The Evaluation of the Pathophysiology of Varicose Veins in Pregnancy
Conditions: Varicose Veins, Varicose Veins of Lower Limb, Chronic Venous Insufficiency, Varicose; Vessel
Sponsor: Imperial College London

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IRAS ID: 277754   
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The evaluation of the pathophysiology of Varicose 
veins in pregnancy 
 
 
 
 
Version 2 
16/05/2021 
 
 
 
 
Main Sponsor: Imperial College London 
Funders: Saudi Cultural Bureau Study coordination centre: Vascular Laboratory 
Sponsor's/protocol number: 20HH5790 
NRES reference: XXXX 
NCT number: IRAS Project ID: 277754 
 
Protocol authorised by: 
 
Name & Role 
Date 
Signature 
Dr Mohammed Aslam, Academic Supervisor               16 .05.21           
 
 
 
 
 
 
 
 
 
 
 
 
 

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Study Management Group 
Chief Investigator: Dr Mohammed Aslam 
 
Co-investigator: Ms Reem Alturki. 
 
Statistician: Joseph Eliahoo  
 
Study Management: Dr Mohammed Aslam 
 
Study Coordination Centre  
For general queries, supply of study documentation, and collection of data, please contact study 
coordinator: Dr Mohammed Aslam 
Address:  
Vascular Department 
Hammersmith Hospital 
London 
W12 0HS 
 
 
 
 
Tel: 020 83831541  
 
 
 
 
 
 
Fax:  02083832083 
 
E-mail:  m.aslam@imperial.ac.uk 
 
Clinical Queries 
Clinical queries should be directed to Dr Mohammed Aslam who will direct the query to the 
appropriate person. 
 
Sponsor 
Imperial College is the main research sponsor for this study. For further information regarding 
the sponsorship conditions, please contact   
 
Ruth Nicholson 
Head of Research Governance and Integrity 
Joint Research Compliance Office 
Imperial College London and Imperial College Healthcare NHS Trust 
Room 215, Level 2, Medical School Building 
Norfolk Place 
London, W2 1PG 
Tel: 0207 594 18 62  
 Email: r.nicholson@imperial.ac.uk  
https://www.imperial.ac.uk/research-and-innovation/research-office/research-governance-
and-integrity/ 
 

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Funder 
Saudi Cultural Bureau  
 
This protocol describes the evaluation of the pathophysiology of Varicose veins in pregnant 
women by taking blood samples for genomic and hormonal testing. Venous blood reflux and 
patency will be evaluated by using Duplex ultrasound and vector flow imaging. This proposed 
project will also assess the limb volume using Truncated cone tool. Following this, iliac vein 
compression will be evaluated using Photoplethymography (PPG) technique. Finally, patient’s 
quality of life will be assessed by using Aberdeen Varicose Veins Questionnaire (AVVQ). 
Every care was taken in its drafting, but corrections or amendments may be necessary. These 
will be circulated to investigators in the study.  Problems relating to this study should be 
referred, in the first instance, to the Chief Investigator. This study will adhere to the principles 
outlined in the UK Policy Framework for Health and Social Care Research It will be conducted 
in compliance with the protocol, the Data Protection Act and other regulatory requirements as 
appropriate. 
 
 
Table of Contents 
1 
Introduction ................................................................................................................... 6 
1.1 
Background ....................................................................................................................... 6 
1.2 
Rationale for current study................................................................................................ 7 
1.3 
Research hypothesis: ......................................................................................................... 7 
2 
Study aim and objectives:.............................................................................................. 7 
3 
Methods ........................................................................................................................ 8 
3.1 
Study design: ..................................................................................................................... 8 
3.2 
Recruiting pregnant women in their first trimester: .......................................................... 8 
3.3 
Study group protocol ....................................................................................................... 10 
3.3.1 
Protocol for gathering patient’s demographic information ......................................................... 10 
3.3.2 
Protocol for Duplex ultrasound scan (DUS) ................................................................................. 11 
3.3.3 
Protocol for Vector flow imaging (VFI): ...................................................................................... 12 
3.3.4 
Protocol for Limb volume assessment ........................................................................................ 13 
3.3.5 
Protocol for Iliac vein compression test (venous outflow function) ............................................. 14 
3.3.6 
Protocol for blood tests (genomic and hormonal blood tests) .................................................... 15 
3.3.7 
Protocol for Quality of life assessment ....................................................................................... 16 
3.4 
Clinical intervention and procedures ............................................................................... 17 
4 
Participant entry ......................................................................................................... 17 
4.1 
Pre-registration evaluation .............................................................................................. 17 
4.2 
Inclusion criteria .............................................................................................................. 17 
4.3 
Exclusion criteria ............................................................................................................. 18 
4.4 
Recruitment and follow up period ................................................................................... 18 

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4.5 
Withdrawal criteria ......................................................................................................... 18 
5 
Adverse events ............................................................................................................ 18 
5.1 
Definitions ....................................................................................................................... 18 
5.2 
Reporting Procedures ...................................................................................................... 19 
5.2.1 
Non-Serious AEs ........................................................................................................................ 19 
5.2.2 
Serious AEs ................................................................................................................................ 19 
6 
Incidental findings: ...................................................................................................... 20 
7 
Assessment and follow up ........................................................................................... 20 
7.1 
Study closure ................................................................................................................... 21 
7.2 
Study outcome measures ................................................................................................ 21 
8 
Statistics and data analysis ......................................................................................... 21 
8.1 
Sample size ...................................................................................................................... 21 
8.2 
Analysis plan.................................................................................................................... 22 
8.3 
Data analysis: .................................................................................................................. 23 
9 
Regulatory issues ........................................................................................................ 23 
9.1 
Ethics approval ................................................................................................................ 24 
9.2 
Consent ........................................................................................................................... 24 
9.3 
Confidentiality ................................................................................................................. 24 
9.4 
Indemnity ........................................................................................................................ 24 
9.5 
Sponsor ........................................................................................................................... 24 
9.6 
Funding ............................................................................................................................ 25 
9.7 
Audits .............................................................................................................................. 25 
10 Study management: .................................................................................................... 25 
11 Publication policy: ....................................................................................................... 25 
12 References ................................................................................................................... 26 
13 Appendix: .................................................................................................................... 29 
13.1 
Complications arising from pregnancy ............................................................................ 29 
 
 
 
 
 
 
 
 
 
 
 
 

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List of Abbreviations 
 
VV 
Varicose veins  
CVI 
Chronic venous insufficiency  
NHS 
National Health Service  
VFI 
vector flow imaging  
PPG 
Photoplethymography 
AVVQ 
Aberdeen Varicose Veins Questionnaire 
DUS 
Duplex ultrasound  
CFV 
common femoral vein  
FV 
femoral vein  
LSV 
long saphenous vein    
SSV 
short saphenous vein 
TO 
Transverse oscillation 
C1 
Circumference at the end of segment length 1  
C2 
Circumference at the end of segment length 2 
L 
Segment length 
VRT 
venous refiling time 
E3 
Estriol 
CEAP 
Clinical-Etiology-Anatomy-Physiology 
 
Study summary 
Title: The evaluation of the pathophysiology of Varicose veins in pregnancy. 
Design: Prospective longitudinal study. 
Aim: The primary aim of this proposed study is to better understand the 
pathophysiology of VV in pregnant women. 
Outcome measures: Venous patency, blood flow direction, peak reflux velocity, 
circumferences (C1, C2) at the end of each segment length (L), BMI, VRT, Genomic 
DNA blood test, Estroil (E3) levels, progesterone serum levels, outcome of quality of 
life.  
Population:  70 Random pregnant women. 
Eligibility: Pregnant women aged between 18-47 years old. 
Duration: 1 year, follow up period will be conducted at the end of first, second and 
third trimester and three months after giving birth. 

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1 Introduction 
 
1.1 Background 
 
Varicose veins (VV) are the early sign of Chronic venous insufficiency (CVI) which are 
characterised by abnormal tortuosity and dilatation of superficial veins of lower limb 
extremities (Naoum et al., 2007). Symptoms of VV are variable and depend on the 
stage and severity of disease. In early stages, symptoms of VV include pain, leg 
heaviness and mild swelling (Adhikari et al., 2000). As the disease advances, 
symptoms include skin changes such as pigmentation, lipodermatosclerosis and 
ulceration (Kendler et al., 2010; Shadrina et al., 2018). Pregnancy is an established 
risk factor for the development of VV (Ismail et al., 2016). Three mains hypotheses for 
this observation have been proposed. First, it was hypothesised that increased size of 
gravid uterus during pregnancy may result in increased venous pressure on iliac veins 
resulting in a disruption of lower limb venous outflow (Mashiah et al., 1991). However, 
VV may develop in the first trimester before the onset of these mechanical factors 
(Dindelli et al., 1993).  Secondly, pregnancy results in an increased blood volume 
which may lead to elevate pressure in lower limb vasculature (Ismail et al., 2016). Both 
theories suggest that high pressures may result in venous valve impairment (Bergan 
et al., 2006). Thirdly, VV in pregnant women were found to be associated with 
increased estrogen and progesterone levels (Mashiah et al., 1991; Ortega, Asunsolo, 
Alvarez-Rocha, et al., 2018). Estrogen hormone elevates coagulation protein levels 
which cause thrombosis. Additionally, estrogen dilates venous valves due to its role in 
relaxation and releasing the chains between collagen fibers. On the other hand, 
progesterone prevents smooth muscle contraction which therefore increase valvular 
insufficiency due to vasodilation which cause lack of connection between two valves 
(Ropacka-Lesiak, et al., 2012). Moreover, large number of studies have discussed the 
associated risk factors that contribute to VV development. However, there are lack of 
studies that consider VV risk factors and pathophysiology in pregnant women. 
Understanding the aetiology of VV in pregnancy may allow us to better identify those 
at risk and manage this risk accordingly.  
 

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1.2 Rationale for current study 
 
Regardless of the underlying pathophysiological cause, VV may have a negative 
social impact on patient’s life and negative economic impact on health care budget.  
VV if left untreated can progress to late stages of chronic venous insufficiency (CVI) 
which include skin changes and ulceration (Adhikari et al., 2000). Ulcers lower 
patient’s quality of life and may result in social isolation, depression, reduce the ability 
to work and patients will be dependent on others (Evans et al., 1994; Cesarone et al., 
2002). Furthermore, venous disease is costly to manage for healthcare service. For 
example, the cost of managing chronic wounds caused by ulcers in the UK National 
Health Service (NHS) during 2005 to 2006 was estimated from 2.3 to 3.1 billion pounds 
which was equivalent to 3% of total UK health care budget (Posnett, J. and Franks, 
2007). Interestingly, the cost was increased between 2012 to 2013 when cost was 
estimated at 5.3 billions of pounds annually which consume 4% of the total health UK 
health care budget (Guest et al., 2015).  
 
1.3 Research hypothesis:  
 
The development of post-partum chronic venous insufficiency is predictable and 
caused by both mechanical and hormonal factors. 
 
 
2 Study aim and objectives:  
 
The primary aim of this proposed study is to better understand the pathophysiology of 
VV in pregnant women. This aim will be achieved by meeting the following objectives: 
Primary objective: 
To develop an antenatal test to predict the risk of developing VV post-partum. 
Secondary aims:   
1. To evaluate mechanical factors causing VV in pregnancy 
2. To evaluate hormonal factors causing VV in pregnancy. 
3. To determine whether lower limb pain during pregnancy is related to changes 
in lower limb venous system.  

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3 Methods 
 
3.1 Study design:  
 
A prospective longitudinal study will be undertaken to assess the development of VV 
among pregnant women. This study will follow up patients for one year, starting by 
following up pregnant women from the first trimester of pregnancy to the end of the 
second and third trimester. Last follow up will be performed three months after giving 
birth. Consequently, this proposed prospective study will be conducting four follow ups 
for each participant.  
 
3.2 Recruiting pregnant women in their first trimester: 
 
Initially, patients will be approached by a member of the direct care team in the 
Maternity outpatients’ departments in the following hospitals: West Middlesex hospital, 
Queen Charlotte’s and Chelsea hospital and St Mary’s hospital. This will be managed 
by experienced research professionals in the area of vascular surgery. If a patient is 
prepared to discuss their potential role in this research, then the researchers will 
discuss the study and recruitment details at the patient’s convenience. Recruitment is 
completely voluntary and will not affect their routine care, this will be made clear during 
the discussion regarding the research, and again during the consenting process. Time 
will be made available for any questions and for the patient to consider recruitment in 
their own time. Patients will be required to consent before any study-related 
procedures are carried out. Then her personal identifiable data will be coded and 
pseudonymised. 
 
The recruited patient will be interviewed to obtain clinical information that is essential 
for robust analysis. This clinical information will include standard demographic form 
which will be used for all patients in each follow up in order to record and gather 
important information which represents the composition of the target population. 
Gathered data will include patients’ details; patients name, day of visit, hospital name, 
ID number, date of birth. Furthermore, this demographic form will record potential risk 
factors which may associate with VV development such as race, number of 

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pregnancies, family history of venous disease, BMI which include weight and height, 
occupational factors, smoking, alcohol consumption and diet. Any complications 
arising from pregnancy will be also recorded (appendix 12.1). Finally, this proposed 
study will only record fetal weight in second and third trimester to correlate increased 
fetal weight with potential venous pathology.  
 
First, patients will be asked to fill the demographic form then will be asked to stand on 
a scale to measure the weight. Height will be recorded by bringing the horizontal 
measure down to rest on the top of the participants head. Following this, patient will 
be asked to sit while measurements are recorded in a relevant source data sheet. 
Next, the patient will be asked to stand on a step facing the sonographer to assess 
the incidence of varicose veins using Duplex ultrasound (DUS) and vector flow 
imaging (VFI). While patient is standing, Truncated cone tool will be performed 
immediately by measuring leg circumferences in each segments of each limb to 
produce accurate measurements of lower limbs volume. Following this, patient will be 
asked to sit on a chair for perform Iliac vein compression test (venous outflow function) 
using photoplethysmography (PPG) technique. Finally, patient’s quality of life 
assessment will be evaluated by using Aberdeen Varicose Veins Questionnaire 
(AVVQ) check list (Klem, Sybrandy and Wittens, 2009). Thereafter, the patient will be 
thanked for their participation and excused.  
Clinical Laboratory tests will be managed and performed by experienced research 
professionals in the area of vascular surgery. Four appointments will be arranged for 
each patient to investigate DNA genomic testing and Estriol (E3) and progesterone 
hormonal testing. First appointment during first trimester will include both genomic 
DNA and hormonal blood tests. The three remaining appointments in the second 
trimester, third trimester and three months after giving birth will include hormonal blood 
samples only. Patients will be entitled to exit the study at any time without providing a 
reason, this is clearly stated on our information sheet and consent form. Verbal 
consent will be requested before all follow-up ultrasound scans. 
Researchers will ensure that demographic data, ultrasound data, lower limb volume 
and iliac vein compression measurements, histopathological results and quality of life 

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assessment results will be collected and stored in such a way that confidentiality is 
preserved. All data will be pseudonymised by replacing names with research codes 
and by keeping any identifying information separate from the main dataset.  
 
The data will be analysed with the help of the Imperial College statistics department. 
The results of this study may be presented at local, national or international meetings 
or submitted for publication in a peer-reviewed journal. From a patient’s perspective, 
all data will be pseudonymous before the statistical analysis phase of the study and 
plans to publish the results of the study are made clear in our patient information sheet.  
Data and all appropriate documentation will be stored for a minimum of 10 years after 
the completion of the study, including the follow-up period.   
 
3.3 Study group protocol  
 
3.3.1 Protocol for gathering patient’s demographic information 
 
Recruited pregnant women will be asked to take a seat to fill a demographic form 
which contains patients’ personal details (name, date of birth, hospital number, date 
of visit and hospital name) and other varicose veins risk factors (see appendix 12.2), 
filling this form may take up to 5 min. Following this, BMI risk factor will be calculated 
by measuring patients’ weight and height.  
First, weight scale will be placed flat on the level ground. Scale will be ensured is at 
zero before commencing. Patient will be asked to remove heavy clothes and empty 
pockets. Preferably, shoes should be removed. Following this, patient will be asked to 
stand, and weight reading will be recorded when it has stabilised. Weight will be 
recorded in kilograms and to the nearest 0.1 kg. Second, height will be measured by 
asking the patient to remove their shoes and stand unsupported with their back to the 
vertical scale. Patients feet should be parallel to each other while standing, toes 
pointing forward and soles flat on the floor. Also, patient should be ensured that she 
is standing as tall as possible looking straight ahead. Horizontal measure will be 
brought down to rest on the top of the participants head. The measurement on the 
vertical scale to the nearest millimetres will be recorded on the relevant source data 

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sheet. Measuring weight and height will take 5 min, which means collecting patients’ 
demographic factors will take 10 min in total.  
 
3.3.2 Protocol for Duplex ultrasound scan (DUS)   
All recruited pregnant women be scanned by duplex ultrasound scan (DUS) to 
investigate the evidence of VV. The examination will be bi lateral for each patient. The 
patient will be asked to remove their clothes to expose the lower limb from groin to 
ankle. Patient will be examined standing to evaluate valves competency against 
gravity. This is because veins are more dilated while standing which make it easier 
and more accurate to assess. This examination is considered intimate; therefore, a 
chaperone will be offered. The scan duration will take 30-45 min and patients may 
sometimes feel dizzy during the examination. To overcome this issue, patients will be 
asked to sit and take a rest during the examination.  
There are two main techniques which will be applied to all venous segments. Firstly, 
B mode technique. B mode technique will be used to evaluate vein patency in 
transverse scan plane by observing the compressibility of veins to rule out any 
possibility of blood clots. Secondly, Spectral (Pulse wave) doppler technique will be 
used to evaluate venous flow characteristics (phasicity, spontaneity and direction flow) 
in longitudinal scan plane by applying manual distal augmentation to assess the reflux 
duration. All scans and measurements will be performed on SuperSonic Imagine’s 
Aixplorer ® ultrasound system. Following this, 9 MHz linear ultrasound probe will be 
placed on the patient skin over the venous blood vessel to be scanned, with the use 
of coupling gel between the skin and the probe. 
The scan will start by evaluating the deep and superficial veins system. Firstly, deep 
veins will be assessed by scanning the Common femoral vein (CFV) at the groin for 
patency and flow. Any abnormality in CFV indicates iliac vein obstruction therefore 
iliac veins and inferior vena cava should be examined. The examination will be 
continued by scanning the length of femoral vein (FV) (proximal to distal), popliteal 
vein and calf veins. Any thrombus or incompetence (Incompetent veins are defined as 
a reflux time more than 0.5 seconds) will be recorded.  

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Secondly, superficial venous veins will be assessed after completing the deep venous 
system examination. This will be achieved by starting again from the groin to evaluate 
the patency and competency of both sapheno-femoral junction and the entire length 
of long saphenous vein (LSV).  This will be coupled with assessing all venous 
perforators and branches. LSV diameters will be recorded in case of any required 
future treatment. After that, examining the sapheno-popliteal junction and short 
saphenous vein (SSV) will be performed by repositioning the patient to assess patency 
and competency. Therefore, VV found in LSV or SSV should be evaluated to detect 
linked sources of reflux such as reflux in perforators. Finally, all recordings will be 
saved and entered in the research database for analysis.  
 
3.3.3 Protocol for Vector flow imaging (VFI): 
 
Ultrasound machines currently use conventional Spectral (Pulse wave) doppler to 
assess the reflux duration in varicose veins protocols. Although spectral doppler is 
widely used, however, it is limited in detecting accurate peak reflux velocity estimation 
due to two main reasons. Firstly, its angle-dependency which means it can only detect 
blood velocities along the direction of ultrasound beam. Secondly, due to the 
anatomical location of lower extremities superficial veins.  To overcome this issue, 
Vector flow imaging (VFI) which is a new ultrasound technique can be used. This 
technique is a beam steering and angle independent technique which can detect 
accurate peak reflux velocity estimation.  VFI has been used recently in evaluating the 
velocity of lower limb veins and obtained promising results (Bechsgaard et al., 2016, 
2017).  
 
While the recruited patient is standing, she will be scanned by VFI which is based on 
transverse oscillation (TO) ultrasound method. VFI software will be installed in the 
same SuperSonic Imagine’s Aixplorer ® ultrasound scanner. Again, 9 MHz linear 
ultrasound probe will be placed in longitudinal scan plane on the patient’s skin over 
the venous blood vessels to be scanned, with the use of coupling gel between the skin 
and the probe. The scan will start by emitting conventional Pulse wave doppler 
estimation. Following this, by using special estimator and changing apodization in 
receive, both axial and transverse velocity components will be found at the same time. 

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Then, blood flow will appear in a colour box, a colour wheel will indicate the direction 
of the blood flow and overlaid arrows in the colour box will make the interpretation of 
flow direction and magnitude easier.  Finally, the patient will be asked to rest and take 
a seat. All recordings will be saved and entered in the research database for analysis. 
 
3.3.4 Protocol for Limb volume assessment 
 
Weight gain during pregnancy is well known. This occur not only due to fetal growth 
and uterus enlargement, but also due to water retention and increased blood volume 
which are required for fetal circulation. This excessive water retention especially in the 
third trimester is coupled with accumulated fat cause swelling (odema) especially in 
both lower limbs. Therefore, measuring limb volume during pregnancy is considered 
significant to estimate water and fat amounts gained during pregnancy and correlate 
these findings with potential VV incidence. This proposed study will assess the lower 
limb volume based on girth measurements using Truncated cone method. This 
method has the advantage of measuring bilateral limb volume oedema where no 
comparator (unaffected) limb is available. This method measures limb volume by 
manual perimetry using tape measure to find limb circumference. Tape measures 
tension may be inconsistent which can change the limb circumference measurement 
by up to 3% (Cheah et al., 1989). Therefore, this study will use spring-loaded 
controlled tension device to keep the tape in closer contact with the skin without 
compressing the underlying tissue.  
 
Measurements will be taken while the patient is standing. Circumferences will be   
measured every 10 cm from the sole up to the seventh measurement. A single 
researcher will measure all patients to prevent differences in tension on the tape. The 
data will be processed using the truncated-cone formula (Campanholi, Duprat Neto 
and Fregnani, 2011):  
 
V= h (C1C2 +C12+C22)/ 12 
V: Volume of each segment (final segment of the limb).  
 
C1 and C2: Circumference measured between the points (at either end of each segment length) 
 

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H: Distance between the circles (C1 and C2 in each segment), measured in centimetres.  
 
Total limb volume: is determined by the sum of the segment volumes.  
 
Finally, Patient will be asked to rest and take a seat on a chair, circumferential 
measurements will be entered into computer software programmes or pre-
programmed calculators to calculate limb volume of each recruited patient.  
 
3.3.5 Protocol for Iliac vein compression test (venous outflow function) 
 
This proposed study will examine iliac vein compression (venous outflow function) in 
pregnancy using photoplethysmography (PPG) technique. This is due to the difficulty 
of examining iliac veins using ultrasound in pregnancy due to increased size of gravid 
uterus. Large size of gravid uterus in pregnancy causes iliac veins compression which 
makes iliac veins hard to investigate. PPG is a non-invasive method used to evaluates 
the severity venous reflux by measuring the venous refiling time (VRT). VRT is 
considered to be abnormal when the refilling time is measured (less than 20 seconds). 
PPG technique has low specificity compared to other imaging tests and it is considered 
not accurate in locating the areas of reflux. However, alongside with its capability of 
evaluating the degree of reflux, it can also predict the treatment outcomes (Cheng and 
Wong, 1995).  
 
The principal of PPG works by using a small light source and a photoelectric cell 
attached to the skin. Produced light will transmit the skin and will be scattered by the 
tissue and partially absorbed. Scattered light will emerge out passing the skin to be 
detected by photoelectric cell. Photoelectric cell will be attached on the side or 
opposite the source of light to monitor blood perfusion based on transmission and 
reflection modes. Following this, the intensity of the light signals detected by 
photoelectric cell will be converted to PPG signals.  
 
In this proposed study, patient will be seated with her both legs dangling on the floor. 
PPG sensor will be attached to the patient’s skin in the medial supramalleolar area of 
the leg (the distal lower medial area of the leg). The exam will start by asking the 
patient to flex and extend one foot (five times), while the other floor placed on the floor. 

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Following this, a traced graph will appear on the machine’s screen. When the trace is 
stable, it means that it is the end of the VRT. The exam will be repeated to evaluate 
the filling time of the second leg. All measurements will be recorded immediately.  
 
3.3.6 Protocol for blood tests (genomic and hormonal blood tests) 
 
Genetic risk factors are reported to play an important role in the aetiology of varicose 
veins. In this proposed study, we aim to estimate the heritability of varicose veins 
among recruited pregnant women by taking a 30-ml blood sample for genomic DNA 
testing. This will be performed once in the first trimester. Results will be entered to the 
research database for analysis and correlation with the UK bio bank study (Fukaya et 
al., 2018).  
 
Along with the genomic blood samples, hormonal blood samples will be taken from 
patients in each trimester and three months after giving birth. This is because elevated 
estrogen and progesterone hormones levels were linked to VV development (Mashiah 
et al., 1991; Ortega, Asunsolo, Leal, et al., 2018). Progesterone serum blood test will 
be conducted to evaluate the progesterone levels during pregnancy and correlate the 
result with the incidence of VV. Normal progesterone serum levels varies according to 
the trimester of pregnancy. For instance, according to the NHS, progesterone levels 
in the first trimester estimates between 6.9 to 470 nmol/L, 71.6 to 300 nmol/L in the 
second trimester and 88 to 770 nmol/L in the third trimester. Therefore, progesterone 
hormone reaches the highest levels in the third trimester which suggests the potential 
effect of this hormone on venous pathology in late pregnancy.  
 
In addition, estrogen hormone blood test will be tested to associate the effect of 
estrogen with VV incidence. Estrogen hormone has three main types which are 
estrone, estradiol and Estriol and each type has different function. Estrone (E1) is the 
main hormone in females which is found after menopause. Estradiol (E2) is a female 
hormone found in non-pregnant women and involved in regulating menstrual cycles. 
Estriol (E3) is a type of estrogen hormone produced with higher amounts in pregnancy. 
This proposed study will investigate Estriol (E3) and progesterone serum blood test in 
each follow up which include first, second, third trimesters and three months after 

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giving birth in order to correlate hormonal changes with any potential venous pathology 
incidence.  
 
Therefore, four appointments will be arranged for each patient to investigate DNA 
genomic testing and Estriol (E3) and progesterone hormonal testing. First appointment 
during first trimester will include both genomic DNA and hormonal blood tests. The 
three remaining appointments in the second trimester, third trimester and three 
months after giving birth will include hormonal blood samples only. At the end of the 
study, these samples will be transferred to research tissue bank to store for future 
research.  
 
3.3.7 Protocol for Quality of life assessment   
 
Several quality assessment tools are used to measure the functional impact of a 
disease on patients’ quality of life as perceived by the patient. Both generic and 
disease specific measures tools are used to estimate patient’s quality of life status. 
This proposed study will use the Aberdeen Varicose Veins Questionnaire (AVVQ) 
quality of life assessment tool. AVVQ is a disease specific quality of life assessment 
tool which is developed in specific for patients with VV. Patients will be asked to 
answer 13 questions which assess patient symptoms such as pain, swelling, itching 
and ulcers. This tool also assesses the impact of VV on social and daily life activities 
and the use of support stocking. Perceived cosmetic impact is taken into consideration 
in AVVQ tool. Each question is designed to reflect the severity of the issue referred to 
in the question. AVVQ questionnaire is scored from 0-100, where 100 represents the 
most severe issues related to VV and lowest quality of life while zero represents the 
best quality of life. Finally, patients will be thanked for their participation and excused. 
 
 
 
 

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3.4 Clinical intervention and procedures 
 
Clinical intervention and procedure 
Time 
 
Consent  
 
Clinical data collection 
 
Blood samples (genomic+ 
hormonal) 
 
DUS 
 
VFI 
 
Truncated cone tool 
 
PPG 
 
AVVQ 
First trimester (0-13 weeks) 
 
Clinical data collection 
 
Blood 
sample 
(hormonal 
only) 
 
DUS 
 
VFI 
 
Truncated cone tool 
 
PPG 
 
AVVQ 
Second trimester (14-26 weeks) 
 
Same as second trimester 
Third trimester (27-40 weeks|) 
 
Same as second trimester 
Three months after giving birth 
 
 
 
 
4 Participant entry  
 
4.1 Pre-registration evaluation  
 
Blood test for pregnancy conformation.  
 
4.2 Inclusion criteria 
 
 
Pregnant women who are aged between 18 to 47 years old  
 
Patients who are willing to be followed up for 1 year and agreeing to give the informed 
consent will be included in the inclusion criteria.  
 

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4.3 Exclusion criteria 
 
 
Any incomplete cases such as intrauterine death. 
 
Participants who fail to attend the scans after their first visit will be excluded.  
 
Patients with congenital vascular abnormalities/syndromes such as Klippel Klippel-
Trenaunay syndrome. 
 
Patients who are unable to provide consent.  
 
Anyone who are taking part in any other research. 
 
Potential participants who might not adequately understand verbal explanations or 
written information given in English, or who have special communication needs.  
 
Patients with signs of acute deep vein thrombosis. 
 
Patient suffered from severe injury of their great-saphenous vein.  
 
4.4 Recruitment and follow up period 
 
Recruitment period will take up to three months. Follow up period will be conducted at 
the end of first, second and third trimester and three months after giving birth.  
 
4.5 Withdrawal criteria 
 
Participants choice  
 
 
5 Adverse events 
 
5.1 Definitions   
 
Adverse Event (AE): any untoward medical occurrence in a patient or clinical study 
subject.   
None are expected during this study. 
Serious Adverse Event (SAE): any untoward and unexpected medical occurrence 
or effect that: 
 
Results in death 
 
Is life-threatening – refers to an event in which the subject was at risk of 
death at the time of the event; it does not refer to an event which 
hypothetically might have caused death if it were more severe 

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 
Requires hospitalisation, or prolongation of existing inpatients’ 
hospitalisation 
 
Results in persistent or significant disability or incapacity 
 
Is a congenital anomaly or birth defect 
 
Medical judgement should be exercised in deciding whether an AE is serious in other 
situations.  Important AEs that are not immediately life-threatening or do not result in 
death or hospitalisation but may jeopardise the subject or may require intervention to 
prevent one of the other outcomes listed in the definition above, should also be 
considered serious. 
 
5.2  Reporting Procedures 
 
All adverse events should be reported.  Depending on the nature of the event the 
reporting procedures below should be followed.  Any questions concerning adverse 
event reporting should be directed to the Chief Investigator in the first instance.   
 
5.2.1 Non-Serious AEs 
 
All such events, whether expected or not, should be recorded.   
 
5.2.2 Serious AEs 
 
An SAE form should be completed and faxed to the Chief Investigator within 24 hours.  
However, death due to eclampsia and any health complications worsened during 
pregnancy does not need reporting as SAEs. 
 
All SAEs should be reported to the North of Scotland Research Ethics Committee 
where in the opinion of the Chief Investigator, the event was: 
 
‘related’, ie resulted from the administration of any of the research 
procedures; and 

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 
‘unexpected’, ie an event that is not listed in the protocol as an expected 
occurrence 
 
Reports of related and unexpected SAEs should be submitted within 15 days of the 
Chief Investigator becoming aware of the event, using the NRES SAE form for non-
IMP studies.  The Chief Investigator must also notify the Sponsor of all SAEs. 
 
Local investigators should report any SAEs as required by their Local Research Ethics 
Committee, Sponsor and/or Research & Development Office. 
 
 
Contact details for reporting SAEs 
jrco@imperial.ac.uk 
m.aslam@imperial.ac.uk 
Fax: 02083832083 attention Dr Mohammed Aslam 
Tel: 02083831541 (Mon to Fri 09.00 – 17.00) 
 
 
 
6 Incidental findings: 
 
 
A lay summary of aggregated results will be provided to all participants. If there is an 
incidental finding such as DVT, which likely have a major impact on patient’s medical 
management then the doctor treating her will be informed and participant will be 
removed/ withdrawn from the study.  
 
 
7 Assessment and follow up 
 
All previously mentioned interventions and procedures including (DUS, VFI, Truncated 
cone tool, PPG, hormonal blood test, AVVQ questionnaire) will be performed four 
times (in first, second, third trimester once and three months after giving birth). DNA 
genomic blood test will be performed once only in the first trimester of pregnancy. No 

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further follow-up required after this time point. The study expected time for each 
participant is 12 months in total. 
 
7.1 Study closure 
 
The study will be closed after one year (9 months pregnancy + 3 months after giving 
birth) the last 12 months appointment of the last recruited patient is completed, and all 
data have been received. 
7.2 Study outcome measures 
 
 
Intervention/procedure 
Outcome measures 
Primary 
outcome 
DUS 
 
Patency 
 
Blood flow 
direction(antegrade/retrograde) 
 
Peak reflux velocity  
Secondary 
outcomes 
VFI 
 
Patency  
 
Blood flow 
direction(antegrade/retrograde) 
 
Peak reflux velocity 
Truncated cone tool  
 
C1 
 
C2 
 
h 
PPG 
Severity of venous reflux by 
measuring Venous refiling time (VRT)  
(Abnormal less than 20 sec) 
Genomic blood test 
Genomic DNA 
 Hormonal Blood test 
Estroil (E3) levels, progesterone 
serum levels 
AVVQ questionnaire 
Outcome of quality of life (0-100). 0 
least severe (good quality of life), 100 
most severe (low quality of life).  
 
 
 
8 Statistics and data analysis 
 
8.1 Sample size 
 

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An exact binomial test with a nominal 5% two-sided significance level will have 91.27% 
power to detect the difference between the null hypothesis proportion, π₀ of 0.25 and 
the Alternative proportion, π₁, of 0.1 when the sample size is 70. 
 
 
     
 
8.2 Analysis plan 
 
All analyses will be performed based on the intention of follow-up. The analysis 
population will include all participants enrolled in the study, including those who 
developed varicose veins during pregnancy and those who do not.  
 
Analysis of primary and secondary outcomes: 
 
Venous patency and Peak reflux time recordings from VFI scan will be correlated with 
Venous patency and peak reflux time recordings from DUS scan. This will be 
performed using descriptive summary statistics. Mixed multivariable logistic 
regression will be used to model venosus patency and peak reflux velocity in each 
scan.  
 
Secondary outcome which include measuring the circumferences (C1, C2) and length 
of segments (h) using Truncated cone method. Calculated volume will be determined 
by summing segment volumes derived from the truncated cone formula. Pearson 
product moment correlations, paired t tests, and linear regression tests will be used to 
assess relative association and absolute differences between calculated volumes in 
each follow up.  
 
Secondary outcome which include PPG will be analysed by comparing the mean value 
for the venous refilling time (VRT) by Student’s t test, agreement between DUS and 
PPG classification was expressed as a kappa (K) value. 
 
Genomic DNA blood samples results will be correlated with the UK biobank study. 
Test statistics will be used to identify genetic variants that influence varicose vein 
susceptibility.  

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Each hormonal blood samples results including Estroil (E3) and progesterone serum 
levels will be correlated with the results of the next follow up to provide comprehensive 
information about increased hormonal levels in pregnancy and varicose veins 
development. 
Outcome of quality of life statistical analysis will be performed using the Statistical 
Package for the Social Sciences (SPSS 23.0) for Windows. Evaluation of AVVQ 
consisted of quality of life in first trimester, second trimester, third trimester and three 
months after giving birth will be done using the Friedman test.  
 
8.3 Data analysis: 
 
The data obtained will be subjected to statistical analysis by an independent 
biostatistician. SPSS Version 23.0 or/ and STATA Version 10.0 will be used for 
statistical analysis. Measures such as the mean and standard deviation or median and 
interquartile range will be used to summarise continuous variables such as age and 
BMI. Tables reporting the numbers and percentages will be used for categorical 
variables such as varicose veins and smoking. Chi square or Fisher’s exact test will 
be used to compare categorical variables such as varicose veins and smoking at the 
final trimester. The t-test or Mann-Whitney test will be used to compare continuous 
variables such as age with varicose veins at the final trimester. The paired t-test or 
Wilcoxon signed-rank test will be used to compare BMI between first and third 
trimester. The Kappa statistic will be used to compare DUS and VFI regarding direction 
and velocity. Longitudinal analysis will be used to look at factors associated with 
varicose veins. Factors include age, BMI, smoking, ethnicity, pregnancies and family 
history. A significance value (p-value) less than 0.05 is considered statistically 
significant. 
 
 
 
 
9 Regulatory issues 
 

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9.1 Ethics approval  
 
The chief investigator has obtained approval from the Research Ethics Committee and 
HRA. This study must also receive confirmation of capacity and capability from each 
participating NHS trust before accepting participants into the study or any research 
activity is carried out. The study will be conducted in accordance with 
recommendations for physicians involved in research on human subjects adopted by 
the 18th World Medical Assembly, Helsinki and later revisions.  
 
 
9.2 Consent 
 
Consent to enter the study must be sought from each participant only after a full 
explanation has been given, an information leaflet offered, and time allowed for 
consideration. Signed participant consent should be obtained. The right of the 
participant to refuse to participate without giving reasons must be respected. After the 
participant has entered the study the clinician remains free to give alternative 
treatment to the specified in the protocol at any stage if she feels it is in the participant’s 
best interest, but the reasons for doing so should be recorded. In these cases, the 
participants remain within the study for the purposes of follow- up and data analysis. 
All participants are free to withdraw at any time from the protocol treatment without 
giving reasons and without prejudicing further treatment.   
 
9.3 Confidentiality 
 
 
The Chief Investigator will preserve the confidentiality of participants taking part in the 
study and is registered under the provisions of the Data Protection Act 2018.  
 
9.4 Indemnity 
 
Imperial College London holds negligent harm and non-negligent harm insurance 
policies which apply to this study. 
 
9.5 Sponsor 
 

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Imperial College London will act as the main Sponsor for this study. Delegated 
responsibilities will be assigned to the NHS trusts taking part in this study.  
 
9.6 Funding 
 
Saudi Arabian Cultural Bureau in London is funding this study. No payment will be 
made to participants and to researchers. 
 
9.7 Audits 
 
The study may be subject to inspection and audit by Imperial College London under 
their remit as sponsor and other regulatory bodies to ensure adherence to GCP and 
the UK Policy Framework for Health and Social Care Research. 
 
10 Study management: 
 
The day-to-day management of the study will be co-ordinated through Dr Mohammed 
Aslam (CI).  
 
11 Publication policy:  
 
The results of this study may be published in peer-reviewed scientific journals or 
conference presentations.  
 
 
 
 
 
 
 
 
 
 
 
 

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12 References  
 
1-Adhikari, A. et al. (2000) ‘The epidemiology of chronic venous diseases’, 
Phlebology. doi: 10.1007/s005230070031. 
2-Azwa, I., Marsh, M. S. and Hawkins, D. A. (2012) ‘Infections in pregnancy’, in 
Neurology and Pregnancy: Clinical Management. doi: 10.1201/b14988. 
Bechsgaard, T. et al. (2016) ‘Blood flow velocity in the popliteal vein using transverse 
oscillation ultrasound’, in Medical Imaging 2016: Ultrasonic Imaging and Tomography. 
doi: 10.1117/12.2216725. 
3-Bechsgaard, T. et al. (2017) ‘Vector and Doppler Ultrasound Velocities Evaluated in 
a Flow Phantom and the Femoropopliteal Vein’, Ultrasound in Medicine and Biology. 
doi: 10.1016/j.ultrasmedbio.2017.06.020. 
4-Bergan, J. J. et al. (2006) Chronic Venous Disease From the Departments of 
Surgery (. Available at: www.nejm.org. 
5-Berger, H. et al. (2016) ‘Diabetes in Pregnancy’, Journal of Obstetrics and 
Gynaecology Canada. doi: 10.1016/j.jogc.2016.04.002. 
6-Campanholi, L. L., Duprat Neto, J. P. and Fregnani, J. H. T. G. (2011) ‘Mathematical 
model to predict risk for lymphoedema after treatment of cutaneous melanoma’, 
International Journal of Surgery. doi: 10.1016/j.ijsu.2011.01.007. 
7-Cesarone, M. R. et al. (2002) ‘“Real” epidemiology of varicose veins and chronic 
venous diseases: the San Valentino Vascular Screening Project.’, Angiology. United 
States, 
53(2), 
pp. 
119–130. 
Available 
at: 
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&PAGE=reference&D=med4&NEWS=N&A
N=11952101. 
8-Cheah, L. H. et al. (1989) ‘Limb volume measurements in peripheral arterial 
disease’, Clinical Physics and Physiological Measurement, 10(1), pp. 75–79. doi: 
10.1088/0143-0815/10/1/008. 
9-Cheng, S. W. K. and Wong, J. (1995) ‘Chronic Venous Insufficiency in a Chinese 
Population: An Anatomic and Functional Study by Continuous-Wave Doppler and 

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Photoplethysmography’, Annals of Vascular Surgery. doi: 10.1007/BF02135287. 
Dindelli, M. et al. (1993) ‘Risk factors for varicose disease before and during 
pregnancy.’, Angiology. United States, 44(5), pp. 361–367. Available at: 
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&PAGE=reference&D=med3&NEWS=N&A
N=8480913. 
10-Evans, C. J. et al. (1994) ‘Epidemiology of varicose veins. A review’, International 
Angiology. 
11-Fukaya, E. et al. (2018) ‘Clinical and Genetic Determinants of Varicose Veins’, 
Circulation. doi: 10.1161/CIRCULATIONAHA.118.035584. 
Guest, J. F. et al. (2015) ‘Health economic burden that wounds impose on the National 
Health Service in the UK’, BMJ Open. doi: 10.1136/bmjopen-2015-009283. 
12-Ismail, L. et al. (2016) ‘A systematic review and meta-analysis of the risk for 
development of varicose veins in women with a history of pregnancy.’, Journal of 
vascular surgery. Venous and lymphatic disorders. United States, 4(4), pp. 518-
524.e1. doi: https://dx.doi.org/10.1016/j.jvsv.2016.06.003. 
13-Kendler, M. et al. (2010) ‘Elevated sex steroid hormones in great saphenous veins 
in men.’, Journal of vascular surgery. United States ([Erratum appears in J Vasc Surg. 
2010 Aug;52(2):538 Note: Tzellos, Thrasivoulos [added]]), 51(3), pp. 639–646. doi: 
https://dx.doi.org/10.1016/j.jvs.2009.07.128. 
14-Klem, T. M. A. L., Sybrandy, J. E. M. and Wittens, C. H. A. (2009) ‘Measurement 
of Health-related Quality of Life with the Dutch Translated Aberdeen Varicose Vein 
Questionnaire before and after Treatment’, European Journal of Vascular and 
Endovascular Surgery. doi: 10.1016/j.ejvs.2008.11.036. 
15-Lauretti, G. R. and McKay, R. S. F. (2008) ‘Infectious diseases in pregnancy’, in 
Obstetric 
Anesthesia 
and 
Uncommon 
Disorders, 
2nd 
Edition. 
doi: 
10.1017/CBO9780511544552.019. 
16-Lefkovits, Y. R., Stewart, Z. A. and Murphy, H. R. (2019) ‘Gestational diabetes’, 
Medicine (United Kingdom). doi: 10.1016/j.mpmed.2018.11.006. 
17-Mashiah, A. et al. (1991) ‘[Etiology and surgery of varicose veins of the lower 
limb].’, 
Harefuah, 
121(5–6), 
pp. 
191–2. 
Available 
at: 
http://www.ncbi.nlm.nih.gov/pubmed/1937260 (Accessed: 1 March 2019). 
18-Naoum, J. J. et al. (2007) ‘Current advances in the pathogenesis of varicose 
veins.’, The Journal of surgical research. United States, 141(2), pp. 311–316. 

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Available 
at: 
http://ovidsp.ovid.com/ovidweb.cgi?T=JS&PAGE=reference&D=med5&NEWS=N&A
N=17070551. 
19-Oettinger-Barak, O. et al. (2005) ‘Severe Pregnancy Complication (Preeclampsia) 
Is Associated With Greater Periodontal Destruction’, Journal of Periodontology. doi: 
10.1902/jop.2005.76.1.134. 
20-Ortega, M. A., Asunsolo, A., Leal, J., et al. (2018) ‘Implication of the 
PI3K/Akt/mTOR Pathway in the Process of Incompetent Valves in Patients with 
Chronic Venous Insufficiency and the Relationship with Aging.’, Oxidative medicine 
and 
cellular 
longevity. 
United 
States, 
2018, 
p. 
1495170. 
doi: 
https://dx.doi.org/10.1155/2018/1495170. 
21-Ortega, M. A., Asunsolo, A., Alvarez-Rocha, M. J., et al. (2018) ‘Remodelling of 
collagen fibres in the placentas of women with venous insufficiency during 
pregnancy.’, Histology and histopathology. Spain, 33(6), pp. 567–576. doi: 
http://dx.doi.org/10.14670/HH-11-948. 
22-Oyelese, Y. and Smulian, J. C. (2006) ‘Placenta previa, placenta accreta, and vasa 
previa’, Obstetrics and Gynecology. doi: 10.1097/01.AOG.0000207559.15715.98. 
23-Posnett, J. and Franks, P. . (2007) ‘SkinBreakdown_MedicalEthics.pdf’, pp. 6–12. 
Ropacka-Lesiak, M., Kasperczak, J. and Breborowicz, G. H. (2012) ‘[Risk factors for 
the development of venous insufficiency of the lower limbs during pregnancy--part 1].’, 
Ginekologia polska. 
24-Ruiter, L. et al. (2015) ‘Systematic review of accuracy of ultrasound in the diagnosis 
of vasa previa’, Ultrasound in Obstetrics and Gynecology. doi: 10.1002/uog.14752. 
25-Shadrina, A. et al. (2018) ‘Genome-wide association study in ethnic Russians 
suggests an association of the MHC class III genomic region with the risk of primary 
varicose 
veins.’, 
Gene. 
Netherlands, 
659, 
pp. 
93–99. 
doi: 
https://dx.doi.org/10.1016/j.gene.2018.03.039. 
26-Silver, R. M. (2015) ‘Placenta Accreta’, in Protocols for High-Risk Pregnancies: An 
Evidence-Based Approach: Sixth Edition. doi: 10.1002/9781119001256.ch53. 
27-Spaight, C. et al. (2016) ‘Gestational diabetes mellitus’, Endocrine Development. 
doi: 10.1159/000439413. 
 
 

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13 Appendix: 
 
13.1 Complications arising from pregnancy (this is a general 
information and won't be given to the participant). 
 
 
 
Some pregnant women may experience complications during pregnancy. These 
complications may affect the mother’s health or fetal health or both. The most common 
pregnancy complications include high blood pressure, gestational diabetes, 
preeclampsia placenta previa and infections. Pregnancy complications will be 
recorded in this study for each participant in order to correlate any potential 
complication with the incidence of VV and to generate comprehensive analysis at the 
end of this study.  
Preeclampsia is one of the main pregnancy complications which cause fetal morbidity 
and mortality. Early signs of preeclampsia include high blood pressure and 
accumulated amounts of proteins in urine which is known as proteinuria after 20 weeks 
of pregnancy (Oettinger-Barak et al., 2005). Untreated preeclampsia may lead to 
eclampsia which involves seizures that may result in coma, brain damage and fetal 
death. Preeclampsia is aggravated by several risk factors including older age, 
nulliparous, chronic hypertension, high BMI>30 and gestational diabetes during 
pregnancy.  
 
High blood pressure and Gestational diabetes mellitus (GDM) are serious pregnancy 
which result in maternal and fetal complications. Hypertension arises when arteries 
carrying blood from the heart to body organs are narrowed which thus cause high 
blood pressure in arteries. In pregnancy, hypertension reduce amount of blood flow 
containing nutrition and oxygen to the placenta. Consequently, this will affect fetal 
growth and further complications such as preterm delivery and preeclampsia. On the 
other hand, GDM is identified as glucose intolerance developed during pregnancy 

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period at any grade and whether insulin or diet adjustment is used for treatment 
(Spaight et al., 2016). Pregnant women with GDM are at higher risk of developing 
diabetic 2 after pregnancy (Berger et al., 2016). Furthermore, GDM is coupled with 
high rates of caesarean delivery, increased obesity, fetal macrosomia which may 
cause birth complications such as stillbirth and shoulder dystocia (Lefkovits, Stewart 
and Murphy, 2019).  
 
Placenta previa, placenta accreta and vasa previa are also critical pregnancy 
complications. Placenta previa occur when the placenta lies low in the uterus less than 
2 cm from the cervix or covers the cervix completely (Oyelese and Smulian, 2006). 
This can lead to further complications such as bleeding at labour and premature 
delivery. Placenta accreta is known as abnormal implantation of the placenta in the 
uterus (Silver, 2015). This condition is vital and cause severe bleeding at labour 
(Oyelese and Smulian, 2006). Vasa previa arise when fetal umbilical cord blood 
vessels is located near or across the cervix internal opening. At labour, umbilical cord 
blood vessels will rapture causing fetal bleeding and critical neonatal mortality and 
morbidity (Ruiter et al., 2015).  
 
Infections may develop during pregnancy and cause further maternal and fetal 
complications. These infections may be caused due to several factors including 
immune system changes during pregnancy, hormone fluctuations, stress and 
mycobiome such as viral, bacteria and fungus (Azwa, Marsh and Hawkins, 2012). 
Infections during pregnancy may be passed from the mother to her fetus causing 
critical complications such as preterm labour, congenital anomalies and maternal 
sepsis (Lauretti and McKay, 2008).