragwanath Academic Hospital at the time, offered for me to take over the head position at the Gestational Endocrine Clinic in 2015. My background as a specialist endocrinologist devoted to full-time service in the state hospital was the perfect platform from which to grow and continue a much-needed service like this. The Gestational Endocrine Clinic was first opened at the hospital in May 1983 by Professor Huddle with the intent of improving maternal and fetal outcomes for women with a diabetic pregnancy. The clinic served an impoverished, predominantly Black African community of Soweto. The impact such a service had on maternal health was profound and became clinically evident when two papers were published in the early 1990s and again in 2000, highlighting the dramatic improvement in both maternal and child health outcomes following a diabetic pregnancy when compared to women who remain undetected and untreated. As you can imagine, I was certainly honoured and delighted to take on this opportunity and further continue to improve maternal and fetal health. Moreover, it is extremely rewarding managing such motivated women who had themselves and their offspring’s best interest at heart. Their enthusiasm is evident in their regular attendance and excellent compliance at the clinic, which ensures their improved immediate outcomes for mother and child when compared to the dire consequences for those left untreated. Sadly, on the contrary, their post-partum follow-up was inexistent, apparent in their poor attendance at their prescribed six-week post-partum oral glucose tolerance test to rule out diabetes. More so, I noted that many of these women returned to our adult diabetic clinic some years later, with full-blown diabetes, unfortunately, some with diabetic-related complications. Data mostly from high to middle income countries has demonstrated that these women are highly vulnerable and are at risk of various cardiometabolic consequences. This highlighted the urgent need to implement platforms for ongoing surveillance among these women. I became pre-occupied with the notion that we as clinicians were afforded an excellent opportunity to change the eventuality of these women. Their diabetic pregnancies can be xiii viewed as a window of opportunity to identify and potentially prevent the development of a burdensome and dreadful chronic illness if simple preventative strategies are employed during the peripartum period, including simple achievable measures which embrace lifestyle interventions like dietary measures and regular exercise. To achieve this goal, I realized that I needed to create a better awareness amongst these at-risk individuals, as well as health care professionals and public health services alike. In order to accomplish this, I needed to conduct research. The opportunity came knocking, when I received a call from Professor Shane Norris in August 2017, affording me the opportunity of joining his team at the MRC/Wits Developmental Pathways for Health Research Unit (DPHRU), to embark on a PhD in the field of diabetes in pregnancy and its intermediate to long-term consequences. Of course, I was elated as this was a topic remarkably close to my heart. Furthermore, Professor Norris is renowned for his knowledge and interest in the field of maternal health. The time had finally arrived to make a difference in the lives of these women and their offspring by exploring their post-partum consequences with the goal of providing evidence to change public health policies to prevent various non-communicable diseases. Now, I have reached the end of a very challenging but certainly rewarding journey and I have learnt that “Life to me is a journey-you never know what may be your next destination” David Russel xiv Structure of the thesis This thesis, which was pursued through three academic papers, can be divided into three main sections as indicated below. The introduction section provides context for the research aims through a thorough discussion of the background literature, and presentation of the conceptual framework and problem statement, as well as an overview of the methodology used. This section is followed by empirical findings presented as papers in chapters 3 through 5. The final section encompasses a general discussion that addresses the implications of the results, the study limitations, and proposes areas for future research. Structure of this PhD thesis Chapter 1: Background and literature review Chapter 2: Methodology Section I: Background and study context Chapter 3: Maternal and neonatal short-term outcomes following a diabetic pregnancy in a high HIV prevalence population. Chapter 4: The maternal and neonatal outcomes following the introduction of oral hypoglycaemic agents for gestational diabetes were comparable to insulin monotherapy within two historic cohorts. Chapter 5: The cardiometabolic outcomes in a cohort of women exposed to hyperglycaemia first detected in pregnancy at 3-6 years post- partum in an urban SA setting. Section II: Empirical chapters Chapter 6: Discussion and conclusion Section III: Discussion and conclusion xv xvi Table of Contents Declaration ii Dedication iii Acknowledgements iv Thesis Material and Contributions vi Funding and Awards vi Presentations and publications vii Abstract ix Preface xii Structure of the thesis xiv List of Tables xx List of Figures xxi Nomenclature xxii SECTION 1 BACKGROUND AND STUDY CONTEXT 1 Chapter 1 2 1.1 Problem statement 2 1.2 Conceptual framework 4 1.3 Background and definitions 7 1.4 Diagnostic criteria 8 1.5 Prevalence 10 1.6 Study context within South Africa 12 1.7 Pathophysiology of gestational diabetes 14 1.8 Health systems: Screening 16 1.8.1 Antepartum screening 16 1.8.2 Post-partum screening 17 1.9 Health systems: Clinical management 17 1.9.1 Antepartum 17 1.9.2 Post-partum 19 1.10 Significance of diabetes in pregnancy 20 1.10.1 Short-term consequences 20 1.10.2 Long-term consequences 21 xvii 1.11 HIV and the risk of dysglycaemia and cardiovascular diseases 34 1.12 Gaps in the literature 35 1.13 Justification for the study 36 1.14 Overall aim 36 1.15 Hypotheses 37 Chapter 2- Methodology 38 2.1 Study setting 38 2.2 Study design and population 41 2.3 Data collection and management 42 2.3.1 Data collection instruments and measurements 47 2.3.2. Sample size 51 2.4 Ethical considerations 52 SECTION 2 53 Chapter 3- Maternal and neonatal outcomes following a diabetic pregnancy within the context of HIV. 54 3. 1 Introduction 55 3.2 Materials and Methods 56 3. 3 Results 57 3. 4 Discussion 60 Chapter 4 – The maternal and neonatal outcomes following the introduction of oral hypoglycaemic agents for hyperglycaemia first detected in pregnancy were comparable to insulin monotherapy within two historic cohorts. 71 4. 1. Introduction 72 4.2. Materials and methods 73 4.2.1 Study population 73 4.2.2 Definitions 73 4.2.3 Clinical Practice 75 4.3 Statistical methods 76 4.4. Results 76 4.4.1 Comparison of the two cohorts of HFDP patients 77 4.4.2 Effect of oral hypoglycaemic agents on maternal and neonatal outcomes within 2010-2014 cohort (Table 4.3) 77 4.5. Discussion 78 Chapter 5- Cardiometabolic outcomes of women exposed to hyperglycaemia first detected in pregnancy at 3-6 years post-partum in an urban South African setting 85 xviii 5.1 Introduction 86 5.3 Sample size calculation 88 5.4 Data collection 88 5.4.1 Questionnaire 88 5.4.2 Anthropometrics 89 5.4.3 Biological samples and OGTT 90 5.4.4 Carotid and femoral imaging 90 5.4.5 Biochemistry and laboratory analyses 91 5.5 Outcomes 91 5.6 Ethics 92 5.6 Statistical analysis 92 5.7 Results 93 5.7.1 Baseline and follow up demographics, maternal factors, anthropometrics, and biochemical parameters between the groups. 93 5.7.2 Stratified analysis of maternal outcomes including diabetes, metabolic syndrome and cardiovascular risk based on exposure to HFDP (Table 5.2) 94 5.7.3 Body composition measures between the groups (Table 5.3) 95 5.7.4 Strength of association of HFDP exposure and progression to diabetes, MetS and high CVD risk (Table 5.4) 95 5.7.5 Predictors of FMI and progression to type 2 diabetes and cardiovascular risk using multiple variable logistic and linear regression. 96 5.7.6 HIV influence on outcomes 96 5.8 Discussion 97 SECTION 3 119 Chapter 6- Discussion and Conclusion 120 6.1 Consolidated findings 120 6.2 Hypotheses revisited 124 6.3 Emerging research themes 124 6.3.1 Underestimation of cardiovascular risk in young women 124 6.3.2. Strengthening health care systems in South Africa 131 6.4 Theoretical relevance 136 6.5 Implications 138 6.6 Strengths and limitations 139 6.7 Future studies 140 6.8 Conclusion 142 xix 7.0 References 143 8.0 Appendices 177 APPENDIX A: AUTHORS/CO-AUTHORS DECLARATION 177 APPENDIX B: MATERNAL INFORMATION AND CONSENT SHEETS 180 APPENDIX C: QUESTIONNAIRE 186 APPENDIX D: PROTOCOLS FOR BIOSPECIMEN COLLECTION 197 APPENDIX E: PROTOCOL FOR PERFORMING OGTT AND PROCESSING OF BLOODS 214 APPENDIX F: PROTOCOL FOR DXA MEASUREMENT 218 APPENDIX G: PROTOCOL FOR CAROTID AND FEMORAL INTIMA MEDIA THICKNESS ASSESSMENT 221 APPENDIX H: ETHICS CLEARANCE CERTIFICATES 226 APPENDIX I: PUBLISHED PAPER I 229 APPENDIX J: PUBLISHED PAPER II 238 APPENDIX K: SUBMITTED PAPER III 243 APPENDIX L: PLAGIARISM DECLARATION 291 xx List of Tables Table 1. 1 Varying diagnostic criteria for HFDP .................................................................. 9 Table 1. 2 IADPSG criteria utilized for the diagnosis of HFDP. .......................................... 10 Table 1. 3 Common risk factors for gestational diabetes mellitus (79) .............................. 17 Table 1. 4 Adverse outcomes associated with uncontrolled hyperglycaemia in pregnancy. ........................................................................................................................................ 21 Table 1. 5 HAPO study primary and secondary outcomes ................................................. 22 Table 1. 6 Long-term consequences following HFDP. ........................................................ 22 Table 1. 7 Summary of systematic reviews for progression to T2DM, CVD and MetS following HFDP. ............................................................................................................... 33 Table 2. 1 Statistical analysis overview ............................................................................ 43 Table 2. 2 Overview of various cardiovascular risk prediction models .............................. 49 Table 2. 3 The sample size required to determine the following outcomes among black SA women with a history of HFDP ......................................................................................... 52 Table 3. 1 Demographic, obstetric, comorbidity, and diabetes characteristics of participants with T1DM, T2DM, and HFDP. ...................................................................... 64 Table 3. 2 Neonatal and maternal outcomes of participants with T1DM, T2DM, and HFDP. ........................................................................................................................................ 65 Table 3. 3 Comparison of baseline characteristics in metformin monotherapy and glibenclamide-exposed groups versus insulin monotherapy. ............................................ 66 Table 3. 4 a. Definition and criteria for diagnosis of diabetes in pregnancy, including T1DM, T2DM, and HFDP. b. Definition of maternal and neonatal outcomes. ................... 67 Table 4. 1 Comparison of maternal characteristics, glycaemic control, and outcomes between 1992-2002 and 2010-2014 HFDP pregnancy cohorts as diagnosed by 100g oral glucose tolerance test and random plasma glucose ......................................................... 81 Table 4. 2 Maternal and neonatal outcomes for the 1992-2002 and 2010-2014 HFDP pregnancy cohorts ........................................................................................................... 82 Table 4. 3 Comparison of oral agents vs. insulin on maternal and neonatal outcomes ..... 83 Table 5. 1 Maternal demographics, characteristics and outcomes at index pregnancy and follow-up. ...................................................................................................................... 100 Table 5. 2 Stratified analysis of maternal outcomes by exposure to HFDP. ..................... 104 Table 5. 3 Maternal body composition outcomes by exposure to HFDP. ......................... 107 Table 5. 4 Frequency of the outcome’s diabetes, metabolic syndrome and CVD event as predicted by FRS and the adjusted odds ratio for the association with a history of HFDP 109 Table 5. 5 Linear regression model for log-transformed FMI. Values are standardized regression coefficients (β) with 95% confidence interval, and p-values. ......................... 110 Table 5. 6 Multivariate analysis of factors associated with risk of progression to diabetes. Values are standardized odds ratios (OR) with 95% confidence interval, and p-value. .... 112 Table 5. 7 Linear regression model for log-transformed carotid intima media thickness (cIMT). Values are standardized regression coefficients (β) with 95% confidence interval, and p-value. .................................................................................................................. 114 xxi Table 5. 8 Definitions of diagnostic criteria for HFDP, maternal pregnancy factors and outcomes ....................................................................................................................... 116 Table 6. 1 Summary table of objectives and findings ...................................................... 121 List of Figures Figure 1. 1 Conceptual model adapted (21) ........................................................................ 6 Figure 1. 2 Timeline of evolution of criteria to diagnose HFDP 1964-present (36) ............... 8 Figure 1. 3 Median (interquartile range) prevalence (%) of HFDP by WHO region, 2005– 2015 (48) ......................................................................................................................... 11 Figure 1. 4 Country-specific prevalence of HFDP according to different diagnostic criteria (47). ................................................................................................................................. 12 Figure 1. 5 Leading causes of mortality with percentages (%), diabetes mellitus being the second commonest (55). .................................................................................................. 13 Figure 1. 6 Changes in glucose homeostasis during pregnancy (66). ................................. 15 Figure 1. 7 Potential major determinants for the progression from HFDP to T2DM and other co-morbidities based on a life course perspective (22) ............................................ 23 Figure 1. 8 Modifiable and non-modifiable risk factors shared by CVD and T2DM (137) ... 26 Figure 1. 9 Natural history of cardiovascular diseases on the background of various risk factors (144) .................................................................................................................... 27 Figure 1. 10 Associations between pre-pregnancy risk factors, adverse pregnancy outcomes, and post pregnancy cardiometabolic risk factors and outcomes (84) .............. 30 Figure 1. 11 Intergenerational cycle of diseases following HFDP. ..................................... 34 Figure 2. 1 Map displaying Soweto, Johannesburg in the Gauteng province. .................... 40 Figure 2. 2 Flow diagram of the study design ................................................................... 46 Figure 3. 1 Neonatal outcomes in metformin monotherapy and glibenclamide-exposed groups versus insulin monotherapy in participants with T2DM and HFDP. ....................... 68 Figure 3. 2 Neonatal outcomes in HIV-infected and uninfected participants with T1DM, T2DM, and HFDP. ............................................................................................................ 69 Figure 3. 3 Flow chart of clinical practice for patients attending the gestational endocrine clinic. ............................................................................................................................... 70 Figure 5. 1 Flow diagram of the study and eligible participants ..................................... 118 Figure 6. 1 Prevention options in women with gestational diabetes mellitus (136). ........ 130 Figure 6. 2 Conceptual framework revisited ................................................................... 137 xxii Nomenclature ACOG American College of Obstetricians and Gynaecologists ADA American Diabetes Association ANOVA Analysis Of Variance aOR Adjusted odds ratio BMI Body mass index CAD Coronary artery disease CCA Common carotid artery CHBAH Chris Hani Baragwanath Academic Hospital cIMT Carotid intima media thickness CS Caesarean section CT Computerised tomography CRP C-reactive protein CVD Cardiovascular disease DIP Diabetes in pregnancy DPHRU Developmental Pathways for Health Research Unit DXA Dual energy X-ray absorptiometry FIGO International Federation of Gynaecology and Obstetrics fIMT Femoral intima media thickness FMI Fat mass index FRS Framingham risk score GDM Gestational diabetes GDP Gross domestic product HAART Highly active antiretroviral therapy HAPO Hyperglycaemia and Pregnancy Outcome HbA1c Glycated haemoglobin HC Hip circumference HDL High-density lipoprotein HDP Hypertensive disorders of pregnancy HFDP Hyperglycaemia first detected in pregnancy HIV Human Immunodeficiency Virus HMIC High to middle income countries HTN Hypertension xxiii hsCRP High sensitivity C-reactive protein IADPSG International Association of Diabetes and Pregnancy Study Group ICAM-1 Intercellular Adhesion Molecule 1 IDF International Diabetes Federation IL-6 Interleukin 6 LBW Low birth weight LDL Low-density lipoprotein LGA Large for gestational age LMIC Low to middle income countries MRC Medical research council MRI Magnetic resonance imaging MetS Metabolic syndrome NCD Non-communicable diseases NDDG National Diabetes Data Group NHANES National Health and Nutrition Examination Survey NICE National Institute for Health Care Excellence OGTT Oral glucose tolerance test OHA Oral hypoglycaemic agents PAI-1 Plasminogen activator inhibitor-1 PIH Pregnancy-induced hypertension PNM Perinatal mortality PWV Pulse wave velocity RCT Randomised controlled trial RDS Respiratory distress syndrome REDCap Research Electronic Data Capture SA South Africa SAT Subcutaneous adipose tissue SBP Systolic blood pressure SES Socioeconomic status SGA Small for gestational age SSA Sub-Saharan Africa T1DM Type 1 diabetes mellitus T2DM Type 2 diabetes mellitus TG Triglycerides xxiv TNF-α Tumour necrosis factor alpha USA United States of America VAT Visceral adipose tissue VCAM-1 Vascular cell adhesion protein 1 WC Waist circumference WHR Waist: hip ratio WHO World Health Organisation xxv Units and Symbols The International System of Units (SI) has been used throughout this thesis. Of note, glucose is reported in mmol/l throughout this thesis. % percentage > greater than < smaller than ≥ greater than or equal to ± plus/minus g gram hr hour kg kilogram kg/m 2 kilogram per square metre km 2 square kilometre mmol/L millimoles per litre mmHg millimetre of mercury 1 SECTION 1 BACKGROUND AND STUDY CONTEXT “Wherever the art of medicine is loved, there is also a love of humanity”. Hippocrates 2 Chapter 1 1.1 Problem statement Worldwide, type 2 diabetes mellitus (T2DM) remains an important area of public health concern since it is one of the major causes of early illness and death. The increasing global prevalence remains concerning as it contributes to an increasing health and financial burden around the world. Estimates from the International Diabetes Federation (IDF) suggests that diabetes affects about 463 million people globally which is projected to increase to 700 million people by 2040 (1), with the prediction that Africa will be subject to the greatest increase in numbers. In 2014, 14.2 million adults aged 20–79 years had diabetes, with over two-thirds of them being unaware of their diabetic status (2). Women are not immune from this with 199 million of them currently living with diabetes, with predictions that this will increase to 313 million by 2040 (1). The prevalence of T2DM in African women doubled, from 4.1% in 1980 to 8.9% in 2014 (3). A recent systematic review exploring the prevalence of dysglycaemia in African women of childbearing age, found the prevalence of T2DM was 7% (4). This growing burden of diabetes, is mostly driven by epidemiologic, demographic, and nutritional changes (5,6). Whilst type 1 diabetes mellitus (T1DM) and T2DM is a state of glucose intolerance occurring outside of pregnancy, gestational diabetes (GDM), recently renamed by the World Health Organization as hyperglycaemia first detected in pregnancy (HFDP), collectively defines it as “any degree of glucose intolerance with onset or first recognition during pregnancy” (7,8).This includes glucose readings that fall within the impaired glucose tolerance (IGT) diagnostic range termed “true” GDM, as well as those within the diagnostic range for diabetes termed “overt” GDM or diabetes in pregnancy(DIP)(9).The prevalence of HFDP, the most commonly encountered hyperglycaemic disorder encountered in pregnancy has doubled in the past 10-15 years owing to the obesity epidemic (10,11) . Sadly, little is known about prevalence rates of HFDP in Africa, with figures varying according to different regions partly due to differences in study populations, survey methodology, and diagnostic methods used (12). A major barrier to appreciating the burden of HFDP in both developed and developing countries is the lack of consensus on the definition of HFDP as well as the absence of universally accepted standards for diagnosis and screening (13). Additionally, HFDP plays a minor role in the shadow of more obvious determinants of maternal and fetal morbidity in low-resource settings. It has only more recently been acknowledged in developing countries as a public health issue given its far- 3 reaching ramifications for mother and child alike. Alongside the well-established adverse short-term outcomes of poorly managed or untreated disease, HFDP, more specifically GDM, can have a marked impact on the future health of both mothers and their offspring, with HFDP playing a significant role in the global diabetes epidemic together with increasing cardiovascular risk and disease (CVD) and development of the metabolic syndrome (MetS) in these young women (14–16). Epidemiological evidence has consistently shown that women with a prior history of HFDP have a 30-84% chance of having a recurrence of HFDP in a subsequent pregnancy (17,18), 20-40% chance of developing MetS within 2-20 years (16)and 17-63% developing T2DM within 5-15years (14). Furthermore, if a woman with diabetes becomes pregnant, her offspring is at an increased risk of developing T2DM, obesity and CVD in adulthood, (19) thereby accelerating the intergenerational risk of T2DM, obesity and CVD. This has implications for the health of the population. Elucidating the causes of these disorders, the prediction and possible prevention or delay of the development of these chronic disease states is of paramount importance. Raising public awareness of this condition and the risks associated with it, remain an important area of public health and this will certainly pave the way for improved maternal and fetal health. A further novel area of interest is the growing problem of diabetes amongst those with human immunodeficiency virus (HIV). Following the successful implementation of combination antiretroviral therapy (ART), and the resultant prolongation of life expectancy, several chronic conditions including diabetes mellitus and CVD has emerged (19). The mechanisms of these are multifactorial and includes shared environmental risk factors for diabetes such as sedentary lifestyle as well as HIV-specific effects encompassing chronic inflammation and ART related factors (20). The risk of developing diabetes together with other metabolic perturbations, such as dyslipidaemia and insulin resistance, places these individuals at risk for CVD. Taken together, HFDP and HIV co-existing in pregnancy potentially identifies a population of young women at risk of T2DM and CVD, which is of particular relevance in low to middle income countries (LMIC) where the heaviest burden of the HIV epidemic is felt. Furthermore, the contribution of HIV on the background of HFDP and the risk of developing diabetes and CVD is not studied or known. In conclusion, the diabetes and obesity epidemics together with increasing CVD among women of reproductive age, poses an enormous public health challenge globally. Both the immediate burden on maternity health services, as well as the transgenerational and population level 4 impact of these metabolic diseases, highlights the need to focus on better antenatal detection and management together with screening and interventional measures during the post-partum period of this condition if we are to curtail this public health impact. 1.2 Conceptual framework The framework for this study depicted in Figure 1.1, which was adapted from Zhang, C. et al. (21), has been designed and structured around the existing body of knowledge in the field of HFDP, mostly extracted from high to middle income countries (HMIC). Our setting which derives from a LMIC, is unique in that our population is predominantly Black African with a high burden of HIV disease. Furthermore, socioeconomic transitions have exposed women to deleterious living conditions, poor dietary habits, and sedentary behaviours. The indirect consequences of both the unique environmental and societal factors among these black women and how they shape the maternal trajectories of the measured outcomes are unknown. On this background, the conceptual framework was designed to incorporate all these distinctive aspects together with acknowledging the significant role of HFDP exposure and how this influences the development of cardiometabolic diseases. The framework adopts a life-course approach and recognises the potential independent influence of non-modifiable and modifiable pre-conception, antenatal and post-partum maternal factors on these outcomes. Non-modifiable factors include ethnicity, age and genetics whilst modifiable pre-conception factors which may interact and influence both immediate and short-term maternal outcomes include maternal body mass index (BMI), co-morbidities including hypertension (HTN), as well as various socioeconomic factors (22,23). Additionally the HIV epidemic which disproportionately affects women in South Africa (SA) 26% (24), is another factor that cannot be ignored; its influence within this context has yet to be explored. Antenatal factors, including disease- related factors including severity of hyperglycaemia and management together with other pregnancy co-morbidities like maternal BMI contribute to these outcomes. Post-partum factors like weight gain and breastfeeding have also been shown to influence the outcomes measured (26–29). For the purposes of this study each component of the life-course model will be explored within the context of exposure to HFDP in terms of its role on the outcomes in an attempt to disentangle various mediators from HFDP exposure. Whilst this study aims to explore and define the post-partum cardiometabolic consequences following HFDP, including progression to T2DM and MetS as well as assessing cardiovascular risk/disease by utilising a life-course approach, the additional interplay of HIV and ART on this relationship will be 5 evaluated, given the known metabolic perturbations including insulin resistance, diabetes and dyslipidaemia in HIV-infected adults since the introduction of effective ART. 6 Ø Ethnicity Ø Parity Ø Pre-pregnancy BMI Ø Socioeconomic factors: SES score and housing density Ø Education Ø Smoking Ø Alcohol Ø Exercise Ø Comorbidities: HIV and HTN Ø Family history: diabetes and CVD Maternal pre-pregnancy factors Maternal pregnancy factors Maternal postpartum factors Progression to the outcomes measured at 3-6 years following index pregnancy. Type 2 diabetes Cardiovascular risk and disease Body composition Metabolic syndrome Ø Breastfeeding Ø Contraception Ø Cardiovascular risk factors Ø Weight gain Ø Exercise Ø Alcohol and smoking Ø Figure 1. 1 Conceptual model adapted (21) Immediate maternal outcomes at delivery Caesarean section Preterm delivery Hypertensive disorders of pregnancy Mortality Ø Age Ø BMI and gestational weight gain Ø Blood pressure Ø Biochemical parameters Ø Therapeutics Ti m el in e: p er ip ar tu m p er io d HFDP exposure 7 1.3 Background and definitions Since the first description of diabetes in pregnancy in 1824 by Bennewitz in Germany (29), the landscape of HFDP has remained a dynamic entity with the prevalence, definitions, screening protocols and diagnostic criteria continuously changing from as early as the 1960’s. The lack of a global approach to HFDP amongst both international and local organisations has created havoc in this field with the ultimate result being variations in prevalence and practice within countries. Further compounding this problem is the fact that LMIC, including sub-Saharan Africa (SSA) often must adopt different strategies given their resource poor infrastructure and unavailability of diabetes care services. There remains a lot of controversy surrounding HFDP, from classification to screening to diagnosis and treatment. The prior definition of GDM as a glucose-intolerant state with onset or first recognition during pregnancy (30) was recently challenged by the International Association of Diabetes in Pregnancy Study Group (IADPSG) (8). They appreciated that a significant proportion of women with undiagnosed pre-existing diabetes (DIP) were being referred to as “GDM” when it was clearly evident that these women have diabetes which incidentally was first detected upon screening during pregnancy. The evidence for this being their degree of hyperglycaemia or their early manifestation (before 24 weeks). The varying terminology adopted for describing HFDP varies among national organisations. More recently, the World Health Organisation (WHO ) (7), American Diabetes Association (ADA) (31) and the International Federation of Gynaecology and Obstetrics (FIGO) (32), have adopted the IADPSG recommendations to differentiate between pregnant females with probable pre-existing diabetes first manifesting in pregnancy, referred to as “overt” diabetes or DIP, versus, a transient manifestation of pregnancy-related insulin resistance, referred to as “true” GDM, which mostly develops during the second half of pregnancy. This new definition broadly grouped together as HFDP intends to differentiate between the varied severities of HFDP, with GDM being distinguished as the milder degree of HFDP and DIP the more serious form. For the purpose of this thesis, the terms pre-gestational diabetes in pregnancy (pre-conceptional type 1 or type 2 diabetes) and HFDP (HFDP) which includes “overt” GDM/ (DIP), and “true” GDM will be used to distinguish the different types of diabetes. HFDP was diagnosed according to the internationally recommended IADPSG criteria following risk-factor based screening, which is in line with the local clinical practice at the Chris Hani Baragwanath Academic Hospital (CHBAH) in Soweto, Johannesburg. It is important to note, that prior to 8 the introduction of the term HFDP, most studies used the term GDM to describe any hyperglycaemia detected in pregnancy, hence most likely including the DIP subgroup. Current standing is that most regional bodies have moved towards adopting the latest WHO guidelines (7). 1.4 Diagnostic criteria The ongoing debate as to the optimal approach for the diagnosis of HFDP has resulted in a variety of diagnostic criteria being endorsed by different organisations (Table 1.1). Historically, diagnostic testing and thresholds for HFDP were in existence from the early 1960’s. The original diagnostic criteria for HFDP by O’Sullivan and Mahan (33) were not based on the perinatal risk but instead were based on the maternal risk of developing diabetes later in life. Since then, there has been a shift to changing the criteria to focus on infant outcomes with two general oral glucose tolerance test (OGTT) tests predominating, one based on a 2-hour, 75g OGTT with two plasma glucose values and diagnostic criteria like those used outside of pregnancy(34) , and another one based on a 100g 3-hour OGTT, with four pregnancy-specific plasma glucose cut-offs (35). Figure 1.2 outlines a historical timeline regarding the introduction of the different diagnostic tests advocated by varying international authorities. Figure 1. 2 Timeline of evolution of criteria to diagnose HFDP 1964-present (36) 9 To date, an absence of a scientific consensus remains regarding an optimal cut-off point to diagnose women with HFDP. Different authorities utilise different glucose challenge tests with varying glucose thresholds, which has led to much confusion and inconsistencies amongst countries resulting in differences in health system practices. After several international workshops and many decades of research, there is still no unified global approach to HFDP. Table 1. 1 Varying diagnostic criteria for HFDP Diagnostic test Blood glucose values (mmol/L) FPG 1hr 2hr 3hr Diagnostic criteria O’Sullivan and Mahan criteria 1964 100g OGTT (3 hour) 5.0 9.2 8.1 6.9 2 or more values above limit O’Sullivan and Mahan criteria 1973 50g OGTT (1 hour) 7.2 If 1hr diagnostic threshold exceeded, to perform 100gm OGTT National Diabetes Data Group (NDDG), 1979 100g OGTT (3 hour) 5.8 10.6 9.2 8.1 2 or more values above limit Carpenter and Coustan,1982 100g OGTT (3 hour) 5.3 10.0 8.6 7.8 2 or more values above limit World Health Organization (WHO),1999 75g OGTT (2 hour) 7.0 7.8 1 value above limit American Diabetes Association (ADA), 2009 100g OGTT/ 75g OGTT (2 hour) 5.3 10.0 8.6 2 or more values above limit IADPSG 2010 75g OGTT (2 hour) 5.1 10.0 8.5 2 or more values above limit WHO 2013 75g OGTT (2 hour) 5.1 10.0 8.5 2 or more values above limit 10 SEMDSA (SA) 2012 75g OGTT (2 hour) 7.0 7.8 As per WHO 1999 SEMDSA (SA) 2017 75g OGTT (2 hour) 5.1 10.0 8.5 As per IADPSG 2010 Abbreviations: IADPSG International Association of Diabetes and Pregnancy Study Group, WHO World Health Organisation, SEMDSA Society for Endocrinology, Metabolism and Diabetes of South Africa, FPG fasting plasma glucose Following the outcomes of the Hyperglycaemia and Adverse Pregnancy Outcomes (HAPO) study, in which a linear relationship between blood glucose and adverse pregnancy outcomes was demonstrated (37), the IADPSG proposed lower diagnostic cut-offs thresholds to indicate a positive diagnosis which has subsequently been adopted by the WHO in 2013 (7) and other organisations. This landmark study investigated the adverse maternal and neonatal outcomes associated with various degrees of hyperglycaemia following a 75g OGTT, in order to work towards an international consensus for outcome-based criteria for HFDP. The resulting IADPSG criteria, shown in Table 1.2 distinguish between GDM and DIP diagnosed in pregnancy. The diagnostic threshold for GDM is lower than previously used criteria such as the Carpenter and Coustan (35)or National Diabetes Data Group (NDDG) criteria for a 100g OGTT(38). Eighteen percent of women would have met criteria for HFDP if we applied the IADPSG criteria retrospectively to the HAPO cohort (39). Table 1. 2 IADPSG criteria utilized for the diagnosis of HFDP. GDM DIP Fasting plasma glucose ≥5.1 mmol/L ≥7.0 mmol/L One-hour plasma glucose ≥10.0 mmol/L Two-hour plasma glucose ≥8.5 mmol/L ≥11.1 mmol/L Random plasma glucose / HbA1c ≥11.1 mmol/L/ ≥6.5% Abbreviations: GDM gestational diabetes, DIP diabetes in pregnancy 1.5 Prevalence Over the years an increase in the worldwide prevalence of HFDP has been observed. This increase appears to parallel the increasing prevalence of T2DM and obesity. Whilst HFDP prevalence is known to be influenced by ethnicity and genetic susceptibility, the nutrition 11 transition is a significant contributing factor towards this increased prevalence (40). According to the International Diabetes Federation (IDF), in 2017 there were approximately 131.4 million live births globally to women aged 20-49 years. Of those births, 16.2% (21.3 million) were affected by hyperglycaemia during pregnancy; 86.4% involved GDM, 7.4% involved overt diabetes first detected in pregnancy (DIP) and 6.2% involved pre-existing diabetes diagnosed before pregnancy (42). Although variabilities exist across countries for HFDP screening (43) and diagnostic cut-off points for the OGTT (44), reported prevalence’s are 2%–6% for Europe (43), 7% for North America (32), and 1%–9% and 4%–24% for white British and South Asian women, respectively, in England and Southern Ireland (45). Of concern is that almost 90% of cases of HFDP occur in LMIC, where access to maternal healthcare is limited (46). This was highlighted in a recent review (47) which included 77 studies representing both HMIC and LMIC in an effort to appreciate the global prevalence of HFDP. Overall, North Africa and the Middle East were found to have the highest prevalence of HFDP with Europe having the lowest (Figure 1.3). Figure 1. 3 Median (interquartile range) prevalence (%) of HFDP by WHO region, 2005– 2015 (48) Since prevalence estimates are subject to diagnostic criteria applied, the same study specified varying diagnostic criteria when giving country specific estimates with notable variations within and between countries (Figure 1.4). Since the introduction and widespread adoption of 12 the IADPSG criteria, in which a lower threshold of glucose is used to diagnose HFDP, the prevalence of HFDP has further increased by two to threefold and even up to sevenfold compared to the previous criteria (49–51). In Africa, limited representation and varying diagnostic criteria limit the accuracy of determining the prevalence as shown by a metanalysis in 2014 which demonstrated that the prevalence is highly variable ranging from 0-14% (11) A more recent metanalysis (52), including 22 studies adopting the latest diagnostic criteria indicated that the pooled prevalence for Africa was 13.6% and 14.2% for sub-Saharan Africa (SSA). Overall, prevalence estimates across populations are fraught with difficulties with numerous factors at play. This includes the employment of varying screening approaches and diagnostic criteria as well as differing inherent characteristics of the study population. Abbreviations: C &C Carpenter and Coustan criteria, IADPSG International Association of Diabetes and Pregnancy Study Groups, NDDG National Diabetes data groups, WHO World Health Organisation, other included International Classification of Diseases codes and local guidelines or criteria Figure 1. 4 Country-specific prevalence of HFDP according to different diagnostic criteria (47) 1.6 Study context within South Africa Urbanisation and recent socio-economic changes in SA have fuelled a rapid nutritional transition and an epidemiological shift especially in the urban black population (53). . The nutrition transition is characterised by a rapid shift towards high fat and sugar consumption, 13 refined foods, acculturation, and increased physical inactivity (54). This is accompanied by a dramatic shift in the burden of disease from communicable disease like HIV to non- communicable diseases (NCD) like diabetes and HFDP. In practice however, countries in transition are found to display a double burden of NCDs and persistent infectious diseases as well as introduction of new or re-emerging infectious diseases such as HIV and tuberculosis. Furthermore, this burden is amplified by a lack of access to diabetes prevention, diagnosis and appropriate management (55), with an estimated 70% of people with diabetes in Africa being unaware of their diagnosis (42). The overall impact of this is highlighted by the fact that diabetes is the second leading cause of death in SA and the top cause of death amongst SA females (56) (Figure 1.5). Figure 1. 5 Leading causes of mortality with percentages (%), diabetes mellitus being the second commonest (55) The knowledge surrounding HFDP is slowly gaining momentum, however, detection of disease and access to specialist care facilities remain limited for the majority of pregnant women with diabetes and as a result these women and their offspring experience poor outcomes and high complication rates. Prevalence rates are limited to those areas with tertiary care facilities with currently only a few local prevalence studies having been conducted for HFDP in SA. Two small studies (56,57) found the prevalence of HFDP to be 8.8% in some regions, 14 though this figure is most likely an under-representation given the lack of universal screening (1)and poor referral of high-risk patients for screening from the local clinics to tertiary centres. Two more recent studies from Johannesburg, employing universal screening and the same diagnostic criteria , found varying prevalence’s of GDM with 25% (1) found at a local clinic, and 9% at a tertiary hospital in the Soweto region(59). Laboratory inaccuracies may have accounted for these varying prevalence’s. Universal screening for HFDP is currently not adopted in SA (60), given our poor and often lacking resources. The detection of HFDP at best, is based on maternal risk-factor identification at the local clinics followed by referral to a specialist centre for the performance of an OGTT. Furthermore, disparities exist in screening methods and selection of risk factors which are used for screening methods throughout the various centres in the country. Needless to say, HFDP in SA remains under-reported (58) and for the most part poorly managed. Though our knowledge surrounding the burden, determinants, and immediate consequences of HFDP in SA is growing, the appreciation of its long-term consequences is poorly explored in our setting. Understanding and appreciating the adverse health consequences of HFDP creates an informed platform for developing health interventions for a condition which is aggressively fuelling the NCD burden. Our study seeks to investigate the cardiometabolic profiles of women with a history of HFDP 3-6 years following their delivery compared to women nonexposed to HFDP on the backdrop of HIV. 1.7 Pathophysiology of gestational diabetes Several metabolic and hormonal adaptations occur during pregnancy to ensure adequate maternal transfer of nutrients to the growing foetus. Physiologically, as the pregnancy progresses, an increase in insulin resistance is naturally observed, peaking during late pregnancy (61,62). This is due to the maternal tissues, particularly skeletal muscle and adipose tissue, becoming less sensitive to insulin and as a result maternal glucose disposal decreasing by approximately 50% (63). As glucose readily crosses the placenta (62,65), the insulin- resistant state prevents or reduces maternal somatic tissues from absorbing the blood glucose thus shunting it to the foetus. In response to the insulin resistant environment, the maternal pancreatic β-cells increase insulin secretion in order to maintain glucose homeostasis; insulin secretion is thought to increase by 200-250% in a normal pregnancy (62). Figure 1.6 illustrates 15 how maternal insulin sensitivity decreases as pregnancy progresses. This results in more insulin production which facilitates glucose being shunted to the foetus. The increased insulin resistance of pregnancy subsides very quickly following delivery suggesting that pregnancy- related hormones are key factors in achieving the insulin-resistant state. Figure 1. 6 Changes in glucose homeostasis during pregnancy (66) Pregnancy can be viewed as a “stress test” for the development of glucose intolerance. The hormonal changes of pregnancy may incite an unmasking of the genetic predisposition for T2DM in women manifesting with GDM. Appearance of many hormones, including human placental growth hormone human placental lactogen, oestrogen, progesterone, prolactin, cortisol, and tumour necrosis factor-alpha (TNF-α), particularly during late pregnancy, triggers a state of insulin resistance by counteracting the effects of insulin thereby increasing insulin requirements. The promotion of a state of insulin resistance and hence a diabetogenic environment is incurred through these changes in the hormonal milieu along with the manifestation of subclinical inflammation (64,67). Exaggeration of this state in a pregnant woman ultimately manifests as GDM. Major determinants for GDM are the result of both pancreatic β-cell insufficiency and increased insulin resistance, with genetic factors and other socioeconomic and lifestyle processes contributing too (68). Gestational diabetes mellitus usually resolves after delivery with approximately 95% of affected women reverting to normal 16 glucose metabolism after delivery of their babies. The removal of the placenta and pregnancy hormones thought to facilitate the insulin-resistant state allows for normal glucose metabolism to be reinstated (69). 1.8 Health systems: Screening 1.8.1 Antepartum screening The issue of universal versus risk-factor based screening remain an area of controversy and is mostly dictated by different organisations based on local prevalence rates and resource availability. Ideally, universal screening should be applied to high-risk pregnant women in order to identify all potential HFDP cases. However, cost-constraints often dictate a more conservative approach with selective screening predominating. A recent study performed in SA where risk-based screening is mostly employed, showed that adopting universal screening locally would significantly increase HFDP prevalence rates (59), which has significant implications in a country with limited resources. The epidemiological studies of risk factors for HFDP are limited and are influenced by numerous confounders (70,71). The comparison of different findings across studies remains challenging given the inconsistencies in diagnostic criteria and measurements of risk factors. Despite this several risk factors emerge consistently and unsurprisingly, overlap with those for T2DM. These include, age, obesity, family history of T2DM and certain ethnic heritages (72) More specific to HFDP are, a prior history of HFDP, previous fetal macrosomia and previous unexplained perinatal death (Table 1.3)(52) . Interestingly one strong risk factor for HFDP includes varying ethnicity (73). These ethnic/racial disparities have best been appreciated in countries like USA, Canada and Australia in which multi-ethnic populations are present (70,71). Women found to have any one of these risk-factors should be offered a screening test for HFDP. Of importance, up to 20% of women diagnosed with HFDP have no risk factors(74) with a recent meta-analysis suggesting that a risk-factor based screening approach is a poor predictor of women who have an abnormal glucose tolerance test (75). The same conclusion was reached by a local study (59). Not all HFDP pregnancies are exposed to these risk factors, and selective screening based on risk factors has been found to miss from 24%-48% of cases that would be identified by universal screening (76,77). Moreover, to further complicate 17 matters, practises with respect to timing of screening also varies, with between 24-28 weeks most commonly being practiced (78). Table 1. 3 Common risk factors for gestational diabetes mellitus (79) 1. Older age 2. High weight 3. Pre-pregnancy BMI of ≥27 kg/m2 4. High parity 5. Having previously given birth to a baby weighing 4 kg or more. 6. A family history of T2DM 7. A previous diagnosis of HFDP 8. Belonging to a particular ethnic group (Hispanic, African, African American, Asian) Abbreviations: BMI body mass index, HFDP hyperglycaemia first detected in pregnancy. 1.8.2 Post-partum screening The increasing prevalence of diabetes and the MetS among all demographics, including women of childbearing age, has challenged the prior notion that HFDP mostly resolves after delivery. A growing body of literature has shown that post-partum follow-up of women with HFDP remains critical since these women are at high risk of developing T2DM and other cardiometabolic diseases (80–87). Nonetheless, a substantial proportion of women fail to return for follow-up (88) owing to either patient or provider-related barriers with this area remaining grossly underprioritized. Studies have identified patient unawareness, lack of time, fear of receiving bad news and other social/economic reasons for patients not adhering to follow-up visits (89,90). Inconsistent guidelines regarding screening intervals, screening methods and lack of patient ownership are a few identified provider-related barriers. In SA, the overburdened health care system is a major rate limiting hurdle in providing post-partum screening with knowledge surrounding additional barriers in LMIC being very poorly explored (91). 1.9 Health systems: Clinical management 1.9.1 Antepartum Since hyperglycaemia, is a major driver of maternal and fetal morbidity, treatment of HFDP is effective for preventing adverse pregnancy outcomes. The best care available for mothers with diabetes in pregnancy, should ideally be offered by a multidisciplinary team of specialists, 18 where three major factors need to be considered, severity of glucose intolerance, maternal response to therapy and effect on fetal growth. Worldwide, the mainstay of therapy remains diet and exercise, which is adequate for the majority of HFDP patients (9,72,92). Whilst the ideal diet remains controversial, a general consensus of limiting excessive carbohydrate intake and distributing it evenly throughout the day is prescribed. A systematic review found that a low glycaemic index diet to be the most effective at managing maternal glucose levels and reducing birth weight in the offspring (93). Frequent self-monitoring of capillary blood glucose levels pre- and post-prandially remains imperative and directs therapy. For those not achieving normoglycaemia despite this (10-30%), pharmacotherapy in the form of oral hypoglycaemic agents or insulin is required. Whilst insulin remains the standard of care since it does not cross the placenta, has not been found to be teratogenic and its long-term safety has been established (32,92), OHA’s have gained a lot of interest over the past two decades as they are inexpensive, and easier to use than insulin making them particularly attractive for LMICs. Currently, no oral agents are licensed for use during pregnancy given the that both agents cross the placenta and their long-term safety data is limited which has translated into restrictions in their usage, particularly in United States of America (USA). Their use in pregnancy is endorsed by some major guidelines (60,71,93–95) with countries like Europe and SA frequently using them as first line agents (96). Over the last while, the efficacy and safety of these agents in the treatment of HFDP have been evaluated through several meta-analyses, the results of which have been inconsistent due to lack of evidence from head-to-head randomised controlled trials (RCT). In one of the more recent meta-analysis (96), glyburide was ranked the worst with the highest incidence of macrosomia, pre-eclampsia, hyperbilirubinemia, neonatal hypoglycaemia, preterm birth, and low birth weight (LBW), despite the fact that it was as effective as insulin, whilst metformin (together with insulin when required), had the lowest risk of macrosomia, pregnancy HTN, large for gestational age (LGA), respiratory distress syndrome (RDS), preterm birth and LBW. Prior meta-analyses have been in accordance with these findings, however the most recent metanalysis which included 41 RCT found that though metformin is safe and effective for the treatment of HFDP, there was a higher incidence of preterm birth when compared to insulin and higher gestational weight when compared to glyburide (97) The frequency of treatment 19 failure has been shown to be similar for both OHA, with 16-17% of women requiring additional pharmacotherapy (32). One review (96) showed that glyburide was more effective at achieving glycaemic control in women with HFDP with lower treatment failure rates. Metformin, with its documented beneficial effects on both glycaemia and immediate pregnancy outcomes, make it a useful substitute during pregnancy, though its postnatal long-term effects are still to be better defined. The beneficial effects of metformin on glycaemia and pregnancy outcomes place it as a good alternative for its use during pregnancy, however the postnatal long-term effects require more evidence (80,81). Some human and animal studies have shown that metformin during pregnancy is associated with excessive postnatal weight gain and obesity in the offspring (98,99). There is no long-term safety data available for the offspring for glyburide exposure in pregnancy. Though the short-term safety of OHA versus insulin has been explored by numerous studies (96,100–107), there is a paucity of research evaluating their long-term effects and safety (104). To date, insulin remains the golden standard of pharmaceutical treatment of hyperglycaemia in pregnancy, given that concerns for the long-term consequences of OHA, including metformin and glibenclamide, remain. Most professional societies (ADA, ACOG) advocate its use as first-line treatment of HFDP after failure of lifestyle modification, whilst others (NICE) promote initial use of OHA following 20 weeks’ gestation prior to switching to insulin. 1.9.2 Post-partum The importance in detecting impaired glucose tolerance other than diabetes, lies in its value in predicting future T2DM as is the case in screening for cardiovascular risk factors in determining cardiovascular risk and subsequent disease. Lifestyle interventions, including weight loss programs and dietary advise remain the cornerstone of therapy in the primary prevention of these consequences. Pharmacologic interventions have also been shown to prevent progression to T2DM (108), whilst the detection and management of each cardiovascular risk factor is advisable in decreasing cardiovascular morbidity and mortality. Encouraging exclusive breastfeeding offers further protection from developing diabetes (109). Contraceptive advice is important since recurrent HFDP pregnancies further increase the risk of progression to T2DM. The optimal contraceptive method for women with a history of HFDP remain unknown, though there are a few studies supporting the use of combined oral contraceptive use (110–112). Hormonal contraception has been shown in large cohort studies 20 to not increase the risk of developing T2DM in healthy women (113,114), though the metabolic changes appreciated with progestins may be magnified in a group of women with a higher risk of T2DM. For the most part, the effect of contraceptive hormones on the subsequent risk of developing T2DM following HFDP is unclear with the current consensus supporting the safety of all contraceptive methods (115). In the setting of obesity, HTN or dyslipidaemia, either mechanical methods or hormonal methods which are vascular neutral are prescribed (116). 1.10 Significance of diabetes in pregnancy Uncontrolled hyperglycaemia during pregnancy, exposes both the mother and child to an increased risk of a range of adverse outcomes (117). The diagnosis of HFDP has implications for the pregnancy and also for the future maternal health as well as their offspring. Furthermore, it contributes indirectly to maternal mortality and morbidity by augmenting the risk of the four main causes of maternal death globally, including haemorrhage, HTN, sepsis and obstruction of labour (118). 1.10.1 Short-term consequences Poorly controlled diabetes in pregnancy, including both pre-existing and HFDP poses adverse perinatal and long-term outcomes for both the mother and foetus (Table 1.4). The risk increases as the maternal fasting plasma glucose increases. Immediate maternal risks include pre- eclampsia (20-30%), polyhydramnios (20%) and CS (30%) which can result in preterm delivery. In the foetus, spontaneous abortions and congenital anomalies may arise if dysglycaemia is present in the first trimester. Later in the pregnancy, elevated glucose levels may give rise to excessive fetal growth. This in the presence or absence of macrosomia and LGA infant can result in concomitant birth trauma. Immediate neonatal complications include respiratory problems and metabolic complications (119). A 20-year audit (1983-2002) performed at the CHBAH gestational diabetic clinic, acknowledged these well-established short-term risks in their “control” group, (those with diabetes in pregnancy who either presented late or only had a maximum of 2 weeks of intervention) (120,121). Needless to say, it is well established that achieving normoglycaemia early on in pregnancy can reduce these complications (122). 21 Table 1. 4 Adverse outcomes associated with uncontrolled hyperglycaemia in pregnancy. Maternal Fetal Neonatal Pregnancy induced hypertension Miscarriage Respiratory distress syndrome Polyhydramnios Stillbirth Hypoglycaemia Pre-term delivery Macrosomia Hyperbilirubinemia Congenital anomalies Hypomagnesaemia/ hypocalcaemia Polycythaemia 1.10.2 Long-term consequences Despite the fact that the majority of women who have HFDP, will experience normalized glucose tolerance in the immediate post-partum period, the development of either subtype of HFDP during pregnancy constitutes a maternal phenotype at increased subsequent risk for developing T2DM, MetS and, with time, overt CVD, including coronary artery disease and HTN (14–16,123). A landmark study conducted, the HAPO study (38), was a large, multinational, ethnically diverse group of women with the main objective to assess four primary outcomes in relation to varying degrees of maternal hyperglycaemia. Short-term maternal and neonatal outcomes measured are listed in Table 1.5. These women after undergoing a 2-hour 75gm OGTT demonstrated that lower glucose levels than those diagnostic of diabetes were associated with adverse pregnancy outcomes along a continuum. A follow-up study (124,125) of the same cohort assessed the long-term risk of any disorders of glucose metabolism among mothers and their offspring, together with adiposity measures among their children 10 to 14 years postpartum. The major findings were that a significant proportion of the mothers with a history of HFDP developed a disorder of glucose metabolism (52.2%) vs. 20.1% of mothers without HFDP (OR 3.44 [95%CI 2.85-4.14]) and higher glucose levels and insulin resistance was noted in the offspring upon exposure to higher glucose levels in utero. This finding was independent 22 of maternal and childhood BMI and a family history of diabetes. Childhood measures of adiposity utilising BMI was not statistically significant between the groups. Table 1. 5 HAPO study primary and secondary outcomes Maternal Neonatal Primary outcomes Caesarean section delivery Birth weight >90 th centile Cord blood C-peptide >90 th centile Neonatal hypoglycaemia Secondary outcomes Premature delivery Shoulder dystocia Birth injury ICU admissions Hyperbilirubinemia Pre-eclampsia The long-term consequences for both mother and child following HFDP, which are mostly cardiometabolic, are outlined in Table 1.6. Table 1. 6 Long-term consequences following HFDP. Maternal Offspring/Children Impaired glucose tolerance Impaired glucose tolerance Diabetes (T2DM and T1DM) T2DM Cardiovascular disease Obesity/MetS Obesity/MetS Cognitive impairment The potential mechanisms by which this progression to the numerous co-morbidities occurs is best depicted in Figure 1.7, in which the interplay of modifiable and non-modifiable factors 23 present both at pre-conception and during conception together with intermediate biological mechanisms during the post-partum period augment the development of these outcomes on the background of glucose intolerance during pregnancy. Abbreviations: GDM gestational diabetes, T2DM type 2 diabetes mellitus Figure 1. 7 Potential major determinants for the progression from HFDP to T2DM and other co-morbidities based on a life course perspective (22) 1.10.2.1 Type 2 diabetes HFDP is fuelling the T2DM epidemic with over a half of mothers with HFDP developing diabetes in ten years, making HFDP one of the strongest predictors of T2DM (14,123). Since HFDP includes undiagnosed pre-existing T2DM as well as GDM, this risk of progression may reflect the underlying T2DM epidemic within the population. An earlier metanalysis involving 20 studies (600, 000 women) spanning 50 years, showed that women with HFDP have an increased risk of developing T2DM compared with those who had a normoglycaemic pregnancy (RR7.43, 95% CI 4.79-11.51) (14). A more recent metanalysis which included 30 cohort studies, incorporating 2,626,905 pregnant women, supported these earlier findings of a substantially increased risk of diabetes with an adjusted risk of 17.92, however it also added that the greatest risk was between 3-6 years following HFDP and amongst the young age group less than 40 years, which is particularly alarming (123). The risk of progression has been shown 24 to vary with ethnic background, obesity and post-partum weight gain, family history of diabetes and the need for insulin during pregnancy amongst others (126). These risk factors vary by study with the most recent metanalysis showing that advanced maternal age and obesity have no role to play (123). Among developing and LMIC countries which are suffering the burden of escalating prevalence’s of HFDP and T2DM, data on the risk of progression to T2DM remains limited and little is known about modifiable factors that may lower this risk. Though a higher risk has been found in black women elsewhere in the world (127), most studies have not included women from Africa, with the applicability of previously recognised racial disparities in progression to T2DM, being uncertain. The variation in either maternal insulin sensitivity or secretion may account for these ethnic differences. This was appreciated in a study which focused on ethnic differences at three months postpartum where 31% of women of non- European ethnicity and 18% of women of European ethnicity were glucose tolerant (128). In SA, two studies in women of mixed ethnic background were found to have high rates of progression to dysglycaemia and T2DM as early as 6-12 weeks post-partum (129) as well as 5-6 years post-partum following a HFDP pregnancy(130). These studies had limited black ethnic representation. The overall need for long-term follow-up and screening for diabetes is essential following all HFDP pregnancies and is generally prescribed at six weeks post-partum followed yearly thereafter (32,61,72). In resource limited settings, since women with HFDP represent a substantial proportion of the population which may be difficult to target as a whole, identifying women at highest risk of progressing to T2DM includes identifying additional factors that may further assist in targeting prevention, with factors like insulin use in pregnancy, higher BMI, recurrent HFDP being important considerations (37) . Advocating simple lifestyle intervention programmes to promote weight loss following HFDP has been shown to reduce the risk of progression (130), though the effectiveness of these interventions remains to be fully established. Furthermore, thiazolididiones and metformin, have been demonstrated to decrease the risk of developing T2DM in HFDP women who are found to have various forms of dysglycaemia postpartum (131,132). 25 1.10.2.2 Cardiovascular disease Cardiovascular diseases are the most common cause of death globally, representing 30% of all deaths (133). The increase in deaths attributable to CVD is largely owing to an increase in risk factors including poor diet, sedentary lifestyle, obesity, hyperlipidaemia and importantly the increasing prevalence of T2DM. Diabetic women in particular have a four-to six-fold increased risk of developing coronary artery disease (CAD) (134). HFDP can be considered a “pre- diabetic state” with studies demonstrating similar cardio-metabolic alterations including obesity, insulin resistance, increased atherogenic lipid profile, increased inflammatory markers like highly sensitive CRP (hsCRP), HTN, and endothelial dysfunction with an increased carotid intima–media thickness (cIMT) as early as a few months postpartum (135). These “common soil” modifiable and non-modifiable risk factors shared by CVD and T2DM are depicted in Figure 1.8. It should be appreciated that there may be unrecognised factors both during and after pregnancy that contribute to the observed association of HFDP and subsequent CVD. These may include associated conditions in pregnancy or latent unrecognised cardiovascular risk determinants. 26 Abbreviations: FFA Free fatty acid; HCL Hepatocellular lipid content; IMCL Intramyocellular lipid content; PIH Pregnancy-induced hypertension. Figure 1. 8 Modifiable and non-modifiable risk factors shared by CVD and T2DM (137) Whereas some studies have shown that women with HFDP, develop CVD if they progress to T2DM, (83,136,137)others have argued that this increased cardiovascular risk can manifest independently of diabetes (138–140). A recent meta-analysis involving more than five million women demonstrated that women with a history of HFDP have a two-fold higher risk of major cardiovascular events in the years following the index pregnancy. In addition, the risk presents as early as the first decade following HFDP and importantly is independent of the development of T2DM (14). The exact pathophysiological mechanisms linking pregnancies complicated by diabetes mellitus with future cardiovascular health are presently unknown. Endothelial dysfunction known to be an important initiating factor in the development of atherosclerosis is an independent predictor of CVD in the general population (141). Studies of women with HFDP have shown that there is functional and biochemical evidence of abnormal vascular endothelial function during pregnancy (142) and up to several years postpartum. Endothelial dysfunction 27 can be measured accurately by several non-invasive techniques which can evaluate large artery function through measures such as flow mediated dilatation or pulse wave velocity, or structural changes, measured by cIMT. More recently various novel biomarkers, including inflammatory markers and oxidative stress related biomarkers, have been found to valuable in predicting CVD risk (143). The natural history of CVD exists along a continuum with a progression from a subclinical state to clinical disease in the presence of various genetic, lifestyle and biochemical markers which act as risk factors (Figure 1.9). The identification of subclinical disease through various surrogate markers is imperative in order to prevent disease. Abbreviations: IMT intima media thickness, LDL low density lipoprotein, HDL high density lipoprotein, CRP C-reactive protein, CVD cardiovascular disease. Figure 1. 9 Natural history of cardiovascular diseases on the background of various risk factors (144) Surrogate markers for cardiovascular disease Carotid intima media thickness (cIMT) detects early atherosclerosis and strongly predicts heart disease and stroke, particularly in women (145). The relationship between HFDP and thickened cIMT is controversial (146). More recently, several studies published on cIMT in 28 women who have had HFDP, found that a history of HFDP can be considered a risk factor for atherosclerosis even before the onset of diabetes or MetS (147–149). A meta-analysis of fifteen studies (2247 subjects) found that HFDP is related to larger cIMT and the relation is stronger in obese HFDP patients with the association already existing at the time of pregnancy and remaining significant years after pregnancy (137). Femoral intima media thickness (fIMT) has more recently been explored as a new marker in evaluating the global cardiovascular risk in patients with ischemic heart disease. The Bogalusa Heart Study found that fIMT, like cIMT is also correlated with the presence and number of cardiovascular risk factors. The usefulness in combining ultrasonography of the femoral artery in conjunction with multiple risk factors profiling for risk stratification in patients with atherosclerotic disease can be helpful (150). 2. Cardiovascular biomarkers Subclinical inflammation is known major risk factor for future CVD in the general population. Inflammatory mechanisms have been linked to a higher risk of CVD later in life for women with a history of HFDP(151), though the mechanisms behind vascular injury and CVD are not well understood (152). More recently, a variety of novel biomarkers reflecting a broad range of pathological events involved in the progression of atherosclerosis, have been reported in association with cardiovascular risk. Certain biomarkers of endothelial dysfunction have been found to be elevated in the early postpartum period in women with a background of HFDP, of which these include intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin (147,153). A further case control study (154)found significantly higher C-reactive protein (CRP), interleukin-6 (IL-6), and plasminogen activator 1(PAI-1) and lower adiponectin concentrations than in control subjects. Though, after adjustment for confounders, only a high CRP and low adiponectin levels were associated with HFDP. Another marker, microalbuminuria, known to be a signal of impaired endothelial function in T2DM (155) has also been reported in women whose pregnancies were complicated by HFDP (156). Although this marker potentially outlines a possible pathophysiological mechanism for future cardiometabolic disease, to date small studies with numerous confounders make the interpretation of these results limited. 3. Pulse wave velocity 29 Arterial stiffness is best assessed utilising carotid to femoral pulse wave velocity (PWV) (157). Pulse wave velocity increases the less distensible a vessel is (158) and this is proportional to the number of cardiovascular risk factors present, such as diabetes or MetS (159,160). A raised PWV has been found in epidemiological studies to be predictive of cardiovascular events (157). During the post-partum period, studies have found that endothelial and vascular smooth muscle reactivity, measured by flow-mediated dilation and arterial pulse wave velocity, were found to be reduced in women with HFDP (161,162). 4. Risk stratification-cardiovascular scores No single cardiovascular surrogate marker provides a highly sensitive and specific recognition of CVD and hence an attractive strategy has been to combine various evaluations to better define the presence of early disease. Scoring systems, much like the Framingham risk score (FRS)(163) has evolved to estimate the statistical risk of disease. This score is now recommended by numerous international guideline committees for CVD prevention since it has been validated across different populations. However, in young women with unique risk factors beyond the traditional risk factors (sex-specific, hsCRP) there is currently a lack of evidence and protocols for implementation of risk stratification strategies. Furthermore, the lack of prospective data in the African setting as well as the absence of locally validated total CVD risk assessment tools, makes risk assessment challenging. The FRS, although validated in white and black populations, has well established limitations in non-white, female and young individuals (164). One study from SA (165) looking the applicability of Framingham laboratory- and non- laboratory-based and National Health and Nutrition Examination Survey (NHANES) I non- laboratory-based equations in black men and women confirmed that the use of non-laboratory scores in clinical practice may be a feasible alternative in predicting cardiovascular risk in our resource-constrained settings. Cardiovascular disease risk prediction tools that include important pregnancy-related factors alongside the traditional CVD risk factors will improve screening for these high-risk individuals, given that an obstetric history of one or more prior pregnancies complicated GDM implies an increased risk for later CVD for the affected woman and should therefore be part of a complete CVD risk factor assessment in women. 30 In order to better assess global risk, integration of multiple biomarkers and hence all risk factors which can assist in incremental risk prediction must be included in diagnosis. This will assist with tailoring therapy and to monitor the effects of therapy in a cost-effective manner. There is compelling evidence to suggest that adverse pregnancy outcomes including HFDP serve as a warning for future CVD events (Figure 1.10) with numerous studies supporting the association of conditions such as preeclampsia, preterm delivery amongst others with CVD (84,166,167). Given that cardiovascular mortality is thus encountered more frequently, these women represent an important and potentially cost-effective subset of the population for diabetes and CVD prevention. Figure 1. 10 Associations between pre-pregnancy risk factors, adverse pregnancy outcomes, and post pregnancy cardiometabolic risk factors and outcomes (84) 1.8.2.3 Body composition and obesity The alarmingly high prevalence of overweight and obesity among women of childbearing age (168) in both developed and developing countries (169,170) is concerning given the immediate and long-term implications for mother and child. Immediate pregnancy complications include HFDP (171), pre-eclampsia, stillbirth, congenital abnormalities, and delivery of LGA infants 31 with the long-term consequences for the mother including T2DM, heart disease and HTN (172) whilst observational studies provide evidence for effects of maternal obesity on the offspring’s risks which include obesity, coronary heart disease, stroke, T2DM and asthma (173,174). Pregnancies complicated by both obesity and HFDP influence these outcomes independently. The long-term effects of HFDP on maternal body composition and obesity is less well explored. Data from a metanalysis of observational studies have shown that various proxies of adiposity such as BMI, weight gain, waist circumference (WC) and hip circumference (HC) and their association with T2DM and CVD risk, is causal (175), independent of overall body fatness. More specifically, body fat distribution, with upper body fat (android and visceral fat) being proportionately correlated with this risk compared to lower body fat depots (gynoid and leg fat) (176). Conventional anthropometric measurements such as waist-hip ratio (WHR) or imaging methods like commuted tomography (CT), magnetic resonance imaging (MRI) and dual-energy-absorptiometry (DXA) have demonstrated this (177). More recently quantifying body fat distribution has been shown to be causally related to CVD risk (178). Observational studies have demonstrated that although conventional anthropometry offers information regarding both fat distribution and total adiposity, it however provides weaker predictions of T2DM and CVD risk factors whereas other imaging platforms such as CT, MRI or DXA are more informative. The greatest limitations in performing CT and MRI remain the cost, radiation exposure and variability in image analysis. DXA has overcome these issues and provides accurate volumetric assessments of both total and regional adiposity (179) with more recent advances in DXA technology enabling accurate quantification of visceral adipose tissue (VAT). 1.10.2.4 Metabolic syndrome The MetS is considered as the concomitant clustering of central obesity, hypertriglyceridemia, low high-density lipoprotein cholesterol, (HDL) hypertension (HTN), and dysglycaemia. Currently there are many MetS definitions in existence with varying criteria being employed by different organizations to define this, which has led to numerous controversies in the field including varying prevalence estimates with some researchers questioning the utility of these criteria and even the existence of the syndrome. In 2009, representatives from various organizations attempted to resolve the differences between the definitions and proposed 32 “harmonizing the MetS” with five components (180) in which there is no obligatory component and three abnormal findings out of five would qualify a person. Though the cut-points were uniformly defined for all components, regional cut-points for waist circumference are advocated. Despite the ongoing debates this constellation of risk factors identifies a group of individuals at high-risk of T2DM and CVD. The rise in the obesity prevalence is considered to be the major determinant of the concomitant increased in obesity related comorbidities such that many women who are obese have T2DM or HTN, which together aggravates the existence of the MetS. HFDP shares several common risk factors (family history of T2DM, increased age, and raised BMI) with T2DM and similarly with MetS. More so, HFDP and MetS are intimately related to each other and develop in the “common soil” of insulin resistance, together with endothelial dysfunction and low-grade inflammation. A meta-analysis of several studies including 5,832 women demonstrated an increased risk of MetS following a HFDP pregnancy (15). Even in the absence of pre-pregnancy obesity, there is a fivefold additional independent risk of developing MetS compared to cont