Iron Deficiency Anaemia in Pregnancy: an Observational Study of Tolerability, Compliance With Oral Iron Therapy and Effects on Haematological/Biochemical Markers
Conditions: Anemia, Iron Deficiency
Sponsor: The Royal Wolverhampton Hospitals NHS Trust

 
 
 
TIAP study protocol version 1.2 04/05/2018  
IRAS number: 244013 
            
                                                              
                                             
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Iron deficiency anaemia in pregnancy: an 
observational study of tolerability, compliance with 
oral iron therapy and effects on 
haematological/biochemical markers 
 
 
 
TIAP 
Treatment of Iron deficiency Anaemia in Pregnancy 
study 
 
 
 
VERSION: 1.2 
DATE: 04/05/2018 
 
 
 
RESEARCH REFERENCE NUMBERS 
ISRCTN: TBC 
IRAS: 244013 
REC: 18/WM/0090 
SPONSOR: TBC 
 
 

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1. SIGNATURE PAGE 
The undersigned confirm that the following protocol has been agreed and accepted and that the 
Chief Investigator agrees to conduct the non-interventional study in compliance with the approved 
protocol and will adhere to the principles outlined in GCP guidelines, the Sponsor’s SOPs, and other 
regulatory requirements as amended. 
 
EU Clinical Trials Directive (2001/20/EC), specifies a non-interventional study is one in which:  
...the medicinal product(s) is (are) prescribed in the usual manner, in accordance with the terms of the 
marketing authorization. The assignment of the patient to a particular therapeutic strategy is not 
decided in advance by a trial protocol, but falls within current practice, and the prescription of the 
medicine is clearly separated from the decision to include the patient in the study. No additional 
diagnostic or monitoring procedures shall be applied to the patients, and epidemiological methods 
shall be used for the analysis of collected data. 
 
I agree to ensure that the confidential information contained in this document will not be used for 
any other purpose other than the evaluation or conduct of the clinical investigation without the 
prior written consent of the Sponsor 
I also confirm that I will make the findings of the study publically available through publication or 
other dissemination tools without any unnecessary delay and that an honest accurate and 
transparent account of the study will be given; and that any discrepancies and serious breaches of 
GCP from the study as planned in this protocol will be explained. 
 
For and on behalf of the Study Sponsor: 
Signature:  
............................................................................................... 
 
Date: 
....../....../... 
 
Name (please print): 
............................................................................................... 
 
 
 
Position: ............................................................................................... 
 
 
 
 
 
Chief Investigator: 
Signature: ............................................................................................... 
 
Date: 
....../....../... 
 
Name: (please print): 
............................................................................................... 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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2. 
 KEY STUDY CONTACTS 
Insert full details of the key study contacts including the following 
Chief Investigator 
Mr D Churchill MD FRCOG, Consultant Obstetrician. The Royal 
Wolverhampton NHS Trust, New Cross Hospital, Wolverhampton, WV10 
0QP. Tel:01902 695153, Fax 01902 695656 
Email: david.churchill1@nhs.net 
Study Coordinator 
Clinical Research Fellows, The Royal Wolverhampton NHS Trust, New 
Cross Hospital, Wolverhampton, WV10 0QP 
Sponsor 
Sarah Glover, The Royal Wolverhampton NHS Trust, Research & 
Development, The Chestnuts (Building 9), New Cross Hospital, 
Wolverhampton, WV10 0QP. Tel: on 01902 695065, Email: 
sarah.glover7@nhs.net     
Funder(s) 
Rotha Abraham Trust, Wolverhampton, Dr E Swarbrick FRCP, 
Wolverhampton Medical Institute, New Cross Hospital, Wolverhampton, 
WV10 0QP  
Key Protocol Contributors 
Mr D Churchill 
Dr Simon Stanworth  
Prof Marian Knight 
Dr H Ali  
Dr M Moussa 
Dr Samaher Sweity   
Dr F Lorencatto 
 
Other collaborators 
 
 
 
Dr H Ali, MB, MRCOG. 
 
Dr M Moussa, MB, 
MRCOG. 
Clinical Research Fellow, The Royal Wolverhampton NHS Trust, New Cross 
Hospital, Wolverhampton, WV10 0QP. Tel:01902 695153, Fax 01902 695656 
 
 
Clinical Research Fellow, The Royal Wolverhampton NHS Trust, New Cross 
Hospital, Wolverhampton, WV10 0QP. Tel:01902 695153, Fax 01902 695656 
MW K Cheshire 
Research Midwife, The Royal Wolverhampton NHS Trust, New Cross Hospital, 
Wolverhampton, WV10 0QP. Tel:01902 698395 
Dr S Stanworth, FRCP 
FRCPath DPhil  
Dr Samaher Sweity  
Consultant Haematologist, John Radcliffe Hospital, Oxford University Hospitals 
Foundation Trust (and Oxford of University), Oxford, OX3 9BQ. 
 
Clinical Trial Advisor, NHS Blood and Transplant, John Radcliffe Hospital, 
Oxford, OX3 9BQ. 
Prof M Knight, DPhil. 
FRCOG. 
Consultant and Professor of Public Health,  National Perinatal Epidemiology 
Unit (NPEU),  Nuffield Department of Population Health, University of Oxford, 
Old Road Campus, Headington,  Oxford,  OX3 7LF 
Dr N Roy, FRCP 
FRCPath DPhil 
Consultant Haematologist. Oxford  University Hospitals NHS Foundation Trust 
and University of Oxford  
Prof M Brookes 
Consultant Gastroenterologist, The Royal Wolverhampton NHS Trust, & 
University of Wolverhampton 
Dr  H Omar  
 
University of Wolverhampton 
Prof J Francis  
Professor of Health Services Research, City University London 
Dr F Lorencatto 
Research Fellow, Centre for Health Services Research, University College 
London 

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3. 
LIST OF CONTENTS 
1. 
SIGNATURE PAGE ....................................................................................................................................................................... 2 
2. 
KEY STUDY CONTACTS ............................................................................................................................................................ 3 
3. 
LIST of CONTENTS ..................................................................................................................................................................... 4 
4. 
LIST OF ABBREVIATIONS ........................................................................................................................................................ 5 
5. 
STUDY SUMMARY ....................................................................................................................................................................... 6 
6. 
STUDY FLOWCHART ................................................................................................................................................................. 7 
7. 
THE NEED FOR A STUDY: BACKGROUND AND RATIONALE ................................................................................... 8 
 
Anaemia ..................................................................................................................................................................................... 8 
 
Treatment.................................................................................................................................................................................. 9 
 
Diagnostic tests to identify anaemia .............................................................................................................................. 9 
8. 
OVERARCHING PURPOSE AND OBJECTIVES ............................................................................................................... 11 
9. 
Aims & Objectives .................................................................................................................................................................... 12 
10. 
STUDY DESIGN AND SETTING ....................................................................................................................................... 12 
11. 
PARTICIPANT ELIGIBILITY CRITERIA ....................................................................................................................... 12 
 
Participant inclusion criteria: ........................................................................................................................................ 13 
 
Participant exclusion criteria:........................................................................................................................................ 13 
 
Study restrictions ................................................................................................................................................................ 13 
12. 
STUDY PROCEDURES, AND ASSESSMENT SCHEDULES ..................................................................................... 13 
 
Identification and recruitment ...................................................................................................................................... 13 
 
Consenting ............................................................................................................................................................................. 14 
 
Data collection: Baseline clinical data ........................................................................................................................ 15 
 
Blood Sampling .................................................................................................................................................................... 15 
 
Symptom and Tolerability Assessment ..................................................................................................................... 16 
 
Participant follow-up and assessments ..................................................................................................................... 17 
 
Follow up and pregnancy outcome data ................................................................................................................... 18 
 
End of study ........................................................................................................................................................................... 19 
13. 
STUDY TREATMENT .......................................................................................................................................................... 19 
14. 
ADVERSE EVENT REPORTING ...................................................................................................................................... 20 
 
Definitions of Adverse Events ........................................................................................................................................ 20 
 
Expected Drug-Related Adverse Events / Reactions ........................................................................................... 20 
 
Adverse Events that Require Expedited Reporting .............................................................................................. 21 
 
Investigators Assessment of SAE .................................................................................................................................. 21 
 
Notification and Reporting of Serious Adverse Events ....................................................................................... 22 
 
Overview of the Safety Reporting Process/Pharmacoviligance responsibilities ..................................... 22 
 
Statutory Reporting ........................................................................................................................................................... 22 
15. 
Ethical and Regulatory Issues ........................................................................................................................................ 22 
 
Compliance ............................................................................................................................................................................ 22 
 
Ethical Compliance ............................................................................................................................................................. 22 
 
Data Protection and Patient Confidentiality ............................................................................................................ 23 
 
Indemnity ............................................................................................................................................................................... 23 
 
Amendments ......................................................................................................................................................................... 24 
 
Access to the Final Study Dataset ................................................................................................................................. 24 
 
Peer Review ........................................................................................................................................................................... 24 
 
Patient & Public Involvement ........................................................................................................................................ 24 
16. 
STATISTICS AND DATA ANALYSIS .............................................................................................................................. 25 
 
Primary outcome measure .............................................................................................................................................. 25 
 
Other Outcomes ................................................................................................................................................................... 25 
 
Statistical Analysis and Overview Analysis Plan .................................................................................................... 25 
17. 
DATA MANAGEMENT ....................................................................................................................................................... 26 
 
Participant Confidentiality and Data Access ............................................................................................................ 26 
 
Archiving ................................................................................................................................................................................ 27 
18. 
DATA MONITORING AND INSPECTION ..................................................................................................................... 27 
19. 
PUBLICATION POLICY ...................................................................................................................................................... 28 
20. 
Protocol Amendments ...................................................................................................................................................... 28 
21. 
REFERENCES: ....................................................................................................................................................................... 29 

 
 
 
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4. 
LIST OF ABBREVIATIONS 
aOR 
adjusted odds ratio 
BCSH 
 British Committee for standards in Haematology   
BSH 
British Society Haematology  
CI 
Confidence Interval 
CI 
Chief investigator  
CRP 
C-reactive protein 
ELISA 
enzyme linked immunosorbant assay  
FBC 
Full blood count  
GCP 
Good Clinical Practice  
GDM 
Gestational diabetes 
GP 
General Practitioner  
Hb 
Haemoglobin  
IFN-g 
Interferon-gamma 
IL 
Interleukin 
IQR 
Interquartile range  
ISF 
Investigator Site File 
IV 
Intravenous  
MA 
Marketing authorisation  
MCH 
Mean cell haemoglobin  
MCHC 
Mean cell haemoglobin concentration  
MCV 
Mean corpuscular volume  
NHS 
National Health Service  
PI 
Principal Investigator  
PIL 
Participant Information Leaflet  
REC 
Research Ethics Committee  
RWT 
Royal Wolverhampton NHS Trust   
TDF 
Theoretical Domains Framework  
TGF-β 
Transforming growth factor-beta 
SMG 
Study Management Group  
TNF 
tumor necrosis factor  
WCC 
 White cell count  
WHO 
World Health Organisation  
WHO-5 
The World Health Organisation- Five Well-Being Index 
WIMM 
Weatherall Institute of Molecular Medicine   
WiP 
Well-being in Pregnancy  
 
 
 
 
 
 
 
 

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5. 
STUDY SUMMARY 
 
Study title 
Iron replacement in pregnant women: an observational study on 
tolerability, compliance with oral iron and effects on 
haematological/biochemical markers of anaemia 
Short title 
TIAP - Treatment of Iron deficiency Anaemia in Pregnancy study 
Clinical Phase  
Observational and mechanistic 
Study Design 
Prospective cohort study 
Study Participants 
Anaemic (iron-deficient) pregnant women receiving oral iron 
replacement therapy 
Planned Sample Size 
120 
Treatment duration 
Initial treatment duration is up to 4 weeks for the assessment period of 
the study. Depending on the response, the iron treatment may continue 
throughout pregnancy or until 3 months after delivery if the anaemia is 
diagnosed in the third trimester, as per local guidelines and under the 
direction of the supervising obstetrician. 
Follow up duration for data 
collection and sampling 
4 weeks for the treatment period and 6 weeks post-delivery for 
obstetric outcome data 
Planned Study Period 
2 years- recruitment period will be 18 months with a 6 month follow up 
period 
Objectives 
1. To describe the symptoms at presentation with anaemia during 
pregnancy 
2. To assess the correlations between symptoms and degree of 
anaemia at presentation 
3. To quantify the clinical symptom response, side effects, tolerability, 
compliance and acceptability with oral iron treatment (using 
recommended national dosing schedules) 
4. To document the haematological changes in haemoglobin, ferritin, 
transferrin saturation, serum hepcidin, other research biomarkers 
of iron metabolism and pro-inflammatory cytokines during a 
treatment course of oral iron 
5. To investigate whether biomarkers, such as serum hepcidin, can 
predict the response to oral iron 
Primary outcome 
 
Secondary Outcomes 
An increase in haemoglobin of at least 10g/L 
 
• 
Tolerability and concordance with treatment 
• 
Quality of life assessments in response to treatment 
Treatment  
Oral elemental iron as per standard care (ferrous sulphate) 
Formulation, Dose, Route of 
Administration 
Ferrous sulphate 100-200 mg up to three-times daily as per standard 
guidelines  
 
 

 
 
 
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6. 
STUDY FLOWCHART 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
  
 

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7. 
THE NEED FOR A STUDY: BACKGROUND AND RATIONALE 
 Anaemia 
The burden of anaemia remains unacceptably high during pregnancy. Over a third of women are 
anaemic by their third trimester of pregnancy. The most common cause is iron deficiency (Friel, 
2014). One key factor is rising iron requirements throughout pregnancy. By the third trimester, body 
iron demand outstrips the amount that can be provided by diet alone. Therefore, iron stores must be 
sufficient at the start of pregnancy (≥300 mg) to make up for that shortfall (Bothwell, 2000). In 
addition, the start of pregnancy is often characterised by sub-optimal iron stores (Bothwell, 2000). 
There are risks associated with anaemia for the mother and infant.  Anaemia in the first and second 
trimester has been significantly correlated with low birth weight and pre-term birth (Haider, 2013), 
and is associated with impaired neurological development of the baby (Congdon, 2012). It also 
increases the risk of intrauterine fetal death (Lone, 2004), and the likelihood of the mother requiring 
blood transfusions during or after delivery (Patterson, 2014).  
 
Our own work is consistent with these findings (Nair et al, 2017). A retrospective cohort of 
anonymised data from 14,001 women was analysed. All were singleton pregnancies ≥24 weeks’ 
gestation giving birth between 2013-15 (7,175 from Royal Wolverhampton National Health Service 
(NHS) Trust, 2013-14 and 6,826 from Guy’s and St Thomas’ NHS Foundation Trust, 2014-15). 
Multivariable logistic regression was used to quantify associations between haemoglobin and 
categories of anaemia (no anaemia ≥110g/L; mild anaemia 100-109g/L; moderate-severe anaemia 
<100g/L) at booking and 28 weeks with stillbirth and perinatal death, adjusting for 11 confounders 
(age, ethnicity, BMI, parity, smoking, gestational diabetes mellitus, antepartum haemorrhage, 
hypertensive disorders, and pre-existing diabetes mellitus, haemoglobinopathies, and other medical 
comorbidities). 
 
Apart from a higher proportion of women from ethnic minorities reflecting local demographics the 
study population compared well to the general population in England. The prevalence of all grades of 
anaemia at the first visit was 7% and the prevalence of moderate / severe anaemia (i.e. <100g/L) 
increased from 2% to 7%, between the first and 28 week visits. Additionally, 26% of women with 
normal haemoglobin at the first visit became anaemic by 28 weeks. In total 46% of women had 
anaemia at some point during their pregnancy. The risk of stillbirth and perinatal death decreased 
linearly per unit increase in haemoglobin concentration at first visit (adjusted odds ratio (aOR) 
stillbirth=0.70, 95% confidence interval (CI) 0.58–0.85; aOR perinatal death=0.71, 95%CI 0.60–0.84). 
Thus, in terms of the population, for every 10g/l increase in haemoglobin there was a 30% reduction 
in the risk of stillbirth. Compared with women with normal haemoglobin concentrations, the risk of 
both stillbirth and perinatal death was four-fold [aOR 4.34 (2.11-8.92)] and three-fold higher [aOR 

 
 
 
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2.91 (1.46 - 5.81)] in women with moderate-severe maternal anaemia at first and at 28 weeks visits, 
respectively. 
 Treatment  
Current approaches to management of anaemia are described in national guidelines, which focus on 
case finding (detection/treatment) of low haemoglobin concentrations (British Committee for 
Standards in Haematology, 2012), and are underpinned by low quality evidence. There is evidence 
that current strategies for anaemia management are not effective. For instance, in a national 
descriptive study of 638 women with anaemia in early pregnancy, it was found that 281 (44%) of 
these women had anaemia at or after 28 weeks (Barroso et al, 2011).  Furthermore; in a recent large 
local audit over two years (2015-16, in Wolverhampton), 343 women had anaemia before 16 weeks 
and of these, 64% still had anaemia at 28 weeks. Current testing to establish a diagnosis of iron-
deficiency involves additional blood tests (ferritin) and it may be several weeks before women are 
counselled and started on oral iron treatment.  
 
In addition, a significant proportion of women who receive oral iron therapy may go on to receive 
intravenous (IV) iron infusions which is costly and requires administration in an observed setting. 
The main indications for receiving iron infusions are when: oral iron therapy fails to improve 
haemoglobin levels after two weeks; the oral iron is not well tolerated (gastro-intestinal side effects); 
the patient is still defined as anaemic after four weeks of oral iron therapy; or when the patient is 
diagnosed as anaemic within four weeks of the estimated delivery date. In addition, women closer to 
term and delivery would be expected to benefit more from IV iron infusion.  
 
Patients with severe anaemia (such as a haemoglobin level < 70g/L) need to be immediately referred 
to secondary care (Pavord, 2012) for assessment as parenteral iron may be required. Blood 
transfusion is reserved for extreme circumstances such as imminent cardiac compromise, severe 
anaemic symptoms which require urgent treatment, or if there is a high risk of further haemorrhage 
(Pavord, 2012). 
 
 Diagnostic tests to identify anaemia 
Anaemia has traditionally been defined by World Health Organisation (WHO) thresholds, which are 
gestation dependent, to reflect the greater proportionate expansion of the plasma volume during 
pregnancy compared to the increase in red cell mass. Further tests for the investigation of iron 
deficiency include: blood film; red cell indices such as mean cell volume, mean cell haemoglobin, and 
mean cell haemoglobin concentration; alongside iron studies such as serum ferritin, serum iron and 
total iron binding capacity/transferrin saturation.  

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All have their limitations and none has proven capable of predicting the response to oral iron therapy 
during pregnancy. These parameters have been analysed for their predictive value in anaemic, non-
pregnant patients. An additional parameter, the iron-regulatory hormone hepcidin, which is not 
routinely tested, has also been included in these analyses. There is now some evidence to suggest that 
in haemodialysis patients, mean cell volume, serum ferritin and hepcidin levels can help to predict 
oral iron therapy responses (Takasawa, 2015). Furthermore, in a wide range of patients diagnosed 
with iron deficiency anaemia, serum hepcidin performed exceptionally well as a predictor of 
response to oral iron therapy, particularly when compared to transferrin saturation and serum 
ferritin levels (Bregman, 2013).  
 
Unfortunately, of the three more promising candidates: mean cell volume, serum ferritin and 
hepcidin, there are still drawbacks to their use in pregnant patients. For example, mean cell volume 
increases during pregnancy and can therefore offset the reduction in volume associated with iron 
deficiency anaemia (Pavord, 2012). These influences may impair its predictive value for response to 
oral iron therapy.  
 
Furthermore, whilst serum ferritin can accurately reflect iron stores in a healthy patient, this 
relationship is lost in acute and chronic infection and inflammation, as serum ferritin is an acute 
phase reactant. Its measurements are further confounded by day-to-day variation, which can be as 
great as 25% (Beard, 1994), and by factors specific to pregnancy such as haemodilution. Serum 
ferritin variation at each trimester of pregnancy also suggests that serum ferritin can dissociate from 
its relationship with iron stores, with one study reporting a rise in serum ferritin levels in the third 
trimester of pregnancy (Asif, 2007). In short, serum ferritin may be a poor informer of body iron 
status in pregnancy.  
 
As a result, interest has been directed towards hepcidin as a possible predictor of response to oral 
iron therapy in pregnant patients. This protein has emerged as a major regulator of iron status. Its 
expression is increased in states of inflammation and iron overload and repressed in iron deficiency, 
hypoxia, and erythroid expansion. Hepcidin binds to ferroportin causing its internalisation and 
degradation. Ferroportin is the sole iron exporter and is expressed in macrophages and duodenal 
cells, allowing iron recycling and absorption. Hepcidin can therefore inhibit iron release from 
macrophages leading to an ‘iron-restricted’ erythropoiesis or from duodenal cells, leading to dietary 
iron deficiency.  
 
Stimulation of immune responses in anemia of inflammation is often associated with upregulation of 
ferritin resulting in intracellular excessive storage of iron. In addition, Interleukin (IL)-6 production 
during these inflammatory conditions stimulates the release of hepcidin which consequently inhibits 
the iron absorption by the enterocytes and downregulates ferroportin therefore increases 

 
 
 
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intracellular storage of iron and leading to a decrease in iron bioavailability. Other cytokines such as 
IL-22 may have the same effect independent of IL-6. Furthermore, inflammatory cytokines down-
regulate transferrin receptors on the cell surface. Reduction in bioavailable iron is an important step 
in limiting iron supply to pathogens but can contribute to the anaemia of inflammation and the 
upregulation of proinflammatory cytokines. These cytokines may also have a role to play in 
pregnancy disorders such as stillbirth. 
 
At present there is a paucity of research into the relationship between clinical symptomatology, 
hepcidin, iron status and pregnancy. In a recent review, Koenig et al (2014) suggested that there is 
growing evidence that maternal hepcidin correlates with maternal iron status during pregnancy. 
However, the data is far from clear-cut. In one recent study focusing on healthy pregnant women, 
there appeared to be a clear reduction in hepcidin concentration during pregnancy, in line with falling 
iron stores, and recovery of hepcidin levels following delivery (van Santen, 2013). However, other 
authors have reported that there is no association between serum hepcidin, body iron status and 
inflammation in similar cohorts (Simavli, 2014). Of further interest is the possible correlation 
between hepcidin and inflammatory conditions such as pre-eclampsia and obesity. In a small study, 
it was found that pre-eclamptic patients had higher plasma hepcidin and ferritin concentrations, and 
lower transferrin, total iron binding capacity, and mean cell haemoglobin concentrations compared 
to healthy pregnant women (Toldi, 2010). Possible associations between maternal obesity, raised 
hepcidin and C-reactive protein (CRP), and reduced fetal iron status have been reported in other 
studies (Dao, 2013), but larger studies are required.   
 
Outside the small field of obstetric research, hepcidin has been heralded as a biomarker for 
distinguishing oral iron therapy “responders” from “non-responders”. It was found that, within the 
subgroup of anaemic African children, hepcidin was superior to serum ferritin in terms of assessing 
anaemia, and for distinguishing between anaemia due to iron-deficiency from anaemia due to 
inflammation, particularly if the two coexist in the same patient. (Pasricha, 2014). In light of 
accumulating and inconclusive evidence for hepcidin assessment during pregnancy, further studies 
are required. 
 
8. 
OVERARCHING PURPOSE AND OBJECTIVES 
To better define the natural history and understand how to use oral iron therapy for iron deficiency 
anaemia in pregnant women. Specifically, the study will document the impact of treatment on 
symptomatology, the induction of side effects, and the utility of several haematological indices; 
haemoglobin, ferritin, transferrin saturation, reticulocyte haemoglobin concentration, alongside 

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changes in new markers of iron homeostasis (hepcidin), which may better predict the success of 
treatment with iron. 
 
9. 
AIMS & OBJECTIVES 
1. To describe the symptoms at presentation with anaemia during pregnancy. 
2. To assess the correlations between symptoms and degree of anaemia at presentation. 
3. To quantify the clinical symptom response, side effects, tolerability, and compliance with oral 
iron treatment (using recommended national dosing schedules). 
4. To document the haematological changes in haemoglobin, ferritin, CRP, transferrin 
saturation, hepcidin and pro-inflammatory cytokines during a treatment course of oral iron. 
5. To investigate whether biomarkers of iron metabolism, such as serum hepcidin, can predict 
the response to oral iron. 
 
10. STUDY DESIGN AND SETTING 
This protocol describes a prospective cohort study where pregnant women identified as anaemic are 
offered treatment and follow up through a dedicated anaemia clinic. Mothers will be invited to 
participate in this study aiming to assess response and tolerability to doses of oral iron, using a 
treatment schedule as described in national guidelines. Additional blood samples will be taken for 
subsequent detailed analysis of pathways of iron metabolism. 
 
Standard practice is ‘reactive’ to anaemia occurring in pregnancy, and will be followed for the 
purpose of this study. All women are offered a full blood count (FBC) to screen for iron deficient 
anaemia at booking (usually 8-12 weeks’ gestation) and 28 weeks’ gestation. Maternity units have a 
system in place to ensure that all routine blood results, including the full blood counts, are reviewed 
promptly, and acted upon. Women identified with iron deficiency anaemia will be offered oral 
elemental iron or IV iron supplementation (and preferably within 28 days). Women will receive 
written information on the correct administration of iron and how to maximise absorption. A full 
blood count will be repeated 2 - 4 weeks following the start of therapeutic iron treatment. 
 
This is a single center study at The Royal Wolverhampton NHS Trust (RWT) supported by the 
laboratory at Weatherall Institute of Molecular Medicine (WIMM), University of Oxford and the 
University of Wolverhampton. The clinical assessment of treatment and collection of samples will be 
carried out at RWT and the hepcidin/specialist iron analyses will be performed in WIMM and the 
University of Wolverhampton. 
 
11. PARTICIPANT ELIGIBILITY CRITERIA 
Participants will be considered eligible for enrolment in this study if they fulfil all the inclusion 
criteria and none of the exclusion criteria detailed below.  

 
 
 
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 Participant inclusion criteria: 
• 
Pregnant women (any stage during pregnancy up to 36 weeks) and women in the puerperium 
(within 6 weeks post-delivery) with anaemia as defined by WHO criteria and described in 
British Society Haematology (BSH)/ British Committee for standards in Haematology (BCSH) 
guidelines. 
o First trimester < 110g/l 
o Second and third trimester < 105g/l 
o Puerperium < 100g/l 
• 
Age: 18-45 years 
• 
Agreement to participate in the study with consenting 
 
 Participant exclusion criteria: 
• 
Anaemic women presenting at or after 36 weeks as there may be insufficient time to delivery 
to assess responses to oral iron) 
• 
Anaemic women affected by a (major) haemoglobinopathy e.g.  B thalassaemia major sickle 
cell disease 
• 
Women with overt clinical signs of sepsis 
• 
Allergies to iron 
• 
Hyperemesis Gravidarum / persistent vomiting 
• 
Women with inflammatory conditions such as Crohns, ulcerative colitis, Systemic lupus 
erythematosus, Rheumatoid arthritis. 
• 
Women with chronic renal failure 
 Study restrictions 
Women can be enrolled in other studies that do not impact on the aims and methods of this study and 
vice versa.  All potential participants will be asked and their care records scrutinised to discover if 
they are participating in any other research projects or programmes. If yes, they will then be assessed 
whether it would be appropriate to include them in this study or not. 
 
 
12. STUDY PROCEDURES, AND ASSESSMENT SCHEDULES 
 Identification and recruitment 
              Eligible women with iron deficiency anaemia will be identified in 3 ways:  

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• 
Women diagnosed de novo in the hospital antenatal clinics and antenatal maternity ward 
with iron deficiency anaemia who have yet to receive treatment.  
• 
Women on the postnatal ward who are found to be anaemic post-delivery. 
• 
From a daily download of blood results from samples taken either at the booking or the 28 
week visit to the antenatal clinics either in the hospital or community setting. 
 
All eligible women will be approached by the research staff (who are also the clinical care team), 
usually the clinical research fellows or research midwives, and given information (verbal & 
written) about the project.  Consent may then be obtained after allowing sufficient time for the 
woman to consider the information and have all her questions answered to her satisfaction. This 
will be depending on each individual’s needs and assessed by health professionals obtaining 
consent, which could vary between a few hours or a few days. In addition, eligible participants 
may be invited to take part in the study via mail by directly mailing them the study cover letter 
and Participant Information Leaflet (PIL). Participants will be followed up by phone and if 
interested patients they can either be consented in their clinic visit.  All participants will be 
invited to attend a dedicated anaemia clinic for the initial visit. 
We aim to recruit women into the study spread evenly throughout three time periods of 
pregnancy/post-partum:  
1. 
Early pregnancy up to 24 weeks gestation  
2. 
Mid – late pregnancy from 24+1 to delivery 
3. 
Postnatally, up to 6 weeks after birth of the baby. 
 Consenting 
The Chief investigator (CI), who is also the Principal Investigator (PI) retains overall responsibility 
for the conduct of research at the site, and this includes the taking of informed consent of participants 
at their site. The PI can delegate this process to medically qualified and midwife delegates. The PI 
must ensure that any person with delegated responsibility to participate in the informed consent 
process is duly authorised, trained and competent to participate according to the ethically approved 
protocol, UK Policy Framework for Health and Social Care Research, principles of International 
Conference on Harmonisation Good Clinical Practice (ICH GCP) and Declaration of Helsinki.  
 
The right of a participant to refuse participation without giving reasons must be respected. The 
participant must remain free to withdraw at any time from the study without giving reasons and 
without prejudicing her further treatment and must be provided with a contact point for the local 
study team (i.e. PI and research fellows/midwives) from whom participants may obtain further 
information about the study. Consent will be established for the use of samples and data collected up 
to the point of withdrawal. 
 

 
 
 
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Informed consent must be obtained prior to the participant undergoing procedures that are 
specifically for the purposes of the study (as described below), and are out-with standard routine 
care at the participating site.  Participants who agreed to take part will sign a Consent Form, which 
will also be signed by the PI or an appropriately delegated professional. The informed consent 
process will be documented in patients’ medical records. The original Consent Form will be stored in 
the Investigator Site File (ISF), one copy will be filed in the participant’s medical record and a second 
copy will be given to participant.  
 Data collection: Baseline clinical data  
Baseline clinical characteristics will be collected from medical notes and participant interviews. 
These include as routine: 
• 
age of the participant 
• 
parity and timing of previous pregnancies  
• 
gestation or post-partum time 
• 
current use of vitamin supplements, make and frequency of administration 
• 
If taking vitamin supplements details on who or what prompted them to take supplements. 
• 
previous use of oral iron supplements 
• 
current use of oral iron supplements 
• 
previous experience of intravenous iron infusions 
• 
number of infusions received if applicable 
• 
number of infusions at the time of blood sampling if applicable 
• 
history of recent red cell units transfused  
• 
significant co-morbidities 
 
Follow up Data Collection 
• 
Routine clinical data will be collected on each participant on the pregnancy outcome including 
the condition of the neonate. See section 7. 
• 
Blood sampling data and data collected through the questionnaires and diary will be 
prospectively collected as the study progresses. 
 
 Blood Sampling 
Blood samples to be collected at baseline are:  
• 
Ferritin, transferrin saturation, reticulocyte haemoglobin concentration, CRP.   

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• 
Full blood count indices - Haemoglobin (Hb), mean corpuscular volume (MCV), mean cell    
haemoglobin (MCH), mean cell haemoglobin concentration (MCHC), white cell count (WCC), 
platelet count  
• 
Renal function – sodium, potassium, urea and creatinine 
• 
Liver function tests – Bilirubin, Albumin 
• 
Total B12 
• 
Hepcidin and biomarkers of iron metabolism (research assay, not part of standard care) 
• 
Cytokine profile (research assay, not part of standard care) 
 
All samples of blood that are not part of standard care will be spun down into 2 aliquots within an 
hour of venesection and the plasma stored at -80 degrees Celsius prior to transporting to the relevant 
laboratory. Apart from the Hepcidin and Cytokine profile the other blood tests are part of routine 
clinical practice. The Hepcidin level and Cytokine profile will be undertaken at the University of 
Wolverhampton and University of Oxford (see below) and are exploratory tests (not used for 
diagnostic or monitoring purposes). Hepcidin levels will be determined before and after iron 
treatment using enzyme linked immunosorbant assay (ELISA) technique. The pro-inflammatory 
cytokines including (IL1, IL-2, IL-6, IL-7, IL-12p70, IL-17, IL-22, IL-12p40, tumour necrosis factor 
(TNF), Interferon-gamma (IFN-)) and the anti-inflammatory cytokines including (IL-4, IL-5, IL-10 
and Transforming growth factor-beta (TGF-β)) will be assessed in the same patients using cytokine 
multiplex bead assays.  
 
Samples of sera will be stored in a -80 degree freezer at the Royal Wolverhampton Hospitals NHS 
Trust. At the end of the study these samples will be transported and analyzed at the Weatherall 
Institute in Oxford, then returned to Royal Wolverhampton Hospitals NHS Trust. At the end of the 
ethically approved timeline of the study all samples will then be destroyed in accordance with the 
Human Tissue Authority’s Code of Practice. 
 Symptom and Tolerability Assessment 
At each visit, participants will be asked to complete Quality of Life questionnaires (The World Health 
Organisation- Five Well-Being Index (WHO-5) (Beck, 2004) and the Well-being in Pregnancy (WiP) 
questionnaire (Alderdice et al. 2017). The frequency and severity of symptoms associated with 
anaemia in pregnancy and the side effects experienced by the women will be assessed using a pre-
piloted and adapted version of a published pregnancy symptomatology tool (Foxcroft 2013). The 
‘Anaemia in Pregnancy Assessment Questionnaire’ has been adapted to investigate the barriers and 
enablers to taking oral iron therapy using the Theoretical Domains Framework (TDF) (Michie et al. 
2005).  
 
At the follow up visits the frequency of side effects, if any, induced by the iron treatment will be 
assessed by examining the pre-piloted tolerability and symptom diary adapted from Peirera (2014). 

 
 
 
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The diary will record the time at which participant takes the iron medication, the symptoms of 
anaemia participant experiences at the time and the side effects participant had suffered since the 
last tablet was taken. Participants will be asked to keep this diary for the period from initiation of 
treatment to the follow up appointment 2 to 4 weeks later. The completed diary will be returned to 
confirm compliance with treatment, symptoms of anaemia, and side effects experienced from the 
treatment itself. The data from the diary will be transcribed ready for analysis.  
 
 Participant follow-up and assessments  
The following sections consider those aspects of care that are standard care and research for this 
study.  
Groups 1-2 Women recruited in the antenatal period. 
Visit 1 (T0) 
• 
Baseline clinical data collection (routine care) 
• 
Blood sampling for routine clinical assessment and research sampling 
• 
Quality of life questionnaires (research) 
• 
Explanation of the tolerability and symptom diary and completion of page 1 as a baseline pre-
treatment assessment (research) 
• 
Prescription given for oral iron (standard care). 
 
At this (and subsequent) visits, the women will have a routine full antenatal check of both their 
health and that of their fetus. After discussion with the mother, these clinic visits may replace any 
scheduled visits to either the consultant or community antenatal clinic so that any inconvenience 
is kept to a minimum. The research clinic will be overseen by a consultant obstetrician with a 
special interest in maternal and fetal medicine and the management of the pregnancy will be 
based upon the clinical need of the woman.  
 
Visit 2 (T0 + 2-4 weeks- routine visit) 
After the initial visit the participant will return for a follow up visit or review appointment between 
2 to 4 weeks after commencing oral iron.  
• 
Clinical data recorded as per normal antenatal care 
• 
Completion of the Quality of life (QOL) questionnaires (research). 
• 
Retrieve tolerability and symptom diary (research) 
• 
Repeat Blood Sampling as above for visit 1 including research samples 
• 
In addition, they will be asked to complete the Anaemia in Pregnancy Assessment 
Questionnaire that further examines the symptoms and side effects they experienced over the 

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initial treatment period and asks about the barriers and enablers to taking the iron treatment 
using the Theoretical Domains Framework (Michie et al. 2005) (research).  
• 
At this visit women will be assessed for response as detailed next (as per standard care): 
 
 
Responders 
For women who respond to treatment, as per routine practice, they will be asked to continue with 
the iron therapy at the dose at which they are tolerating side effects for a further 3 months or until 
delivery if this is sooner as per standard care. They will be reviewed again at the end of this period. 
(During the 3 months’ period the diary will not be required but the symptomatology and side effects 
profile will be formally assessed at a final visit. Also QOL questionnaires and research blood samples 
will be required at this visit (research)).  
 
Non-Responders (women who do not have the expected rise in haemoglobin) 
The reasons for non-responding will be explored with these women. If the non-response is due to 
side effects a modified regimen of twice daily administration will be tried. They will be asked to 
continue with modified treatment (as per standard care) and the diary (research) and then given an 
appointment to be reviewed in the clinic in a further 2 weeks (Visit 3) when the same process will be 
followed as Visit 2. As per routine practice, women who continue to be non-responders, when 
assessed at visit 3, will be offered intravenous iron to treat their anaemia. 
 
Worsening anaemia 
Women, whose anaemia worsens between any of the visits, will be offered intravenous iron as per 
standard care. All women who receive intravenous iron will be asked to return to the clinic after 2 
weeks for repeat blood sampling (including research blood samples and QOL questionnaires) to 
ensure that they have responded and their haemoglobin has increased. The number of visits will 
depend upon clinical need in these patients as per national BSH guidelines.  
 
Group 3 Women recruited in the postnatal period. 
These women will participate in visits 1 & 2, as described above. 
For those women who are post-delivery and remain anaemic at the second visit a referral will be 
made to their General Practitioner for further follow up and on-going investigation as they feel 
appropriate and as per local guidelines. 
 
 Follow up and pregnancy outcome data 
Haematological and tolerability data will be collected in line with the detailed schedule described 
above and extracted from the data collections systems at the end of the study. 
 

 
 
 
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Pregnancy outcome data will also be collected 8 weeks after birth to allow for complete data 
collection in the puerperium. These data are collected routinely for this group of women. Broad 
headings for the items are shown below: 
 
 
Maternal 
• 
Haemoglobin (Hb) concentration, MCV and MCH nearest to the point of delivery 
• 
Hb, MCV, MCH in the puerperium 
• 
Antenatal Obstetric complications e.g. pre-eclampsia, intra-uterine growth restriction, 
gestational diabetes mellitus, infections and sepsis, etc. 
• 
Induction process if required 
• 
Mode of delivery 
• 
Complications associated with the delivery process. e.g. haemorrhage, perineal trauma, intra-
operative trauma at caesarean section,  
• 
Complications in the puerperium, e.g.  secondary postpartum haemorrhage, sepsis, 
psychological disorders, etc. 
Fetal 
• 
Birth weight 
• 
Gestation at delivery 
• 
Condition at birth, e.g. 5 minutes Apgar score, cord blood gases, need for resuscitation and 
techniques used. 
• 
Admission to higher level of neonatal care 
• 
Neonatal complications and diagnoses. 
 
 End of study 
The study will be closed 30 days after 8 weeks post-delivery of the last participant enrolled, to 
allow sufficient time for completion of baby outcomes data collection. The Chief Investigator will 
notify the REC of the end of the study or earlier if the study ended prematurely. 
 
13. STUDY TREATMENT 
Standard practice will be followed and enrolled women will be issued with a prescription for oral 
iron therapy as per the BSCH guidelines for iron deficiency anaemia in pregnancy. The women will 
be given verbal instruction on how to take the treatment. This will be backed up with written 
information. The BCSH guideline recommend 100-200mg elemental iron per day; this is traditionally 

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prescribed as 200mg ferrous sulphate three times a day (65mg elemental iron per tablet x 3 = 
195mg), and local RWT policies describing this will be followed. 
 
Iron has a marketing authorisation (MA) in the UK and is being used in its marketed presentation and 
packaging bearing the MA number. 
 
14. ADVERSE EVENT REPORTING 
The principles of ICH GCP require to follow specific procedures when notifying and reporting adverse 
events in clinical trials. These procedures, relevant to this non-interventional study, are described in 
this section.  
 Definitions of Adverse Events  
The definitions to be applied to adverse events recorded in this study are given in Table 9a below.  
 
Table 14a: Definitions 
Term 
Definition 
Adverse Event (AE)
  
Any untoward medical occurrence affecting a trial participant during the 
course of a clinical trial.  
Adverse Reaction (AR) 
Any adverse event when there is at least a possibility that it is linked to a 
trial drug or intervention. 
Unexpected Adverse 
Reaction (UAR) 
An unexpected occurrence of an adverse reaction  
Serious Adverse Event 
(SAE)  
 
A serious adverse event that:  
• results in death* 
• is life-threatening** 
• requires hospitalisation or prolongation of existing hospitalisation*** 
• results in persistent or significant disability or incapacity 
• is a congenital anomaly/birth defect 
• other important medical event(s)**** 
Serious Adverse 
Reaction (SAR) 
An SAE that is thought to be causally linked to a trial drug or intervention. 
Suspected Unexpected 
Serious Adverse 
Reaction (SUSAR) 
An unexpected occurrence of a SAR; there need only be an index of 
suspicion that the event is a previously unreported reaction to a trial drug 
or a previously reported but exaggerated or unexpected frequent adverse 
reaction. 
* Death due to the underlying disease or associated conditions will not be reported as an SAE.  
**The term ‘life-threatening’ in the definition of ‘serious’ refers to an event in which the patient 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. 
***Hospitalisation is defined as an inpatient admission, regardless of length of stay, even if the hospitalisation is a 
precautionary measure for continued observation.  Hospitalisations for a pre-existing condition (including elective 
procedures that have not worsened) do not constitute an SAE.  
 
 
 Expected Drug-Related Adverse Events / Reactions 
 

 
 
 
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Ferrous Sulphate tablets is a marketed medicinal product in licensed indication with a well-known 
safety profile, therefore adverse reactions which are expected (assessed against the Summary of 
Product Characteristics (https://www.medicines.org.uk/emc/medicine/27767 and not serious do 
not require recording, as these will not improve the knowledge of the safety profile of the drug. 
 
 
 
 
 Adverse Events that Require Expedited Reporting 
 
          Serious Adverse Events (SAEs) 
 
All adverse events which meet the definition of serious, occurring from the start of study treatment 
until 28 days post-study treatment must be recorded on the Serious Adverse Event Form and sent to 
the Sponsor within 24 hours of the research staff becoming aware of the event.  
 Investigators Assessment of SAE 
The Principal Investigator is required to consider seriousness (according to the definitions given in 
table 14a), causality in relation to the study treatment and expectedness of any SAE. Causality will be 
assessed according to the following categories:  
 
Table 14b: Definitions of Causality 
Relationship 
Description 
Unrelated 
There is no evidence of any causal relationship 
Unlikely 
There is little evidence to suggest there is a causal relationship (e.g. the 
event did not occur within a reasonable time after administration of the 
trial drug or intervention).  There is another reasonable explanation for 
the event (e.g. the patient’s clinical condition, other concomitant 
treatment). 
Possible 
There is some evidence to suggest a causal relationship (e.g. because the 
event occurs within a reasonable time after administration of the trial 
drug or intervention).  However, the influence of other factors may have 
contributed to the event (e.g. the patient’s clinical condition, other 
concomitant treatments). 
Probable 
The evidence is clearly in favor of attributing the adverse reaction to the 
trial drug or intervention 
Definitely 
There is conclusive evidence beyond reasonable doubt attributing the 
adverse reaction to the trial drug or intervention. 
 
The Investigator must assess the expectedness of each SAE using the ferrous sulphate SmPC – see Section 14.2   
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. 
 

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 Notification and Reporting of Serious Adverse Events  
Serious Adverse Event (SAEs) that are considered to be ‘related’ and ‘unexpected’ (SUSAR) are to be 
reported to the sponsor within 24 hours of learning of the event and to the Research Ethics 
Committee (REC) within 15 days in line with the required timeframe.  
 
 Overview of the Safety Reporting Process/Pharmacoviligance responsibilities 
The Serious Adverse Event Form must be completed by the Investigator (the Consultant named on 
the delegation of responsibilities log who is responsible for the patient’s care).  
The SAE should be reported to the CI within 24 hours of becoming aware of aware of the event 
The Investigator must follow-up all reported SAEs until resolution or the event is considered stable. 
 
The CI has the overall pharmacovigilance oversight responsibility and has a duty to ensure that 
pharmacovigilance monitoring and reporting is conducted in accordance with the sponsor’s 
requirements. All reported serious adverse events will be reviewed by the Chief Investigator (or a 
medically qualified delegate).  
 
 Statutory Reporting  
Reporting suspected adverse reactions after authorisation of the medicinal product is important. It 
allows continued monitoring of the benefit/risk balance of the medicinal product. Healthcare 
professionals are asked to report any suspected adverse reactions via the Yellow Card Scheme at: 
www.mhra.gov.uk/yellowcard  
15. ETHICAL AND REGULATORY ISSUES  
 Compliance  
This study complies with the Declaration of Helsinki 2013. It will also be conducted in compliance 
with the approved protocol, the principles of GCP, the UK Data Protection Act and the NHS UK Policy 
Framework for Health and Social Care Research  
 Ethical Compliance  
• 
Before initiation of the study, the protocol, informed consent, participant diary, 
questionnaires and any information to be provided to the prospective participant will be 
submitted to an NHSREC for ethical approval. Any subsequent amendments will be submitted 
to, and approved by, the same REC.  
• 
The protocol and study documents will be submitted by those delegated to do so to the Health 
Research Authority (HRA) for approval. 

 
 
 
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• 
For any amendment to the study, the Chief Investigator or designee, in agreement with the 
sponsor, will submit information to the appropriate body in order for them to issue approval 
for the amendment. The Chief Investigator or designee will work with site (R&D department) 
so it can put the necessary arrangements in place to implement the amendment to confirm 
their support for the study as amended. 
• 
Substantial amendments that require review by NHS REC will not be implemented until that 
review is in place and other mechanisms are in place to implement at site.   
• 
All correspondence with the REC will be retained. 
• 
The Chief Investigator will produce the annual reports as required. 
• 
The Chief Investigator will notify the REC of the end of the study. 
• 
An annual progress report will be submitted to the REC within 30 days of the anniversary date 
on which the favourable opinion was given, and annually until the study is declared ended. 
• 
If the study is ended prematurely, the Chief Investigator will notify the REC, including the 
reasons for the premature termination. 
• 
Within one year after the end of the study, the Chief Investigator will submit a final report with 
the results, including any publications/abstracts, to the REC. 
 Data Protection and Patient Confidentiality  
• 
All study team must comply with the requirements of the Data Protection Act 1998 with regards to 
the collection, storage, processing and disclosure of personal information and will uphold the Act’s 
core principles.  
• 
The confidentiality standards will be maintained by coding each participant enrolled in the study 
through assignment of a unique participant identification number. 
• 
Consent will be sought from participants to inform their General Practitioner (GP) of their 
enrolment in the study. Medical information may be given to a participant’s GP or other appropriate 
medical personnel responsible for the participant’s welfare, for treatment purposes.  
• 
Data generated by this study must be available for inspection upon request by representatives of 
the REC and other relevant national and local health authorities, as appropriate. 
 Indemnity 
This is an NHS-sponsored research study, and the NHS indemnity scheme therefore applies. If there 
is negligent harm during the study when the NHS body owes a duty of care to the person harmed, 
NHS indemnity covers NHS staff, medical academic staff with honorary contracts, and those 
conducting the study. The NHS indemnity scheme does not cover non-negligent harm. The liability 

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of the manufacturers of ferrous sulphate is strictly limited to those claims arising from faulty 
manufacturing of the commercial product and not to any aspects of the conduct of the study. 
 
 Amendments   
The sponsor will decide whether an amendment is substantial or non-substantial for the purposes 
of submission to the REC. If the sponsor wishes to make a substantial amendment to the REC 
application or supporting documents, the sponsor will submit a valid notice of amendment to the 
REC for consideration. The REC will provide a response regarding the amendment within 35 days 
of receipt of the notice. Amendments will also be notified to the participating organisation (R&D 
department and local research team), to assess whether the amendment affects the NHS permission 
for the study site. 
 
Once an amendment has been approved, the version number of the protocol will be updated. All 
superseded versions of the protocol will be kept in the Site File, scored through to indicate their 
replacement. 
 Access to the Final Study Dataset 
The SMG will have access to the final study dataset. Custody of the final data set will reside with the 
CI and the Sponsor.  
 Peer Review  
The study protocol has been subject to independent scientific peer review by two independent 
experts, by the sponsor (as part of sponsorship assessment), and by the NHDBT CTU research team. 
Feedback has been incorporated within the protocol and relevant study documents. 
 Patient & Public Involvement 
This proposed study has been discussed at several meetings of the Nottingham Maternity Research 
Network (PPI network) to assess acceptability. PPI group who highlighted that anaemia often treated 
as ‘routine’ and not seen as significant; a need for anaemia to be resolved prior to labour due to the 
challenges of treating anaemia after birth; and the need to understand the follow up of the outcomes 
of anaemia. The PPI group will be consulted during the dissemination process and whether posters 
and other materials are required.  
 

 
 
 
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16. STATISTICS AND DATA ANALYSIS 
 Primary outcome measure 
The proportion of pregnant women who meet the criteria for a clinical response, defined as an 
increase in haemoglobin concentration of 10g/l, 2-4 weeks after the onset of iron therapy. 
 
 Other Outcomes 
1. The change in frequency and severity of symptoms associated with anaemia in pregnancy using a 
diary. (As assessed by our pre-piloted version of a published pregnancy symptomatology tool 
(Foxcroft 2013). 
2. The frequency and severity of side effects induced by iron therapy to treat anaemia using a diary. 
(As assessed by a published tolerability tool (Peirera 2014), undertaken alongside the pregnancy 
symptomatology questionnaire) 
3. A measurement of compliance with currently recommended oral iron regimens prescribed in 
pregnancy. (As assessed by patient reported compliance using a diary and the need for dose 
adjustments in line with BSCH guidelines) 
4. To document the longitudinal changes in haemoglobin, red cell indices, iron, transferrin, ferritin, 
CRP, during a treatment course of oral iron.  
5. To investigate the changes in research biomarkers, such as serum hepcidin, and whether they can 
predict the response to oral iron.  
 
 Statistical Analysis and Overview Analysis Plan 
We aim to recruit approximately 120 patients into this study split equally between the three chosen 
time phases of pregnancy and the puerperium. This number divided between the cohorts represents 
a pragmatic decision based on estimated numbers of anaemic women seen in the antenatal period in 
Wolverhampton, and to support exploratory analyses to meet the objectives. 
 
Data will be collected on paper Case Report Forms (CRFs), which will bear no patient identifiable 
data. Each patient will receive a unique CRF number for identification. Data will be stored using a 
system that complies with the requirements of the FDA CFR part 21 using a secure hospital server in 
accordance with the UK Data Protection Act 1998 and transferred onto statistical software for 
analysis.  
 
Physiological recovery from anaemia will be defined in this study as in increase in Hb by >10 g/L by 
2-4 weeks and these patients will be classed as ‘responders’. Differences between responders and 

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non-responders will be explored by comparing baseline demographic data, haematological, 
biochemical and iron parameters. Data which do not follow a parametric distribution will be 
transformed. Geometric means will be calculated for logarithmically transformed data and mean 
values compared using the t-test. Statistical significance was defined as p value <0.05. 
 
The primary outcome will be a response; an increase in the haemoglobin concentration of >10g/L, to 
iron treatment. This will be described as a percentage for the group as a whole and by each cohort, 
early pregnancy, late pregnancy and post-delivery. 
 
Quantitative variables will be described using percentages and exact binomial 95% confidence 
intervals, continuous variables using median and interquartile range (IQR).  
 
Logistic regression may be used to investigate associations of maternal anaemia and responses to 
treatment, as a binary outcome for the primary outcome.  
Data from the study questionnaire and participants’ diary will be analysed using descriptive 
statistical analysis.  
 
Statistical analyses will be performed using statistical analysis software (SAS/STAT, Version 9 of the 
SAS System for Windows, SAS Institute, Inc., Cary, NC). 
 
17. DATA MANAGEMENT 
 Participant Confidentiality and Data Access 
Individual participant data for the TIAP study will be captured on a Case Report Form (CRF). Only the 
data required by the protocol will be captured in the CRF. All paper documents including the CRFs 
will be stored securely and only be accessible by study staff and authorised personnel. Electronic data 
will be stored on secure servers with secure back up arrangements. The servers, CRFs, and other data 
stores will be held in secure research office at RWT. Only clinical care teams will have access to 
patient identifiable data. 
The CI/PI and delegated team at site will keep a record of all participating patients with sufficient 
information to link records, including CRFs, hospital notes and samples, and all original signed 
informed consent forms.  
The study will comply with the Data Protection Act, which requires data to be anonymised before 
transferring to CRF and transferring outside clinical care team. Any electronic data processed off the 
hospital site will have been anonymised before leaving the site and stored in a password-protected 
system. 

 
 
 
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Data will be collected by staff trained in high quality handling and secure procedures. All other 
information and data will be stored electronically. The security of the database is maintained by the 
following principles: 
• 
Access to clinical and personal data in the database will be limited to the local care team. 
Access will be secure and password protected. The research individuals will maintain 
confidentiality. 
• 
The number of staff having access to all fields of the database will be limited.  
• 
Direct access will be granted to authorised representatives from the Sponsor (i.e. host 
institution) and the regulatory authorities to permit study related monitoring, audits and 
inspections in line with participant consent. Responsible members of the University of 
Oxford, the Royal Wolverhampton NHS Trust Department of Research and Development or 
the University of Wolverhampton may be given access to data for monitoring and/or audit of 
the study to ensure the study is complying with regulations. 
 
 Archiving 
Archiving will be authorised by the Sponsor following submission of the end of the study report. All 
study documentation will be archived for 5 years after completion of the study as per study Sponsor's 
policy and procedures. Destruction of these documents after the 5 years point will require 
authorisation from the Sponsor.  
 
The site must keep the signed Informed Consent forms, all study documentation, and source 
documents collected during the study in a secure location (e.g. locked filing cabinets in a room with 
restricted access).  
 
All data must be accessible to the competent authorities and the Sponsor with suitable notice for 
inspection. In addition, the Investigator must not discard or destroy any study specific materials 
unless otherwise instructed by the Sponsor. 
18. DATA MONITORING AND INSPECTION 
A Study Management Group (SMG) comprising the Chief Investigator, other lead investigators, and 
members of the research team. The TMG will be responsible for the day to day running and 
management of the study. It will meet at least four times a year, more often during set up and close 
down phases of the study. At least one face to face meeting will be held each year.  

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The safety data will be reviewed periodically by a nominated agent of the sponsor and 
discussed with sponsor to determine patterns and trends of events, or to identify safety issues, 
which would not be apparent on an individual case basis.  
 
The host R&D department reserve the right to conduct site audits, either as part of its on-going 
audit programme, or in response to adverse observations occured. 
 
 
19. PUBLICATION POLICY 
• 
Reporting will be in compliance with CONSORT recommendations.  
• 
The CI will approve any publications arising from this study  
• 
No data may be made public before publication and never without agreement from the CI 
and Sponsor. 
• 
Funder will be acknowledged in all publications/presentations. 
• 
The final results of the study will be submitted to a peer-reviewed journal for publication. 
Authors will be the CI, the co-investigators and other members of the research team.  
• 
A study identifier will be included on all presentations and publications. (ISRCTNxxxxx)  
• 
For all papers or posters/presentations, we will use the International Committee of Medical 
Journal Editors of definitions of Authorship and the Contributorship 
(http://www.icmje.org/ethical_1author.html). 
 
 
20. PROTOCOL AMENDMENTS 
Revision History:  
Protocol  
Version  
Author  
Date  
Reason for revision  
 1.2 
Various  
04/05/2018 
Administrative changes  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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21. REFERENCES: 
Alderdice F, McNeill J, Gargan P, Perra O (2017). Preliminary evaluation of the Well-being in Pregnancy 
(WiP) questionnaire. Journal of Psychosomatic Obstetrics & Gynecology, 1743-8942 DOI: 
10.1080/0167482X.2017.1285898. 
Asif, N. (2007). Comparison of serum ferritin levels in three trimesters of pregnancy and their 
correlation with increasing gravidity. Int J Path , 26-30. 
Beard, J. (1994). Iron Deficiency: assessment during pregnancy and its importance in pregnant 
adolescents. Am J Clin Nutr , 502S-508S. 
Bech P (2004) Measuring the Dimension of psychological general well-being by the WHO-5.Quality 
of Life Newsletter 32 15-16. 
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