Cardiovascular Risk in Infants Exposed to Pre-eclampsia in Utero

An increasing number of scientific publications show that high blood pressure is being described in younger and younger children of African ancestry. It therefore makes sense to seek for the causes of this raised blood pressure in the in utero events. Recent studies have attributed and increased risk to cardiovascular disease (CVD) risk factors to the gestational environment. Pre-eclampsia (PE) is associated with endothelial dysfunction and oxidative stress in the mother which may influence how the developing foetus interacts with the external environment later on in life. Indeed scientific literature suggests that the foeto-placental vascular endothelial dysfunction may cause epigenetic alteration in the intrauterine environment of the foetus which may be at the origin of chronic diseases in children, thus predisposing them to risk factors of CVD. However, very few studies in women of African ancestry have been carried out to investigate whether or not children born of pre-eclamptic mothers of African ancestry are at risk of developing CVDs. Hence, the aim of this study is to investigate the relationship between pre-eclampsia and cardiovascular risk in children born to pre-eclampsic mothers in a rural South African population. A prospective case-control control design recruiting pre-eclamptic and normotensive pregnant women and their offspring subsequently will be used. CVD risk will be accessed in the pregnant women at 30 weeks of gestation and in the offspring at birth and then six weeks later. The difference in CVD risk between children born to these two groups of women will be assessed and the correlation between maternal and offspring risks for CVDs determined. It is expected that results obtained from this project will provide information on the cardiovascular effect of in utero exposure to PE in a population of African ancestry. This knowledge will advise policy on the management of women with pre-eclampsia with a view of preventing cardiovascular diseases in the offspring. Furthermore, the project will afford the opportunity for scientific research capacity building in students in Walter Sisulu University and foster collaboration between clinical and fundamental researchers.

Study design This will be a prospective case-control study involving babies born to pre-eclamptic (cases) pregnant women as the case and babies born to normotensive mothers (control). The mothers will be recruited at the Nelson Mandela Academic Hospital, Mthatha, Eastern Cape Province of South Africa. This is a tertiary hospital to which all specialist cases are referred from the peripheral hospitals. Even though the prevalence of PE in South Africa is just over 5% nationally, 18% of all obstetric cases consulted in the Nelson Mandela Academic Hospital in Mthatha are PE cases (unpublished data). It will therefore not be a great challenge to recruit the desired number of PE patients over the study period.

Sample size calculation The study done by Amini et al, (2010) showed blood pressure (BP) differences between neonates from normotensive and pre-eclamptic mothers5. The difference in blood systolic BP (SBP) was very high (normotensive: 49.85±5.49 versus pre-eclamptic: 68.2±149). Because the SBP variance and standard deviation were very high in the pre-eclamptic group, we decided to rather use the diastolic blood pressure values (normotensive: 30.17±11.89 versus pre-eclamptic: 42.11±11.49) for sample size calculation. The software - R was used: alpha = 0.01, mean difference = 11.94 and standard = 0.05 respectively were used. The results showed that total sample size is 97 (48 cases and 49 controls). We make the assumption that not all participants would return for the follow-up. Thus we adjusted our sample size by 20% to compensate for possible loses to follow-up. Therefore the total sample will be 97 + (97x20/100) = 117 neonates. However the study will start with the recruitment of 117 pregnant women (58 pre-eclamptic women and 59 normotensive pregnant women who meet the selection criteria).

Ethical approval Ethical approval and permission to carry out the research project has been applied for from Faculty of Health Sciences Research and Ethics Committee (HRSEC) at Walter Sisulu University (WSU). When the project is approved the Provincial and District Departments of Health will be approached for clearance and approval.

Informed consent The purpose of the study will be explained thoroughly to potential participants attending the antenatal clinic in the Nelson Mandela Academic Hospital, Mthatha, Eastern Cape Province. Pregnant women who meet the selection criteria and are willing to participate in the study will be required to sign informed consent forms to participate in the study and to allow their children to participate in the study from birth.

Research Protocol Pregnant women who meet the selection criteria will be recruited between weeks 20-26 of gestation while data will be collected in week 30 of gestation. Participants will be reminded of the hospital visit for data collection in advance to enable them make all necessary preparation to spend some extra time at the hospital.

1. Baseline information: Maternal demographic, obstetric and medical history will be collected from participant using a questionnaire.
2. Anthropometric measurements: Height and weight will be determined as per recommendations of the NHANES, 2009-2010 recommendations24
3. Confirmation of pre-eclampsia status: Hospital files will be used to confirm pre-eclampsia status.
4. Blood pressure measurements: Office blood pressure will be measured as described by Putner et al., (2019)25
5. Flow mediated dilatation. A Mobile Esaote MyLabTM Five portable ultrasound device (Genoa, Italy) with an Esaote Doppler probe (LA523, 12 MHz) connected to computerized software with edge detection technology (Quipu Cardiovascular Suite (CVS)™; Pisa, Italy) will be used. Endothelial function will be measured as described by Strijdom et al (2017)26
6. Gestational ultrasound will be performed to determine placental morphometry, architecture and vascularization. Uterine arteries, umbilical artery and middle cerebral artery will be assessed and uterine artery mean pulsating index determined. Foetal cerebroplacental ratio and anthropometry will be calculated when possible.
7. Measurement of markers of oxidative stress : Fasting blood will be collected for determination of oxidative stress in the maternal environment to which the foetus is exposed.

At birth: The Gestational age at delivery and mode of delivery will be noted. The APGAR score (1-5 minutes), will be determined, weight and height/length of the child will be measured. Cord blood will be collected for determination of titres of CVD risk as indicated below.

1. Markers of endothelial function: Endocan and asymmetric dimethyl arginine (ADMA), markers of endothelial function will be assayed using ELISA kits as per manufacturers' instructions.
2. Markers of oxidative stress: Lipid peroxidation, total antioxidant capacity and 8-hydoxyl-2-deoxyguanine (8-OHdG) will be determined using ELISA kits as per manufacturer's protocols.
3. Lipid profile : Total cholesterol, triglyceride, LDL cholesterol and HDL cholesterol, and oxidised LDL-cholesterol will be measured as per manufacturer's protocol using colorimetric kits as per manufacturers' instructions
4. Insulin Resistance: Fasting glucose, glycated haemoglobin and insulin concentrations will be determined as per manufacturer's methods. The HOMA-IR formula will be used to calculate insulin resistance from fast glucose and insulin as described by Mather et al, 1985)27.
5. Renal function indices: glomerular filtration rate (GFR), creatinine and albumin will be determined using colorimetric kits as per manufacturers' instructions
6. Blood pressure measurements: The oscillometric technique (Dinamap 8100) will be used for BP measurement according to the standard protocol for assessment of BP measurement in newborns by Nwankwo et al. (1997)28.
7. Anthropometric measurements: All measures were done as reported by Meldere et al. (2013) 29.
8. Arterial stiffness will be assessed by measuring Brachio-femoral pulse wave velocity (bfPWV) using an oscillometric device (Vicorder, Skidmore Medical) as reported by Alwan et al., 201530.

Eligibility Criteria:
Inclusion Criteria:

Pre-eclamptic or normotensive pregnant women with singleton uncomplicated ≥ 20 week old pregnancies and their offspring will be recruited for the study.

Exclusion Criteria:

Pregnant women with chronic hypertension, type 2 diabetes, gestational diabetes, renal and cardiovascular diseases or any critical health condition will be excluded from the study.