The Effect of in Utero Hyperglycaemia, Maternal Overnutrition and Interaction With Postnatal Lifestyle on Cardiometabolic Risk at Young Adulthood - Extension of HAPO Follow-up Study
Conditions: Hyperglycemia, Pregnancy, Overnutrition, Maternal Obesity During Childbirth, Diabetes Mellitus, Cardiovascular Diseases, Gestational Diabetes Mellitus in Pregnancy, Hypertension
Sponsor: Chinese University of Hong Kong

 
 
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Title: The effect of in utero hyperglycaemia, maternal overnutrition and interaction with postnatal 
lifestyle on cardiometabolic risk at young adulthood – extension of HAPO follow-up study 
 
Principal Investigator: 
1Wang, Chi-chiu, Professor 
Co-Investigators: 
2Tam, Wing-hung, Clinical Professor (Honorary), 
3Chan, Michael Ho-ming, Consultant 
 
4Chan, Ruth Suk-mei, Senior research fellow 
 
4Ma, Ronald Ching-wan, Professor 
 
4Tam, Claudia Ha-ting, Postdoctoral Fellow 
 
 
 
1Department of Obstetrics and Gynaecology, Prince of Wales Hospital, The Chinese University of 
Hong Kong 
2 MBChB (CUHK), MD(CUHK), FRCOG, FHKAM(O&G), Cert HKCOG(Maternal Fetal Med),  
Department of Obstetrics and Gynaecology, Faculty of Medicine, The Chinese University of Hong 
Kong 
3Department of Chemical Pathology, Prince of Wales Hospital, The Chinese University of Hong 
Kong 
4Department of Medicine & Therapeutics, Prince of Wales Hospital, The Chinese University of 
Hong Kong  
 
 
 
 
 
 
 
 
 
 
 
 

 
 
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The prevalence of diabetes mellitus (DM) and cardiovascular disease (CVD) escalate remarkably 
worldwide and obesity becomes an epidemic disease. The main contributing factors are 
undoubtedly the contents of our food and lifestyle. These primarily adult diseases now extend 
across to the younger generation including children. Using Hong Kong as an example, 20% of type 
2 DM were diagnosed as young onset below the age of 40 years.1 Moreover, type 2 DM in children 
had increased by 10 times between 1997 and 2007, and likewise, the prevalence of childhood 
overweight and obesity has risen steadily over time in the last decade.2,3 
The concept of fetal origin of disease was proposed by David Barker, who had challenged the 
traditional belief that the causes of non-communicable diseases, like DM and CVD, were 
predominantly explained by a combination of bad genes inherited from our parents and unhealthy 
lifestyles. Barker proposed that their origins could lie in the earliest stages of one’s life. This 
Barker’s hypothesis was subsequently supported by work on animal models and epidemiology 
studies. The most cited work was the long-term outcome of the Dutch famine birth cohort, and 
their persistent epigenetic changes in association with prenatal exposure of undernutrition.4,5 
Furthermore, these persistent epigenetic changes can be transmitted to the next generation.6 
Over the last 2 decades, the model of Developmental Origin of Health and Disease (DOHaD) has 
extended from the spectrum of in-utero undernutrition to overnutrition. Through the follow-up 
study of mother-child pair from the “Hyperglycemia and Adverse Pregnancy Outcome” (HAPO) 
study participants, our group had demonstrated that maternal gestational diabetes mellitus (GDM) 
increased offspring’s risk of DM, obesity and hypertension at 7 years of age, independent of 
various confounding factors, like maternal obesity and maternal DM status.7 Similar findings were 
reproduced by the multi-centre HAPO follow-up study funded by NIH and NIDDK, where our 
group shared our study design and was also one of the 10 study centres, when the children were 
11-14 year of age.8 At the latest HAPO follow-up visit, 18% of 740 children assessed in the HK 
centre were diagnosed with prediabetes (based on their OGTT and HbA1c level) at their early 
teens.  
The prevalence of maternal obesity and GDM are increasing worldwide. Up to 20-25% of pregnant 
mothers were affected by GDM in Hong Kong. A recent nation-wide study covering 31 provinces 
in mainland China discovered that 1.4% and 13% of women planning to get pregnant were 
diagnosed DM and prediabetes respectively, of whom, 8% were either overweight or obese.9 
Taken together, the DOHaD model might help to explain the worldwide escalating prevalence of 
these metabolic diseases on top of our gene and lifestyle. The key concept of DOHaD is that the 
fetal and early infancy period is a window of plasticity, whereby the in utero programming can 
alter our gene expression.  
 
Early studies in the 1980’s on Pima Indian women had shown that the children and adolescent 
offspring of women with abnormal glucose tolerance during pregnancy were more obese and 
glucose intolerant, independent of their birth weight.10,11 It, therefore, suggested that maternal 
GDM was associated with long-term effects on obesity and glucose tolerance in the offspring. It 
has also been shown that offspring of type 1 DM mothers have an increased risk of developing DM 
in later life.12 Study of amniotic fluid insulin by amniocentesis has demonstrated that excessive 
insulin secretion in utero was a strong predictor of impaired glucose tolerance (IGT) in the children 

 
 
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of DM mothers.13 This may explain the relationship between in-utero overstimulation of pancreatic 
cells and the subsequent reduction in insulin secretion in their later life.14 
The findings on the relationship between the in-utero exposure to hyperglycaemia and the future 
development of childhood overweight or obesity are still inconsistent due to the heterogeneous 
methodology of confounders adjustment, importantly the pre-conceptional maternal or paternal 
BMI.15,16 A multinational study including 7,372 children of 9-11 ages from 12 countries shows that 
maternal GDM is positively associated with increased risk of children’s obesity, but results are 
attenuated when adjusted by maternal BMI. Maternal obesity is considered as an essential 
confounding factor for the association between maternal hyperglycaemia and the obesity in 
offspring that predispose to chronic cardiometabolic disorders in the future.17 
Excessive maternal gestational weight gain (GWG), which reflects the in-utero overnutrition 
environment, is found to be positively associated with the adiposity in the offspring,18 while some 
studies observe a U-shaped relationship.19,20 A few studies also demonstrated that excessive GWG 
predicted obesity-related metabolic factors including fasting plasma glucose (PG), insulin 
resistance and blood pressure (BP) in both childhood and adulthood.21,22 
 
In the early 2000’s, our group has reported an 8-year follow-up study on a cohort of women with 
previous history of GDM diagnosed between 1991 and 1994.23 We found that offspring exposed to 
mild GDM had significantly higher BP and worse lipid profile compared to offspring of mothers 
with normal glucose tolerance.23 Elevated umbilical cord insulin and C peptide levels at birth was 
associated with increased risk of glucose intolerance in children at 8 years, obesity and metabolic 
syndrome at 15 years, as well as increased arterial stiffness in adolescents, highlighting the 
persistent effects of hyperglycaemia and hyperinsulinaemia in utero on subsequent obesity, 
glucose intolerance and cardiovascular risk in the offspring.23-25 
Since women with GDM in the cohort had received dietary and/or insulin treatment during 
pregnancy, the data from this cohort cannot address whether this intervention had led to any 
improvement in the risks of future DM and metabolic syndrome amongst the offspring. 
Through the follow-up study of nearly 970 mother-child pairs from the HAPO study, our group 
discovered that maternal GDM increased their children’s risk of prediabetes and DM by 3-fold, 
overweight and obesity by 50%, and higher BP by 10% at 7 years of age.7 Moreover, we also 
demonstrated a graded effect of increasing risks of prediabetes/DM, overweight/obese and 
prehypertension/hypertension in association with maternal PG level at pregnancy.7 These 
associations were independent of BMI before pregnancy, childhood obesity or status as large for 
gestational age. 
The above study had sparked off an international multicentre HAPO follow-up study in 2013-2016, 
supported by NIH and NIDDK. Our research team was one of the 10 study centres collaborating 
this project. Similar associations discovered by our team could be reproduced by the multicentre 
HAPO follow-up study result, showing that maternal GDM increases childhood obesity by 50% 
after adjusting for maternal obesity and other confounders in 4,775 children.8 A graded effect of 
maternal glucose level in pregnancy on childhood obesity was also reiterated. The association of 
maternal GDM and glycaemic level at pregnancy with children’s glycaemic level and abnormal 

 
 
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glucose regulation were also presented at the ADA meeting in 2017 with the manuscript submitted 
for publication. 
In the HAPO follow-up study, we also discovered that both inadequate and excessive GWG, which 
could be surrogate indicators of maternal undernutrition and overnutrition respectively, increased 
children’s risk of overweight/obesity at 7 years of age. We also demonstrated that children had 
higher BP, greater insulin resistance and compensatory rise in insulin secretion in association with 
either inadequate or excessive GWG.26 
 
Our research team has just completed measuring maternal and umbilical cord serum Vitamin D 
level in the HAPO cohort funded by GRF 2016-2017. Preliminary analysis on the 7-year-old 
follow-up data showed that low umbilical cord blood vitamin D level was independently 
associated with children’s hyperglycaemia as well as arterial stiffness, which is the precursor of 
hypertension in later life. Two abstracts on the preliminary analysis were accepted in 2 
international conferences.27,28 
The available data would have great potential to explore the effect of in utero vitamin D status and 
long-term cardiometabolic risk at early teens and young adulthood. Our preliminary finding is 
consistent with the previous report showing vitamin D insufficiency in adult was associated with 
increase arterial stiffness and endothelial dysfunction. 29 Experimental studies did suggest that 
vitamin D modulated the endothelial cell function by reducing the endothelium-dependent 
contraction and regulating the calcium influx. 30,31 Vitamin D may affect the vascular wall through 
the renin-angiotensin-aldosterone axis and takes part in the lymphocyte and monocyte/macrophage 
differentiation and secretion of inflammatory cytokines which determines the monocyte infiltration 
and cholesterol retention in the vascular wall. 32,33 
 
Our research team has been investigating the effect of maternal hyperglycaemia, under- and 
overnutrition on the children’s long-term cardiometabolic risk in the HAPO study cohort over the 
last 10 years. We have the most comprehensive data among all HAPO study centres, including 
offspring’s BP, BMI, skinfold thickness, 5 point OGTT with insulin levels to explore their 
trajactories, plus the pre-pregnant BMI and information on GWG  
Now that the HAPO cohort children are reaching young adulthood. We have archived important 
antenatal, childhood data, maternal serum and cord blood serum, and cord blood DNA material to 
determine these associations and their possible mechanism. Epigenome-wide association study 
(EWAS) was already performed to identify differentially methylated loci and regions in cord blood 
DNA in offspring which was supported by previous GRF. 
Since the mothers have not been subjected to antenatal treatment on the various degree of maternal 
hyperglycaemia in pregnancy, this is a unique cohort that enables us to determine the effect of 
various degree of maternal hyperglycaemia below the level of overt DM, on children’s 
cardiometabolic risk in Chinese population.  
Our research team has successfully followed up more than 70% children from the original HAPO 
cohort, even though we only started to re-contact them a few years after the completion of the 
HAPO study; we are keeping them in contact for future assessment. Among those we have been in 

 
 
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touch with, we expect a higher response rate among those who have attended the last two follow-
up studies and estimate a response rate of 80% in the proposed study. 
 
Objectives 
1. To assess maternal glycaemic levels, gestational weight gain and pre-pregnancy BMI in the 
prediction of offspring’s risk of DM, hypertension and obesity at 18-20 years of age. 
2. To determine the trajectory of insulin resistance and pancreatic beta cell functions at 7 years 
and 11-14 years of age in association with corresponding BMI to the development of young-
onset type 2 DM at 18-20 years of age. 
 
Research plan and methodology  
i) Subjects 
Our unit is one of the centres contributing to the International multicentre HAPO study. We 
recruited 1760 Chinese pregnant women between 2000 and 2005. The HAPO study investigated 
whether any adverse outcome was associated with mild degree of GDM. All mothers underwent a 
75-gram OGTT at 24-32 weeks gestation, but clinicians were blinded to the results as long as the 
fasting PG was ≤ 5.8 mmol/L & 2-hour PG ≤ 11.1 mmol/L. The maternal serum C-peptide and 
HbA1c, cord serum C-peptide and early neonatal PG, pregnancy outcome and the neonatal 
anthropometric parameters are available for future study. This is so far the largest cohort in a 
Chinese population who has been investigated for glycaemia during pregnancy, but with OGTT 
results remained undisclosed to subjects and clinicians. This unique cohort can allow us to study 
the effect of in-utero hyperglycemia on the cardiometabolic risks at childhood, adolescence and 
adulthood. 
Eligible subjects are all mother-child pairs participating in the original HAPO study. Children born 
preterm before 37 weeks of gestation, non-Chinese and whose mother’s OGTT result were 
unblinded during pregnancy will be excluded.The family (the child and the mother) will be invited 
for a third follow-up assessment.  
 
ii) Data collection 
Questionnaire, anthropometry and BP measurement 
Detail clinical information on maternal characteristics during pregnancy (mothers’ smoking history, 
alcohol use and smoking habit, pregnancy complications, mode of delivery and gestational age at 
delivery), children’s characteristics at birth, at 7 and 11-14 years of age were available in our 
dataset from the original HAPO study and the two follow-up studies.7.8 
In the present study, the mother-child pairs will be asked to complete a questionnaire to review any 
significant medical history, family history of DM and hypertension, dietary pattern and activity 
level. We will also capture the menstrual history and endocrine information in both mother and 
female offspring, as well as reproductive history of mother. Signs of androgen excess in female 
offspring and mothers will be noted in the physical examination and graded according to the 

 
 
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Ferriman-Gallwey score or the frequency of shaving per week. Offspring’s dietary pattern will be 
assessed by using a newly derived short version food frequency questionnaire (FFQ), which was 
designed on the basis of previously validated FFQ used by the local population, as well as expert 
opinions from dietitians and nutritionists experienced in nutritional epidemiology.34 Research staff 
will administer the FFQ to capture the participant’s usual intakes over the past one year on the 
food groups and type of cooking style.  
 
For female offspring, their skinfold will be assessed by skinfold calipers at 4 sites (biceps, triceps, 
subscapular and suprailiac) in order to predict the amount of body fat. In all offspring, body fat 
percentage will be evaluated by electrical bioimpedance. A subset of offspring will be further 
evaluated for non-alcoholic fatty liver disease (NAFLD) by using Fibroscan®  (Echosens). 
 
Offspring’s physical activities will be recorded and analysed by short form of International 
Physical Activity Questionnaire (IPAQ-S) developed for the physical activity surveillance.35 
Metabolic Equivalents (METs) will be used for the analysis of IPAQ data to express the intensity 
of physical activities. 
 
Their body height and weight will be measured to the nearest 0.1 cm and nearest 0.1 kg 
respectively. BP will be measured three times in the non-dominant arm using an automated BP 
monitor, at one minute intervals, after five minutes of rest. The average readings will be used for 
analysis. 
 
Arterial stiffness of mothers  
Mothers will be lying on their back with their head supported and their arms relaxed by their sides.  
Three ECG electrodes will be applied to their chest walls.  Augmentation index and pulse wave 
velocity will be acquired by using applanation tonometry (SphygmoCor® PVx). 
 
• 
Augmentation index: Tonometer will be placed and gently compressed onto the radial 
artery with the subject’s wrist in neutral position.  A series of peripheral blood pressure waveforms 
are then captured by holding the tonometer for 10 seconds.  Central aortic blood pressures and 
augmentation index will be computed by SphygmoCor® PVx after using the mathematical transfer 
function.  
• 
Pulse wave velocity: Tonometer will be applied to carotid artery, radial artery and femoral 
artery in the same manner to obtain the peripheral blood pressure waveforms.  The speed of BP 
wave which travels along the carotid, radial and femoral arteries are determined with reference to 
the ventricular contraction recorded from the ECG.  The distances between the suprasternal notch 
(the notch at the top of the sternum) to the various tonometry points, namely carotid, radial femoral 
in order to compute the carotid-radial and carotid-femoral PWV.  The acquisition of the data is 
non-invasion and the device has been approved by FDA. 
 
 
Biochemical assay 

 
 
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Mothers will have fasting blood collected for the measurement of glucose, lipid profile, HbA1c, 
female hormonal profile (LH, FSH, estradiol, total testosterone, androstenedione, 17-OH and 
AMH) and renal function profile. The offspring will undergo a 75-gram OGTT with PG and 
insulin at 0, 30 and 120 minutes. Fasting blood will be taken from all offspring for lipid profile and 
renal function profile, whereas female hormonal profile will be assessed in female offspring only. 
HbA1c and lipid profile of the offspring will also be assessed by fasting blood. Blood for 
extraction of DNA will also be collected from offspring for genetic and epigenetic studies, 
including validation of established genetic risk scores for cardiometabolic traits. 
 
Primary and secondary outcomes 
The primary outcome is the rate of DM in the young adult offspring. 
The secondary outcomes are their rate of impaired glucose regulation, obesity and adiposity, 
hypertension and various insulin indices (insulin sensitivity, pancreatic -cell function, oral 
disposition indices). 
 
iii) Statistical analysis 
Between-group comparison of outcomes between offspring of mothers with GDM and those of 
mothers with normal glucose tolerance will be made by using the Student’s t-tests and the Chi-
square/Fisher’s exact tests, as appropriate. Group comparison of outcomes among offspring of 
mothers with inadequate, appropriate and excessive GWG will be made by using ANOVA test. 
Linear and logistic regression analyses with the adjustments for potential confounders will be used 
to assess the associations for continuous and binary outcome variables, respectively. Any sex 
difference in the associations will be performed in subgroup analysis. Data will be presented as 
beta with standard error for continuous outcomes, and odds ratios (ORs) with 95% confidence 
intervals for binary outcomes. 
The data from FFQ will be analysed by using the nutrition analysis software Food Processor 
Nutrition analysis and Fitness software version 8.0 (ESHA Research, Salem, USA) with specific 
focus on local foods consumption from Mainland and Hong Kong. Dietary patterns of the studied 
population will be generated by principal component analysis.  
 
iv) Sample size and power calculation 
We estimate the sample size and calculate the power using the objective 1.  
Assuming an 50% response rate of the eligible subjects, around 900 mother-child pairs will be 
recruited. Our previous findings detected an adjusted OR of 1.8-2.0 in children’s abnormal glucose 
regulation with per SD rise in maternal fasting, 1-hour and 2-hour PG at OGTT during pregnancy. 
We estimate the overall prevalence rate of type 2 DM as 2% in this young age group, the sample 
size will be able to detect a similar odds ratio of 1.8 per SD rise in glycaemic levels, at 80% power 
and a significance level of 0.05 by using logistic regression analysis.36 
 

 
 
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v) Duration of study 
We estimate two and half year to complete the recall of all the HAPO mother-child pairs Another 6 
months will be required for the data analysis. 
 
This protocol is in compliance with the Declaration of Helsinki. 
 
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