Normal Development of the Human Fetus and the Influences and Mechanisms by Which That Development Occurs and is Perturbed
Conditions: Fetal Developmental Abnormality, Fetal Development, Human, Life Style
Sponsor: University of Aberdeen

Protocol V2 12/01/2017 
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Scottish Advanced Fetal Research Study 
Full Title: Normal development of the human fetus and the 
influences and mechanisms by which that development 
occurs and is perturbed. 
Sponsor: University of Aberdeen  
Funders:  
Medical Research Council,  
 
Society for Endocrinology,  
 
NHS Grampian 
 
Chief Investigator: Prof. Paul Fowler 
 

Protocol V2 12/01/2017 
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Study Management Group 
 
Chief Investigator:  Professor Paul Fowler BSc Hons, PhD, FRSB 
 
 Institute of Medical Sciences, 
School of Medicine, medical Sciences & Nutrition, Foresterhill 
 
 Aberdeen AB25 2ZD 
 
 Scotland 
 
 United Kingdom 
Telephone no:  
 01224 437528 
E-mail: 
 p.a.fowler@abdn.ac.uk 
 
 
Sponsor:  
The Sponsor for this study is the University of Aberdeen/NHS Grampian.  
 
For Further information the sponsor contact is: 
 
 
Ms Patricia Burns 
 
 
Research Governance Management 
 
 
Foresterhill House Annexe 
 
Foresterhill 
 
Aberdeen 
 
AB25 2ZBTelephone no:  
01224 551123 
Fax no:  
01224 550559 
E-mail:  
patriciaburns@abdn.ac.uk 
 
Funder: 
 
MRC 
 
Polaris House 
 
North Star Avenue 
 
Swindon 
 
SN2 1ET 
 
 
Society for Endocrinology 
 
22 Apex Court 
 
Woodlands 
 
Bristol 
 
BS32 4JT 
 
 
NHS Grampian 
 
Research & Development 
 
Foresterhill House Annexe 
 
Aberdeen 
 
AB25 2ZB 
 
 

Protocol V2 12/01/2017 
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This protocol describes the “Normal development of the human fetus and the influences and 
mechanisms by which that development” study and provides information about procedures for 
entering participants.  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 directed, in the first instance, to the Chief Investigator. This protocol will adhere to the principles 
outlined in the NHS Research Governance Framework for Health and Social Care (2nd edition).  It will be 
conducted in compliance with the protocol, the Data Protection Act and other regulatory requirements as 
appropriate. 
 
Table of Contents 
 
1. Introduction 
1.1 Overview 
1.2 Drug use during pregnancy 
1.3 Social deprivation 
1.4 The placenta is at risk from adverse maternal lifestyle factors 
1.5 The human lifestyle and the fetus are very different from the rodent 
1.6 Importance of fetal liver development for adult health (currently 
funded by the Medical Research Council) 
1.7 Normal human fetal development remains poorly understood 
1.8 Summary 
 
2. Study Objectives 
2.1 Objectives  
2.1.1 Primary Objectives 
2.1.2 Secondary Objectives 
2.2 Outcomes 
2.2.1 Primary Outcomes 
2.2.2 Secondary Outcomes 
 
3. Study Design 
 
4. Study Population 
4.1 Number of Participants 
4.2 Inclusion criteria 
4.3 Exclusion criteria 
 
5. Participant Selection and Enrolment 
5.1 Identifying participants  
5.2 Consenting participants 
5.2.1 Post-consent stages in the ward 
5.2.2 Post-consent stages in the laboratory 
5.3 Participant withdrawal 
 
6. Data Collection and Management 
6.1 Data Collection 
6.2 Data management system 
 
7. Statistics and Data Analysis 
7.1 Proposed Analysis 

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8. Study Management 
 
9. Inspection of Records 
 
10. Good Clinical Practice 
 
10.1 Ethical Conduct of the Study 
10.2 Confidentiality 
10.3 Data protection 
10.4 Insurance and Indemnity 
 
11. Study Conduct Responsibilities 
11.1 Protocol Amendments, Deviations and Breaches 
11.2 End of Study 
 
12. Reporting, publications and notifications of results 
12.1 Authorship Policy 
12.2 Publication 
12.3 Peer Review 
 
13. Bibliography 

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List of Abbreviations:  
 
SAFeR:  
Scottish Advanced Fetal Research study 
ToP 
 
Termination of Pregnancy 
PoC 
 
Products of Conception 
SOPs  
Standard Operating Procedures 
 

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PROTOCOL APPROVAL 
Signatures 
By signing this document I am confirming that I have read, understood and 
approve the protocol for the above study. 
 
 
Prof Paul A. Fowler 
 
 
 
 
05/112015 
Chief Investigator 
 
Signature 
 
Date 
 
 
 
 
 
Dr Alex Douglas 
 
 
05/11/2015 
Individual 
Responsible 
for 
Statistical Review 
 
Signature 
 
Date 

Protocol V2 12/01/2017 
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1.0.  
Introduction 
 
1.1 
Overview 
In-utero exposure to drugs and chemicals through maternal smoking, alcohol use, drug abuse, 
prescription medicines and occupational/lifestyle exposures is widespread. Such exposures can 
alter fetal development and programming, leading to the effects becoming “locked in” from birth 
and causing long-term adverse consequences for the individual. These include costly and 
widespread conditions such as obesity, hypertension, metabolic syndrome and infertility (Cupul-
Uicab et al. 2012, Hackshaw et al. 2011, Reynolds et al. 2011, Snijder et al. 2011, Snijder et al. 
2012). The weight of evidence linking these conditions to fetal recreational drug or environmental 
chemical exposures, including cigarettes, alcohol, air pollution, food contact materials, is 
overwhelming. What is lacking is an understanding of how fetal drug exposure translates to adult 
ill-health and this is due, largely, to our inability to study the problem directly in affected human 
fetuses.  
 
1.2 
Drug use during pregnancy 
While the overall incidence of smoking in the UK has fallen over the last 20 years, significant 
numbers of women continue to smoke: typically around 30% of women aged <30 years. In 
Aberdeen, for example, 31% of women are smokers when first pregnant and typically fewer 
than 4% of pregnant women stops smoking during pregnancy (Tappin et al. 2010). In the UK, 
roughly 80% of young women drink (40% heavily) and up to 20% use recreational drugs, with 
smokers more likely to also drink. Women who use drugs tend to stop taking ecstasy when they 
become pregnant but 50% continue to use cannabis (Moore et al. 2010). Questionnaire 
sampling suggests that alcohol use also declines when women become pregnant but in the UK 
more than 20% continue to drink during pregnancy (6% heavily) (Mehta et al. 2009). 
Unfortunately, fetal alcohol exposure is difficult to measure reliably (Joya et al. 2012), especially 
below levels inducing fetal alcohol syndrome. Thus national exposure levels may be higher than 
assumed and exposure for the individual neonate is generally unknown. It should be noted that 
second hand (side-stream) cigarette smoke exposure is also associated with a significant 
increase in congenital defects and post-natal health deficits (Meeker and Benedict 2013). 
 
1.3 
Social deprivation 
Social deprivation is a significant risk factor for childhood health (Weightman et al. 2012) and 
some of this may come from programming during fetal life (Reynolds et al. 2013). Furthermore, 
as we have seen in our studies of the human fetal liver exposed to maternal cigarette smoking 
(Drake et al. 2015, Filis et al. 2015, O'Shaughnessy et al. 2011), there is also sexual dimorphism 
in the growth response to maternal stress (Giesbrecht et al. 2015). The sex of the fetus is 
important in terms of the placenta (Muralimanoharan et al. 2015) and this also holds true for 
the effects of another adverse maternal lifestyle factor, obesity. Furthermore, adverse lifestyle 
choices such as drinking and smoking are more prevalent amongst the most deprived women 
(e.g. (Raisanen et al. 2014)). This means that the fetus and placenta will be subjected to multiple 
adverse environmental factors in some women. Distinguishing between those fetuses and 
placentas that are affected significantly by such multiple “hits” and those who are not is a 
significant health challenge. 
 
1.4 
The placenta is at risk from adverse maternal lifestyle factors 
Both maternal smoking (Kawashima et al. 2014, Shinjo et al. 2014) and, to a lesser extent, 
alcohol use (Lui et al. 2014, Wang et al. 2014) are implicated in reduced placental and fetal 
development, although the effects become more evident later in pregnancy (Anblagan et al. 
2013). The placenta has been studied as a potential matrix with which to detect adverse 
exposures using biomarkers, such as environmental heavy metal or polycyclic aromatic 
hydrocarbons. Furthermore, there are emerging studies showing that specific biomarkers in the 
placenta can highlight specific risks (e.g. Arrebola et al. 2015). However, the use of the placenta 

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as a matrix with which to monitor diseases in a more advanced clinical perspective has been 
hampered by lack of focused experimental and epidemiological data. In addition, sex 
differences in placental responses to adverse conditions (Brown et al. 2014) mean that the sex 
of the new-born will be an important criteria in biomarker searches. 
1.5 
The human lifestyle and the fetus are very different from the rodent 
Maternal intake of chemicals, including pollutants and drugs, occurs via multiple routes of 
exposure (Fig.1), with maternal biotransformation (metabolism, modification, conjugation, 
sulphation) in the gastro-intestinal tract, liver and placenta. The fetus will be exposed to 
xenochemicals (e.g. nicotine metabolite cotinine (Fowler et al. 2008) through the feto-maternal 
interface at the placenta. Fetal blood from the placenta travels through the umbilical vein and 
supplies 70% of the blood to the fetal liver. The fetal liver is, therefore, exposed to high 
concentrations of maternally-derived xenochemicals/drug metabolites. Our own data shows 
that maternal smoking increased total polycyclic aromatic hydrocarbons (PAHs, a significant 
health-damaging component of air pollution) in the female human liver 5.7-fold (P=0.011) 
(Fowler et al. 2014). Unlike the rodent, the human 2nd trimester fetal liver expresses many 
xenochemical sensing and metabolising transcripts (O'Shaughnessy et al. 2011), which will act 
to protect the fetus from exposure. We demonstrated that expression of these transcripts 
change in responses to chemicals in cigarette smoke (O'Shaughnessy et al. 2011), probably due 
to increased levels of compounds activating the xenosensors AHR, PXR and CAR. We have also 
carried out a pilot proteomic study (Filis et al. 2015) of a closely matched subset of fetal livers 
previously published (O'Shaughnessy et al. 2011, O'Shaughnessy et al. 2013). Proteins involved 
in cellular homeostasis (males) and gastrointestinal diseases (females) were dysregulated 
differently between the sexes if the mother smoked. Maternal smoking, therefore, causes sex-
specific changes in the human fetal liver proteome, affecting diverse pathway and potentially 
laying the foundations for disease later in the offspring’s life.  
 
KEY: red outline = major 
biotransforming organs; GI 
= gastrointestinal tract 
Chemicals are primarily 
lost via maternal 
exhalation, urination and 
defecation.  
 
Fetal urination and drinking 
passes chemicals back and 
forth between fetus and 
amniotic fluid, which acts as 
a chemical reservoir. 
Fig.1: How exogenous chemicals reach the fetus. Maternal routes of ingestion/absorption 
pass via maternal liver and the placenta to the fetus where further biotransformation occurs.  
 
One of the effects of fetal xenochemical exposure is to alter fetal liver function, and this is likely 
to have knock-on effects to other aspects of fetal growth. We have shown that fetal liver function 
is sexually dimorphic, probably through androgen receptor expression (O'Shaughnessy et al. 
2013), thus playing a role in the differential growth of male and female fetuses. Furthermore, 
the human fetal liver expresses an extensive range of steroidogenic and steroid metabolising 
enzymes enabling a significant modulation of fetal circulating steroids (O'Shaughnessy et al. 
2013). This makes the fetal liver capable of altering steroid hormone signalling from the 
placenta, the mother and other fetal organs (e.g. adrenal glands) and suggests that it is a key 
endocrine organ during human fetal development. Overall, therefore, the fetal liver is likely to 
play a dual critical role in human pregnancy, protecting the fetus from xenotoxicant exposure by 
oxidation/reduction/conjugation reactions and by regulating steroid hormone exposure. 

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Liver development is an on-going complex interaction of factors likely to have significant knock-
on effects on fetal development in general. However, the fetal liver is also a target for these 
xenotoxicants, with consequences for its function. Our own data (Drake et al. 2015) shows that 
maternal smoking disturbs sex differences in the IGF system, significantly reducing hepatic IGF2 
in male fetuses, one way in which growth may be dysregulated in-utero, thus programming the 
fetus towards IUGR and adulthood metabolic syndrome. We also found that maternal smoking 
altered DNA methylation at a number of genes susceptible to the in-utero environment, 
including the imprinted gene IGF2 and glucocorticoid receptor (NR3C1). Reduced male fetal 
hepatic DNTM1 expression demonstrates a sex-specific way by which maternal smoking alters 
CpG residue methylation. 
 
1.6 
Importance of fetal liver development for adult health (currently funded by 
the Medical Research Council) 
There is strong evidence to suggest that dysregulaton of human fetal liver development has 
consequences for adult health. One link is through impaired fetal growth, which can lead to fetal 
programming of metabolic syndrome in adulthood (Latini et al. 2004). A down-stream 
consequence of increasing metabolic syndrome levels is increased non-alcoholic fatty liver 
disease, which affects 17-30% of the population in Western countries (Kneeman et al. 2012) 
and which also has roots in disturbed fetal growth. Programming of metabolic syndrome may 
also be partially affected by alterations in steroid enzyme activity (Nyirenda et al. 2009) and 
there is strong evidence for greater risk of fetal programming of dysregulated hepatic lipid 
metabolism in males (Choi et al. 2007). The link between fetal environment (including 
nutritional variation), the liver and adult disease is poorly understood (e.g. (Morrison et al. 
2010)) and further research into the factors leading from dysregulated fetal liver development 
to disturbed adult metabolic processes is required. 
 
1.7 
Normal human fetal development remains poorly understood 
We have previously (approved by the NHS Grampian Research Ethics Committees (REC 
04/S0802/21)) reported adverse effects of maternal cigarette smoking on the fetal testis 
(Fowler et al. 2008, Fowler et al. 2009), ovary (Anderson et al. 2014, Fowler et al. 2014), 
urogenital tract (Fowler et al. 2011b) and liver (Drake et al. 2015, Filis et al. 2015, 
O'Shaughnessy et al. 2011, O'Shaughnessy et al. 2013). However, there are many aspects of 
human fetal development that remain very poorly understood, in part because researchers have 
relied on studies of rodents and other animals and only a limited number of studies have used 
either miscarried fetuses (therefore of suspect normality) or electively terminated fetuses from 
normal pregnancies. We have also contributed significantly to understanding of normal fetal 
development, and sex differences, in terms of the gonads, urogenital tract and the liver (Drake et 
al. 2015, Filis et al. 2015, Fowler et al. 2009, Fowler et al. 2009, Fowler et al. 2011a, Fowler et al. 
2011b, Fowler et al. 2014, O'Shaughnessy et al. 2007, O'Shaughnessy et al. 2011, O'Shaughnessy 
et al. 2011, O'Shaughnessy et al. 2013). It is very important to continue this process of 
characterising the processes of normal human fetal development in order to better understand 
how that development is controlled, how it can be disturbed (e.g. by maternal lifestyle or 
pollution) and how this can be detected. Furthermore, health-risks posed by chemicals and 
medicines (e.g. plastics like bisphenol A, pain-killers), to both the mother and the fetus (e.g. 
paracetamol (Snijder et al. 2012, van den Driesche et al. 2015)) requires better fundamental 
understanding of how the fetus and its placenta develop and how that process is regulated. A 
current example is a study we are preparing for publication: we have found that the human 
male fetus has at least 2,000 times higher levels of oestrogens circulating in its blood than an 
adult man. This will likely have significant implications for the risk posed to the developing male 
fetus by environmental oestrogens (e.g. chemicals from the pill found in water), which are 
considered a major cause for concern in the environmental toxicology field. Out other on-going 
studies include research on the fetal endocrine systems focussing on the fetal adrenal and 
thyroid glands, both in terms of normal development and the effects of maternal smoking. 

Protocol V2 12/01/2017 
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1.8 
Summary 
We, and others, have shown that human fetal development, which lays the foundations of adult 
health and function (fetal programming), is quite different from the rodent and frequently 
exhibits surprising aspects. It has become evident that the close interconnectivity of the 
developing fetal organs and also the placenta, means that a much more holistic approach to 
research aiming to understand human fetal development and the challenges posed to 
programming for a health adulthood is critical. To that end we are requesting a carefully 
considered expansion in the numbers and ages range (from 11-21 to 7-20 weeks of gestation) of 
fetuses we can study and in the range of organs, body fluids and maternal information collected. 
Specific project areas are highlighted in the associated SAFER project REC application. 
 
2.0  
Study Objectives 
 
2.1 
Objectives  
Our overarching objective is to intensively and systematically study the human fetus during a 
normal pregnancy and pregnancies where aspects of maternal lifestyle and environment will 
challenge the fetus. We aim to provide fundamental information to better understand the 
mechanisms involved and to detect and treat or ameliorate adverse effects during pregnancy 
(such as maternal smoking/drinking, deprivation, exposure to pollution). In the long term 
findings from this research will be important for future studies aimed at enabling better health 
in later life. 
 
2.1.1 Primary Objective 
To provide fundamental information to better understand the mechanisms involved and to 
detect and treat or ameliorate adverse effects during pregnancy. 
 
2.1.2 Secondary Objectives 
The secondary objectives of this study are al aimed at better understanding of normal processes 
during development and how maternal lifestyle and environment can affect it. Our research will 
involve a wide range of laboratory, cell and tissue culture and xenografting as well as 
microscopic techniques to study chemicals, hormones, proteins, genes and DNA in the fetal 
tissues. We have developed sophisticated approaches to minimise how much tissue we require 
to carry our as many different measurements as possible in the same sample. It should be noted 
that the proposed studies are collaborative with different research groups since no single 
research group could carry out all the studies in isolation. Furthermore, the proposed studies 
will inform back to animal and cell studies, enabling more accurate interpretation of their own 
findings in light of mechanisms and specific conditions actually at play in the human fetus. 
 
(1) Human fetal endocrine system 
We aim to investigate how the different hormone systems in the fetus develop and are affected 
by adverse maternal factors such as cigarette smoking and obesity. These include the testis, 
ovary, adrenal gland, thyroid gland, kidney and the hypothalamus, pineal gland and pituitary 
gland in the brain. In addition, we also aim to study the placenta, including its endocrine 
function, and how it is affected by maternal environment, since it is a key organ of pregnancy. 
Changes in programming that establishes the endocrine system can have lifelong consequences 
for health and wellbeing. 
 
(2) Human fetal reproductive and urogenital system 
We aim to investigate how the ovary, testis, urogenital tract (including prostate gland, breast 
buds and uterus), genitalia and kidney develop in the human fetus. There is considerable 
evidence that maternal smoking and over the counter medicine use can have negative impacts 
on this system and we will investigate when such impacts begin and what mechanisms are 

Protocol V2 12/01/2017 
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involved. In addition, common cancers, such as testis cancer, may have fetal origins and 
certainly events in fetal life may predispose the resulting adult to develop cancers. In the case of 
testis, ovary, breast buds and prostate gland, an additional aim is to determine whether and 
how events between 7 and 20 weeks of gestation increase the likelihood of cancers in these 
organs in adulthood. 
 
(3) Mechanistic studies, Epilepsy and other brain disorders  
We aim to investigate development of the fetal central nervous system, including the brain. The 
first study planned is to investigate changes in genes and the structure of DNA in the brain 
related to epilepsy and other neurodevelopmental disorders linked with maternal cigarette 
smoking during pregnancy. A second aim is to use the smoke-exposed fetuses to better 
understand the role vitamin A plays in normal fetal development, including development of the 
central nervous system. Similar concerns are expressed in the literature with respect to 
maternal alcohol consumption and by accessing data from the FAST alcohol questionnaire that 
is already collected at clinic, we will be able to address this issue systematically with respect to 
all our studies, including fetal brain development. 
 
(4) Metabolism and liver disease 
We aim to continue our studies (funded by the Medical Research Council) of the human fetal 
liver and other organs important for metabolism, including pancreas and fat cells and cells that 
will become fat cells (precursor cells). Programming of metabolism during life in the womb is 
important for adult health and better understanding of the development of the metabolic 
systems and how they are perturbed by factors such as maternal smoking and deprivation is an 
important basis of developing strategies for wellbeing post-natally. A critical disease of growing 
importance in these studies is non-alcoholic fatty liver disease, which is increasing in incidence, 
especially in obese individuals. Given that the human fetal liver is active and responding to 
maternal environment, some of the basis of this disease may be established in fetal life. In 
addition, the development of thyroid and adrenal glands is also essential in establishing normal 
metabolic processes. Cutting edge techniques, such as large-scale metabolomic and lipidomic 
studies, including analysis of a wide range of dietary metabolites, will also be used. 
 
(5) Heart and cardiovascular system 
The development of the heart and the major artery, the aorta, between 7 and 20 weeks of 
gestation will be characterised. The effects of adverse in-utero environment of the 
developmental processes will then be investigated. 
 
(6) Immune system development 
The thymus is critical and large organ in fetal development that is important to establish the 
immune system. The spleen manufactures antibodies and, in fetal life, red blood cells (as does 
the fetal liver) and recycles red blood cells. Both organs have important role in maintaining the 
immune system. We aim to investigate whether adverse maternal lifestyle and/or environment 
can alter thymus and spleen development and function. 
 
(7) Bone, cartilage and joints 
Very little is known about normal bone and cartilage development in the human fetus. 
Importantly, it is not even know when the cells involved in bone, cartilage and joint 
development begin to alter their behaviour into a more “adult” format. The reason this is 
important is that in musculoskeletal disease in the elderly, especially osteoarthritis, early stages 
of disease is usually characterised by renewed tissue growth – i.e. a return to a more “fetal” 
pattern of cell behaviour. The role of cell programming during fetal life and better 
understanding of the correct development and maintenance of bone, cartilage and joints, and 
how programming is affected by the fetal environment, is therefore one of our aims. 
 
 

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(8) The development of the placenta between 7 and 20 weeks of gestation 
We know a great deal about the term placenta because it is so easy to obtain and to relate 
directly to the accompanying new-born and its parents. However, the placenta is a dynamically 
developing organ that is vital for normal pregnancy and that is affected by maternal lifestyle 
(e.g. placental growth is reduced if the mother continues to smoke while pregnant). We aim 
therefore to better understand how the placenta develops during this window in gestation and 
how it is affected by maternal environment. 
 
(9) Nerve repair 
Spinal cord injury is a massive health problem globally. Recently it has been shown that human 
fetal central nervous system cells or embryonic nerve stem cells have the potential to assist in 
novel treatment strategies to repair spinal cord injury.  This final secondary objective is 
focussed on a proof of concept study to determine whether culturing severed nerves in the 
presence of human fetal nerve cells assists in correct and functional nerve regrowth and re-
joining across the induced gap. Any further studies, if the proof of concept aim is realised in a 
satisfactory manner would require a separate ethic application. 
 
(10) Maternal emotional health and wellbeing effects on the fetus 
Maternal stress is a well-known negative influence on fetal development. Our aim is to perform 
a pilot study relating indices of fetal development, such as morphology, endocrine status, to 
maternal records of emotional health and wellbeing and abuse. The principal outcome will be an 
indication as to whether maternal stress is affecting fetal development at a general level 
between 7 and 20 weeks of gestation. Placental biomarkers identified in other studies listed 
above will also be related to measures of maternal stress in order to establish whether there 
may be placental biomarkers of early fetal response to maternal stress.  
 
(11) Fetal lung 
While it is well known that the adverse effects of maternal smoking include increased 
incidences of respiratory problems, such as asthma. However, there are also data suggesting 
that the offspring exposed to cigarette smoke may be more likely to develop lung cancer. Our 
aim is to determine whether maternal smoking disturbs fetal lung development early in 
pregnancy and if this includes signs of increased likelihood of cancer development, such as 
polycyclic aromatic hydrocarbon-DNA adducts in the fetal lungs. 
 
(12) Quantifying human fetal exposure to pollutants, drugs, pharmaceuticals and other 
chemicals  
It is simple to measure chemicals in the blood or urine of the mother, or in amniotic fluid 
collected during amniocentesis. However, amniocentesis is not performed for research 
purposes because of not inconsiderable risk to the fetus. This means that we have to reply on 
animal studies of such chemicals getting to the fetus but this is rather inadequate due to major 
species differences. Therefore we aim to measure chemicals and drugs of potential health 
concern (such as bisphenol A which is in plastics and over the counter drugs taken by mothers 
which may affect the fetus, such as paracetamol) in the fetal tissues, including blood and urine 
where available. This is the only way to accurately assess how much of these compounds 
actually get into the fetus itself. This is important since the fetus is considered much more 
vulnerable than the adult to toxic chemicals. In addition, we aim to also measure these 
compounds in the placentas (and cord blood where available) from the same fetuses because 
the placenta is readily available at term and we aim to investigate in the placenta biomarkers of 
fetal exposure to adverse compounds (e.g. alcohol) that are detected in the second trimester 
fetal liver. 
 
(13) Non-genetic programming of the fetus for adult life 

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Epigenetic changes are changes that involve marks being put onto DNA that affects how genes 
are activated. Therefore, an epigenetic change can change, sometimes greatly, the link between 
the amount of a gene present in cell and its effect. This linkage can be quite large with a gene 
being practically silenced in terms of its effect, even though there may be plenty of the gene 
present in the cell. Conditions within the womb, such a maternal smoking, can have epigenetic 
effects. We aim therefore to better understand how the environment experience by the fetus can 
affect development and programming of adult health and disease by epigenetic mechanisms. 
 
2.2 
Outcomes 
 
2.2.1 Primary Outcomes 
The provision of an extensive dataset on the human fetus during normal pregnancy and 
pregnancies where aspects of maternal lifestyle and environment will challenge the fetus, 
informing on future heath and function. 
 
2.2.2 Secondary Outcomes 
The headline secondary objectives of the study, applying to a wide range of body systems and 
organs, are: 
 
(1)  
The identification of key stages and regulators required for the normal development of 
the human fetus and its placenta. 
 
(2)  
The quantification of maternal lifestyle factors, including body burdens of toxicant 
chemicals (e.g. from smoking, from air pollution, from food contact material) and 
determination of their relationship with adverse fetal and placental outcomes. 
 
(3)  
Mechanistic insights into how fetal human development is controlled and how this control 
can be disturbed by maternal factors. 
 
(4)  
Establishment of the risks of adverse in-utero environment for future generations. This is 
epigenetic and focuses on ways in which fetal event, without any changes in genes 
themselves, can mis-programme the development of future generations. 
 
3.0  
Study Design 
 
3.1 Study Description 
Normally, for a study, a specific question is addressed by prospectively recruiting a carefully 
considered, statistically validated, number of patients of specific types. Effects or differences are 
then measured as detailed in the ethics application. For our study this approach is simply not 
possible. There are several reasons for this, most importantly: 
 
(1)  
It is impossible to predict the categories of pregnancies that become available, including 
maternal characteristics, sex and age of the fetus, normality of the fetus and whether the 
mother will withdraw from the study. 
(2)  
It takes months and even years to collect a statistically valid number of specific fetuses 
suitable to answer secondary research questions. 
(3)  
It is evident that there are very clear sex differences during development and for the 
majority of studies it is essential to have matching numbers of male and female fetuses. 
(4)  
In our experience of the last 10 years of such studies, new data very frequently leads to 
unexpected new secondary questions. 
 
For these reasons, the only viable strategy is to collect every fetus that becomes available with 
written, informed, consent and store the tissues and data collected in ways appropriate for a 

Protocol V2 12/01/2017 
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range of different research methods to be used. We have consulted with the Grampian 
Biorepository with respect to this application. Thereafter, once appropriate numbers of fetuses 
best matching a specific sub-project become available, the sub-project can begin. Essentially this 
is a type of retrospective design but this is the only scientifically and statistically valid manner 
to proceed under the circumstances (see 3.2 Study Design Flowchart below). 
3.2 Study Design Flowchart 
 
4.0  
Study Population 
4.1 Number of Participants 
Based on analysis of recent figures, approximately 800 and 1,400 fetuses ≥7 weeks of gestation 
are terminated medically in Aberdeen and Glasgow respectively. Of these some 5%10% are 
expected to consent. At a highly conservative estimate, over 10 years, this would be 1,200 
fetuses Aberdeen and Glasgow combined should be able to achieve this target. For reasons laid 
out here and in the full ethics application, we cannot have a priori finalisation of sample size. 
Therefore, we request approval to aim to collect 1,200 fetuses, enabling sufficient numbers to 
perform appropriately powered sub-studies. 

Protocol V2 12/01/2017 
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Protocol V2 12/01/2017 
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4.2      Inclusion criteria 
• 
Women at 7-20 weeks of gestation (critical stage of fetal development). 
• 
Women aged 16 years and older, deemed capable of making a rational decision. 
• 
Absence of fetal anomaly at ultrasound scan (only normal fetuses are required). 
• 
Women who are fluent English speakers. This is in order to ensure the woman 
understands that fetal tissues will be collected. 
 
4.3     Exclusion criteria 
• 
Women exhibiting considerable emotional distress. 
• 
Fetal anomalies identified at ultrasound scan (it is essential to ensure fetal normality). 
 
5.0  
Patient selection and enrolment 
 
NB: see SAFeR Consent to Disposal flowchart (page 19) in conjunction with sections 5 and 
6 below 
 
5.1 
Identifying participants 
 
Women seeking a termination will attend clinic to receive their first medication around 36-48 
hours prior to ward admission as part of standard care. The study will be explained to potential 
participants during this appointment. Patients will in no way be persuaded, induced or coerced 
into participating in this study. The researchers and laboratory staff play not part what so ever 
in participant identification. The SAFeR Study Information Sheet will be offered to women 
who fit the inclusion criteria by members of the direct clinical team. The women will be able to 
take this information sheet home with them and will have at least 24 hours prior to ward 
admission for reflection. Where a woman is approached, this is recorded in her clerking sheets. 
 
The following data are routinely collected by clinical staff and recorded on the clinical record of 
each woman attending: 
 
1. Age 
2. Weight 
3. Height 
4. LMP 
5. Previous pregnancies or ToPs 
6. Medication use (both over-the-counter and prescribed) 
7. Recreational drug use, including cigarette and ecigarette use 
8. FAST alcohol score (validated questionnaire) 
9. Postcode*  
10. Intimate partner abuse (short list of questions) 
11. Emotional health and wellbeing (short list of questions) 
12. Normality and stage of the pregnancy based on ultrasound scan 
 
*note the postcode will never be supplied to any researcher involved in the study 
 
These data are noted on the Clerking Sheet for each woman, which is routinely passed on to the 
nurses if the woman is progressing to termination of pregnancy (ToP). The women are not 
asked any additional questions for the SAFeR study.  
  
5.2 
Consenting participants 
This is performed by a member of the direct clinical team when patients are admitted to the 
appropriate ward. The clinical staff both in the clinics and in wards will independently assess 

Protocol V2 12/01/2017 
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the women according to inclusion and exclusion criteria. Members of the clinical team will be 
GCP trained and will have experience of this vulnerable patient group. 
 
 
In the ward, women will be asked if they have read the SAFeR Study Information Sheet 
(provided at clinic at least 24 hours previously) and whether they are willing to take part in the 
study. If they consent, they will be asked to sign the SAFeR Consent Form. The nurses will 
remain with the women throughout the day and will be available to answer questions at any 
time. The participants are able to sign the consent form and change their mind at any 
stage, right up to the point where the products of conception are removed from the ward. 
Once the consent form is signed, the nurses will notify the laboratory staff by telephone. The 
laboratory staff will then open a new entry in the electronic SAFeR MONITORING LOG, which is 
held in a secure and restricted, password-protected, filespace on a University of Aberdeen 
server (therefore continuously backed-up) and begin recording the entire process. 
 
5.2.1 Post-consent stages in the ward 
The ToP proceeds in exactly the same manner as it would if the women did not consent. The 
nurses also check that all approved data from the clerking sheets is recorded in the Maternal 
Datasheet. The clerking sheets remain confidential and are never seen by the research team. 
Once the products of conceptions (fetus and placenta) are delivered by the mother there are 
two possible scenarios: 
 
CONSENT IS WITHDRAWN: 
In this case the ward proceeds with the disposal of the products of conception to the mortuary 
according to current legislation. The nurses notify the laboratory to enable logging of the 
withdrawal of consent. The process is then at an end. 
 
CONSENT IS MAINTAINED: 
In this case the nurses clamp the umbilical cord, place the products of conception in the 
appropriate sealed opaque container and telephone the laboratory staff. They also enter the 
SAFER number and date (only) on the Fetal Datasheet. No maternal identifiers are recorded in 
either the Fetal or Maternal Datasheet. When the products of conception are collected by the 
laboratory staff, the nurses record this in their logbook. This acts as an independent cross-check 
for whether the fetus was removed from the ward. Once the products of conception have been 
removed from the ward, the nurses also note in the TOPS coding sheet that the pregnancy has 
entered the SAFeR study. This then enables the Data Management Team (under Dr Katie Wilde), 
University of Aberdeen, who host the Grampian TOPS database, to record this fact and to send 
Professor Fowler monthly reports on the 5% category of the SIMD score that each woman 
(whose products of conception have been taken to the laboratory) falls within. This enables the 
relative deprivation status of the mother to be used in the research project in a manner that will 
have absolutely no impact what so ever on her absolute anonymity (e.g. avoids the need for 
postcode data to be used by the researchers).  
 
Before leaving the ward the laboratory staff check that they have all relevant documentation 
(copy of CONSENT FORM and SAFeR MATERNAL and FETAL DATASHEETS and the NHS 
MORTUARY FORMS). The laboratory staff then transport the products of conception to the 
laboratory. On the way they stop at the relevant NHS Grampian Biorepository office to pass 
on the SAFeR CONSENT FORM and thus register the PoC and the SAFeR study number with the 
Biorepository. The consent forms are available at the Biorepository for NHS audit purposes. The 
laboratory staff then continue to the laboratory. In the event that the Biorepository is shut (i.e. 
after hours working), the SAFeR Consent Form will be taken to the Biorepository on the 
earliest available working day.  
 

Protocol V2 12/01/2017 
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5.2.2  Post-consent stages in the laboratory 
Laboratory staff collects the products of conception from the ward as soon as possible, after 
being notified by the nurses, and transport them back to the laboratory in the sealed opaque 
container carried in a sealable opaque cold box (i.e. 3 layers of containment of the PoC). On the 
way to the laboratory (working hours) or as soon as possible on the following working day the 
CONSENT FORM must be lodged with the Biorepository. Upon arrival in the laboratory, 
remaining available documentation from the ward (SAFeR MATERNAL and FETAL 
DATASHEETS and the NHS MORTUARY FORMS) must be re-checked and any irregularities not 
observed in the ward queried with the nurses and logged. There are two possible scenarios that 
affect exactly how the products of conception are handled in the laboratory: 
 
PRODUCTS OF CONCEPTION DELIVERED WITHIN 8 HOURS OF ToP TREATMENT ONSET: 
In this case the samples are collected and processed in a manner suitable for molecular biology, 
cell/tissue culture and histology/morphology, requiring rapid processing of samples for 
molecular biology to liquid nitrogen and then storage at -80°C and/or processing to culture 
conditions and processing for histology/morphology 
 
PRODUCTS OF CONCEPTION DELIVERED MORE THAN 8 HOURS OF ToP TREATMENT ONSET: 
In this case the samples are collected and processed in a manner suitable for 
histology/morphology or other measurements less affected by length of time after fetal death, 
such as measurement of stable pollutants. Processing is less urgent and if necessary can occur 
the following day. Products of conceptions that are not processed within 48 hours of delivery 
must be disposed of immediately. 
 
SAMPLE PROCESSING: 
A detailed protocol for dissection (SAFeR Tissue Collection Protocol ) is used by laboratory 
staff to guide the processing of the products of conception. The processing is conducted as 
rapidly as possible in an isolated laboratory using a class 2 cabinet to ensure laboratory staff 
safety. The laboratory has opaque windows and is allocated to Prof Fowler’s research group 
uniquely. Any staff present, who is not involved in the SAFeR study, will leave the laboratory 
immediately, prior to the transport bag being opened. This ensures that the process is not 
observed and is conducted with as much respect and sensitivity for the fetus as possible.  
 
Mr Paul Brown, Consultant Paediatric Pathologist, NHS Grampian is a named collaborator 
and is available to inspect fetuses should the laboratory staff have cause for concern with 
respect to normality. He is also able to provide additional training and advice to the laboratory 
staff harvesting the tissue samples. 
 
During dissection and processing the SAFeR Fetal Datasheet must be completed fully. Once all 
the samples have been processed, the person undertaking the processing must update the 
SAFeR Monitoring Log. 
 
DISPOSAL OF PRODUCTS OF CONCEPTION: 
Once all sample processing is completed the SAFeR Mortuary Receipt Form must be 
completed and the fetus and the NHS Mortuary Forms must be taken to the mortuary the next 
working day for disposal according to current legislation. The NHS Mortuary Forms are 
retained by the mortuary. The fetuses must be disposed of according to the Scottish CMO 
guidance 2012 update (SGHD/CMO(2012)7) on “SOHHD/DGM (1992)4: Sensitive disposal of 
fetuses and fetal tissues following termination of pregnancy” with a minimum standard of 
disposal being disposal at a crematorium. At this point the laboratory staff have no access to any 
patient identifiers and no record of these will be retained by the laboratory staff. Any enquiries 
requiring identifiers must thereafter be conducted via the Biorepository. 
 

Protocol V2 12/01/2017 
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The SAFeR Mortuary Receipt Form must be signed by the ARI mortuary staff and retained for 
the SAFeR Site File. The SAFeR Monitoring Log has a specific entry to record this. 
 
5.3  Participant withdrawal 
In this case, the ward proceeds with the disposal of the products of conception to the mortuary 
according to current legislation. The nurses notify the laboratory to enable logging of the 
withdrawal of consent. The process is then at an end. Once the products of conception leave the 
ward, the woman is no longer able to withdraw, 
 
6. Data Collection and Management 
The PI then maintains the SAFeR Site File, cross-checks and closes the SAFeR Monitoring Log 
for that fetus and enters fetal and maternal data into the SAFeR database. This is the research 
database and contains no identification information other than the unique study (SAFeR) 
number assigned to the products of conception by the nurses when the mother was consented. 
Electronic copies of the completed SAFeR Maternal and FETAL datasheets are then emailed to 
the Biorepository 
 
The SAFeR database and the SAFeR Monitoring Log are located in a secure folder with highly 
restricted, password-protected access, backed-up University server. New staff joining the team 
will complete the Good Clinical Practice (GCP) course before taking part in the research or 
having access to these two files. Data will be stored in accordance with the University of 
Aberdeen Data Protection Policy and the Data Protection Act (1998). The research described 
here will comply with University of Aberdeen SOPs.  
 
Site-specific Note: each site has its own SAFeR Monitoring log, but the SAFeR database is located 
in a secure folder with highly restricted, password-protected access, backed-up University of 
Aberdeen server. Named team members at each site will have access to this database only with 
prof Fowler’s specific approval. These individuals will be recorded and counter-signed in the 
Aberdeen Site file 
 
 

Protocol V2 12/01/2017 
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Protocol V2 12/01/2017 
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7. 
Statistics and Data Analysis 
7.1 Proposed Analysis 
 
The SAFeR study core personnel include Dr Alex Douglas, a highly experienced bio-medical 
statistician and data modeller. This is necessary because of the complexity of some of the data 
and statistical analyses to be performed. 
 
The SAFeR project (like its successful predecessor, the FEGO project) has an unusual design. 
This is simply because of the nature of the patients recruited (women undergoing elective 
terminations of pregnancy) and the samples obtained (products of conception). In addition, 
there is a steady incidence of withdrawals of consent once the patient see the products of 
conception. We are not able to have any information on the fetus prior to recruitment and 
successful return of the samples to the laboratory. This means that it is simply not possible to 
prospectively adjust numbers of fetuses falling into four principal groups (i) male, (ii) female, 
(iii) non-exposed, (iv) stage of gestation (7-20 weeks). There is certainly no prior planning 
possible with respect to which fetuses will have high or low levels of different exposures or 
different maternal lifestyle factors. This means that recruitment and collection of samples has to 
be conducted without specific target numbers until the point sufficient numbers of fetuses 
appropriate for specific sub-studies are available. Therefore the studies themselves have to be 
conducted retrospectively. Building up an extensive collection of fetuses also gives us the 
flexibility the flexibility to collaborate in a range of projects to answer study questions by 
bringing in world-leading expertise. Again, these could not be arranged prospectively. 
 
Examples of power analyses (accepted as suitable by the MRC when funding our study) are as 
follows (but each sub-study will require its own power analysis):  
 
FOR RNA-SEQ: The power of and RNA-seq experimental design to detect differential gene 
expression was assessed using the Scotty software package. Since there are no RNA-seq pilot 
data on the human fetal liver available, the power analysis was based on a publicly available 
human liver RNA-seq dataset, which enabled us to compare two treatment groups. Although our 
dataset will be more extensive, this allowed a preliminary power analysis to be performed. A 
total of 60 fetuses would have 85% power to detect a 2-fold change difference in gene 
expression assuming a sequencing depth of 30 M reads, a type I error rate of 1 % and equal 
number of fetuses in each group. 
 
FOR qPCR: To establish the likely power of the study we examined a representative outcome 
based on existing data from the 2nd trimester human fetus, using our most constrained 
population size. Based on the liver expression of GSTP1 (expressed relative to house-keeping 
genes), a study of 60 fetuses would have 80% power to detect differences in gene expression of 
700 units between fetuses from mothers who used drugs compared with those who do not, 
assuming a common standard deviation of 951 units, a type 1 error rate of 5% and equal 
numbers of fetuses in each group. 
 
The statistical approach depends upon the specific sub-project, its study design, its hypotheses, 
questions and objectives, the numbers of suitable fetuses available and the specific 
methodologies to be used. Examples of statistical analytical methods (accepted as suitable by 
the MRC when funding our study) are as follows (but each sub-study will require its own 
tailored statistical analytical approach):  
 
The RNA-seq data will be quality controlled using FastQC, quality filtering applied and the data 
aligned to the current reference human genome using BWA on the University of Aberdeen 
Maxwell computer cluster. Thereafter, HTSeq will be used in conjunction with current 
annotation to determine the number of reads aligning to each gene, feeding into the expression 

Protocol V2 12/01/2017 
22 
analysis. Following quantification of reads aligning to each gene, genes with altered transcript 
numbers will be identified using an empirical Bayes moderated generalised linear modelling 
approach as implemented in the Bioconductor package edgeR. This approach permits the 
efficient estimation of gene-specific biological variation and will allow us to identify gene 
transcripts that are significantly differentially expressed according to fetal sex, level of maternal 
drug exposure and gestation period. Type I error rates (false discovery rates, FDR) will be 
addressed: (i) non-specific filtering of genes with low numbers of reads across all treatment 
groups will be performed prior to statistical modelling to reduce the number of transcripts 
tested and therefore reduce the potential FDR. (ii) P-values associated with the differential 
expression analysis of each transcript will be adjusted using the Benjamini & Hochberg 
algorithm to control the FDR at <0.05. Co-expression of these significant transcripts will be 
further investigated by clustering transcripts based on expression profiles across treatment 
groups using hierarchical clustering techniques. 
 
8. 
Study Management 
 
The CI will have the overall responsibility of the study, assisted by the Glasgow site specific PI. 
Any queries will be resolved by the CI or a delegated member of the study team. A study-specific 
Delegation Log will be prepared for each site, detailing the responsibilities of each member of 
staff working on the study. 
 
9. 
Inspection of Records 
 
The CI, PIs and all institutions involved in the study will permit study-related monitoring, 
audits, and REC review. The CI agrees to allow the Sponsor direct access to all study records and 
source documentation. Due to the nature of this study, the consent process and tissue storage 
will be monitored frequently by NHS monitors who work closely with Research Governance.  
 
10.  Good Clinical Practice 
 
10.1 Ethical Conduct of the Study 
The study will be conducted in accordance with the principles of good clinical practice (GCP). In 
addition to Sponsorship approval, a favourable ethical opinion will be obtained from the 
appropriate REC and appropriate NHS R&D approval will be obtained prior to commencement 
of the study. 
 
10.2 Confidentiality 
All laboratory specimens, evaluation forms, reports, and other records will be identified in a 
manner designed to maintain participant confidentiality. All records will be kept in a secure 
storage area with limited access to study staff only. Clinical information will not be released 
without the written permission of the participant, except as necessary for monitoring and 
auditing by the Sponsor or its designee. The CI and study staff involved with this study will not 
disclose or use for any purpose other than performance of the study, any data, record, or other 
unpublished, confidential information disclosed to those individuals for the purpose of the 
study. Prior written agreement from the Sponsor or its designee will be obtained for the 
disclosure of any said confidential information to other parties. 
 
10.3 Data protection 
The CI and study staff involved with this study will comply with the requirements of the Data 
Protection Act 1998 with regard to the collection, storage, processing and disclosure of personal 
information and will uphold the Act’s core principles. The CI and study staff will also adhere, if 
appropriate, to the current version of the NHS Scotland Code of Practice on Protecting Patient 
Confidentiality.  Access to collated participant data will be restricted to the CI and appropriate 

Protocol V2 12/01/2017 
23 
study staff. Computers used to collate the data will have limited access measures via user names 
and passwords. Published results will not contain any personal data that could allow 
identification of individual participants. 
 
10.4 Insurance and Indemnity 
The University of Aberdeen and Grampian Health Board are Co-Sponsoring the study. 
Insurance:  The University of Aberdeen will obtain and hold a policy of Public Liability 
Insurance for legal liabilities arising from the study. 
Indemnity: The Sponsor does not provide study participants with indemnity in relation to 
participation in the Study but has insurance for legal liability as described above. 
 
11. Study Conduct Responsibilities 
11.1 Protocol Amendments, Deviations and Breaches 
The CI will seek approval for any amendments to the Protocol or other study documents from 
the Sponsor, REC and NHS R&D Office(s). Amendments to the protocol or other study docs will 
not be implemented without these approvals. In the event that a CI needs to deviate from the 
protocol, the nature of and reasons for the deviation will be recorded in the CRF, documented 
and submitted to the Sponsor. If this necessitates a subsequent protocol amendment, this will 
be submitted to the Sponsor for approval and then to the appropriate REC and lead NHS R&D 
Office for review and approval. In the event that a serious breach of GCP is suspected, this will 
be reported to the Sponsor immediately using the form “Breach Report Form”. 
 
11.2 End of Study 
The end of study is defined as last termination date. The Sponsor, CI and/or the TSC have the 
right at any time to terminate the study for clinical or administrative reasons.  The end of the 
study will be reported to the Sponsor and REC within 90 days, or 15 days if the study is 
terminated prematurely. The CI will ensure that any appropriate follow up is arranged for all 
participants. A summary report of the study will be provided to the Sponsor and REC within 
1 year of the end of the study. 
 
12. Reporting, publications and notifications of results 
12.1 Authorship Policy 
Ownership of the data arising from this study resides with the study team and their respective 
employers. On completion of the study, the study data will be analysed and tabulated, and a 
clinical study report will be prepared. 
 
12.2 Publication 
The clinical study report will be used for publication and presentation at scientific meetings. 
Investigators have the right to publish orally or in writing the results of the study. Summaries of 
results will also be made available to Investigators for dissemination within their scientific and 
clinical areas (where appropriate and according to their discretion). 
 

Protocol V2 12/01/2017 
24 
 
13. Bibliography 
 
Anblagan D, Jones NW, Costigan C, Parker AJ, Allcock K, Aleong R, Coyne LH, Deshpande R, Raine-Fenning 
N, Bugg G et al. Maternal smoking during pregnancy and fetal organ growth: a magnetic resonance 
imaging study. PLoS One 2013:8:e67223. 
Anderson RA, McIlwain L, Coutts S, Kinnell HL, Fowler PA and Childs AJ. Activation of the aryl 
hydrocarbon receptor by a component of cigarette smoke reduces germ cell proliferation in the human 
fetal ovary. Mol Hum Reprod 2014:20:42-48. 
Arrebola JP, Molina-Molina JM, Fernandez MF, Saenz JM, Amaya E, Indiveri P, Hill EM, Scholze M, Orton F, 
Kortenkamp A et al. A novel biomarker for anti-androgenic activity in placenta reveals risks of 
urogenital malformations. Reproduction 2015:149:605-613. 
Brown ZA, Schalekamp-Timmermans S, Tiemeier HW, Hofman A, Jaddoe VW and Steegers EA. Fetal sex 
specific differences in human placentation: a prospective cohort study. Placenta 2014:35:359-364. 
Choi GY, Tosh DN, Garg A, Mansano R, Ross MG and Desai M. Gender-specific programmed hepatic lipid 
dysregulation in intrauterine growth-restricted offspring. Am J Obstet Gynecol 2007:196:477.e1-477.e7. 
Cupul-Uicab LA, Skjaerven R, Haug K, Melve KK, Engel SM and Longnecker MP. In Utero Exposure to 
Maternal Tobacco Smoke and Subsequent Obesity, Hypertension, and Gestational Diabetes Among 
Women in the MoBa Cohort. Environ Health Perspect 2012:120:355-360. 
Drake AJ, O'Shaughnessy PJ, Bhattacharya S, Monteiro A, Kerrigan D, Goetz S, Raab A, Rhind SM, Sinclair 
KD, Meharg AA, Fowler PA. In utero exposure to cigarette chemicals induces sex-specific disruption of 
one-carbon metabolism and DNA methylation in the human fetal liver. BMC Med 2015:13:18-014-0251-
x. 
Filis P, Nagrath N, Fraser M, Hay DC, Iredale JP, O'Shaughnessy P and Fowler PA. Maternal smoking 
dysregulates protein expression in second trimester human fetal livers in a sex-specific manner. J Clin 
Endocrinol Metab 2015:jc20143941. 
Fowler PA, Anderson RA, Saunders PT, Kinnell H, Mason JI, Evans DB, Bhattacharya S, Flannigan S, Franks 
S, Monteiro A et al. Development of Steroid Signaling Pathways during Primordial Follicle Formation in 
the Human Fetal Ovary. J Clin Endocrinol Metab 2011a:96:1754-1762. 
Fowler PA, Bhattacharya S, Flannigan S, Drake AJ and O'Shaughnessy PJ. Maternal Cigarette Smoking and 
Effects on Androgen Action in Male Offspring: Unexpected Effects on Second-Trimester Anogenital 
Distance. J Clin Endocrinol Metab 2011b:96:E1502-1506. 
Fowler PA, Bhattacharya S, Gromoll J, Monteiro A and O'Shaughnessy PJ. Maternal Smoking and 
Developmental Changes in Luteinizing Hormone (LH) and the LH Receptor in the Fetal Testis. J Clin 
Endocrinol Metab 2009:94:4688-4695. 
Fowler PA, Cassie S, Rhind SM, Brewer MJ, Collinson JM, Lea RG, Baker PJ, Bhattacharya S and 
O'Shaughnessy PJ. Maternal Smoking during Pregnancy Specifically Reduces Human Fetal Desert 
Hedgehog Gene Expression during Testis Development. J Clin Endocrinol Metab 2008:93:619-26. 
Fowler PA, Childs AJ, Courant F, MacKenzie A, Rhind SM, Antignac JP, Le Bizec B, Filis P, Evans F, 
Flannigan S et al. In utero exposure to cigarette smoke dysregulates human fetal ovarian developmental 
signalling. Hum Reprod 2014:29:1471-1489. 
Fowler PA, Flannigan S, Mathers A, Gillanders K, Lea RG, Wood MJ, Maheshwari A, Bhattacharya S, Collie-
Duguid ES, Baker PJ et al. Gene expression analysis of human fetal ovarian primordial follicle formation. 
J Clin Endocrinol Metab 2009:94:1427-1435. 
Giesbrecht GF, Campbell T, Letourneau N and APrON Study Team. Sexually dimorphic adaptations in 
basal maternal stress physiology during pregnancy and implications for fetal development. 
Psychoneuroendocrinology 2015:56:168-178. 
Hackshaw A, Rodeck C and Boniface S. Maternal smoking in pregnancy and birth defects: a systematic 
review based on 173 687 malformed cases and 11.7 million controls. Hum Reprod Update 2011:17:589-
604. 
Joya X, Friguls B, Ortigosa S, Papaseit E, Martinez SE, Manich A, Garcia-Algar O, Pacifici R, Vall O and 
Pichini S. Determination of maternal-fetal biomarkers of prenatal exposure to ethanol: A review. J 
Pharm Biomed Anal 2012:69:209-222. 
Kawashima A, Koide K, Ventura W, Hori K, Takenaka S, Maruyama D, Matsuoka R, Ichizuka K and 
Sekizawa A. Effects of maternal smoking on the placental expression of genes related to angiogenesis 
and apoptosis during the first trimester. PLoS One 2014:9:e106140. 
Kneeman JM, Misdraji J and Corey KE. Secondary causes of nonalcoholic fatty liver disease. Therap Adv 
Gastroenterol 2012:5:199-207. 

Protocol V2 12/01/2017 
25 
Latini G, De Mitri B, Del Vecchio A, Chitano G, De Felice C and Zetterstrom R. Foetal growth of kidneys, 
liver and spleen in intrauterine growth restriction: "programming" causing "metabolic syndrome" in 
adult age. Acta Paediatr 2004:93:1635-1639. 
Lui S, Jones RL, Robinson NJ, Greenwood SL, Aplin JD and Tower CL. Detrimental effects of ethanol and its 
metabolite acetaldehyde, on first trimester human placental cell turnover and function. PLoS One 
2014:9:e87328. 
Meeker JD and Benedict MD. Infertility, Pregnancy Loss and Adverse Birth Outcomes in Relation to 
Maternal Secondhand Tobacco Smoke Exposure. Curr Womens Health Rev 2013:9:41-49. 
Mehta C, Masson G, Iqbal Z, O'Mahony F and Khalid R. Prevalence of excessive alcohol consumption in 
pregnancy. Public Health 2009:123:630-631. 
Moore DG, Turner JD, Parrott AC, Goodwin JE, Fulton SE, Min MO, Fox HC, Braddick FM, Axelsson EL, 
Lynch S et al. During pregnancy, recreational drug-using women stop taking ecstasy (3,4-
methylenedioxy-N-methylamphetamine) and reduce alcohol consumption, but continue to smoke 
tobacco and cannabis: initial findings from the Development and Infancy Study. J Psychopharmacol 
2010:24:1403-1410. 
Morrison JL, Duffield JA, Muhlhausler BS, Gentili S and McMillen IC. Fetal growth restriction, catch-up 
growth and the early origins of insulin resistance and visceral obesity. Pediatr Nephrol 2010:25:669-
677. 
Muralimanoharan S, Guo C, Myatt L and Maloyan A. Sexual dimorphism in miR-210 expression and 
mitochondrial dysfunction in the placenta with maternal obesity. Int J Obes (Lond) 2015:39:1274-1281. 
Nyirenda MJ, Carter R, Tang JI, de Vries A, Schlumbohm C, Hillier SG, Streit F, Oellerich M, Armstrong VW, 
Fuchs E et al. Prenatal programming of metabolic syndrome in the common marmoset is associated 
with increased expression of 11beta-hydroxysteroid dehydrogenase type 1. Diabetes 2009:58:2873-
2879. 
O'Shaughnessy PJ, Baker PJ, Monteiro A, Cassie S, Bhattacharya S and Fowler PA. Developmental Changes 
in Human Fetal Testicular Cell Numbers and Messenger Ribonucleic Acid Levels during the Second 
Trimester. J Clin Endocrinol Metab 2007:92:4792-801. 
O'Shaughnessy PJ, Monteiro A, Bhattacharya S and Fowler PA. Maternal Smoking and Fetal Sex 
Significantly Affect Metabolic Enzyme Expression in the Human Fetal Liver. J Clin Endocrinol Metab 
2011:96:2851-2860. 
O'Shaughnessy PJ, Monteiro A, Bhattacharya S, Fraser MJ and Fowler PA. Steroidogenic enzyme 
expression in the human fetal liver and potential role in the endocrinology of pregnancy. Mol Hum 
Reprod 2013:19:177-187. 
O'Shaughnessy PJ, Monteiro A and Fowler PA. Identification of stable endogenous reference genes for 
real-time PCR in the human fetal gonad using an external standard technique. Mol Hum Reprod 
2011:17:620-625. 
Raisanen S, Kramer MR, Gissler M, Saari J, Hakulinen-Viitanen T and Heinonen S. Smoking during 
pregnancy was up to 70% more common in the most deprived municipalities - a multilevel analysis of 
all singleton births during 2005-2010 in Finland. Prev Med 2014:67:6-11. 
Reynolds JN, Weinberg J, Clarren S, Beaulieu C, Rasmussen C, Kobor M, Dube MP and Goldowitz D. Fetal 
alcohol spectrum disorders: gene-environment interactions, predictive biomarkers, and the relationship 
between structural alterations in the brain and functional outcomes. Semin Pediatr Neurol 2011:18:49-
55. 
Reynolds RM, Labad J, Buss C, Ghaemmaghami P and Raikkonen K. Transmitting biological effects of 
stress in utero: implications for mother and offspring. Psychoneuroendocrinology 2013:38:1843-1849. 
Shinjo A, Ventura W, Koide K, Hori K, Yotsumoto J, Matsuoka R, Ichizuka K and Sekizawa A. Maternal 
smoking and placental expression of a panel of genes related to angiogenesis and oxidative stress in 
early pregnancy. Fetal Diagn Ther 2014:35:289-295. 
Snijder CA, Brouwers MM, Jaddoe VW, Hofman A, Roeleveld N and Burdorf A. Occupational exposure to 
endocrine disruptors and time to pregnancy among couples in a large birth cohort study: the Generation 
R Study. Fertil Steril 2011:95:2067-2072. 
Snijder CA, Kortenkamp A, Steegers EA, Jaddoe VW, Hofman A, Hass U and Burdorf A. Intrauterine 
exposure to mild analgesics during pregnancy and the occurrence of cryptorchidism and hypospadia in 
the offspring: the Generation R Study. Hum Reprod 2012:27:1191-1201. 
Tappin DM, MacAskill S, Bauld L, Eadie D, Shipton D and Galbraith L. Smoking prevalence and smoking 
cessation services for pregnant women in Scotland. Subst Abuse Treat Prev Policy 2010:5:1. 
van den Driesche S, Macdonald J, Anderson RA, Johnston ZC, Chetty T, Smith LB, McKinnell C, Dean A, 
Homer NZ, Jorgensen A et al. Prolonged exposure to acetaminophen reduces testosterone production by 
the human fetal testis in a xenograft model. Sci Transl Med 2015:7:288ra80. 

Protocol V2 12/01/2017 
26 
Wang N, Tikellis G, Sun C, Pezic A, Wang L, Wells JC, Cochrane J, Ponsonby AL and Dwyer T. The effect of 
maternal prenatal smoking and alcohol consumption on the placenta-to-birth weight ratio. Placenta 
2014:35:437-441. 
Weightman AL, Morgan HE, Shepherd MA, Kitcher H, Roberts C and Dunstan FD. Social inequality and 
infant health in the UK: systematic review and meta-analyses. BMJ Open 2012:2:10.1136/bmjopen-
2012-000964. Print 2012.