Effect of Iron Supplementation During Pregnancy on Neurodevelopmental Status of Babies
Conditions: Iron Deficiency Anemia, Neurodevelopmental Abnormality, Autism Spectrum Disorder
Sponsor: Thomas Jefferson University

 
 
 
 
 
 
 
 
RAPIDIRON-KIDS Study 
 
RESEARCH PROTOCOL 
Version 2.3 
September 20, 2022 
 
 
 
 
 
 
 
 
 
 
 
 
 
TJU IRB Control Number: 22G.387 
US PI: Richard J. Derman, MD, MPH 
Belagavi PI: Mrutyujaya B. Bellad, MD 
ClinicalTrials.gov number: NCT05504863 
 

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Table of Contents 
RAPIDIRON-KIDS Study Investigators & Key Personnel ............................................................................. 3 
Version Tracking ........................................................................................................................................... 6 
Acronyms ...................................................................................................................................................... 7 
Abstract ......................................................................................................................................................... 8 
A. 
Specific Aims ......................................................................................................................................... 9 
B. 
Background and Significance ................................................................................................................ 9 
C. 
Research Team and Preliminary Studies ........................................................................................... 11 
D. 
Research Design and Methods ........................................................................................................... 13 
E. 
Statistical Methods .............................................................................................................................. 18 
F. 
Gender/Minority/Pediatric Inclusion for Research............................................................................... 21 
G. 
Human Subjects .................................................................................................................................. 21 
H. 
Data and Safety Monitoring Plan ........................................................................................................ 24 
I. 
Literature Cited .................................................................................................................................... 26 
 
 
 

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RAPIDIRON-KIDS Study Investigators & Key Personnel 
Thomas Jefferson University: 
Richard J. Derman, MD, MPH,  
Principal Investigator 
Associate Provost for Global Affairs 
Richard.Derman@jefferson.edu 
Rupsa Boelig, MD, MS 
Perinatologist 
Assistant Professor, Maternal-Fetal Medicine 
Rupsa.Boelig@jefferson.edu  
Benjamin Leiby, PhD 
Biostatistician 
Professor, Pharmacology, Physiology, and Cancer Biology 
Benjamin.Leiby@jefferson.edu 
Vanessa Short, PhD, MPH 
Perinatal Epidemiologist 
Assistant Professor, Obstetrics & Gynecology 
Vanessa.Short@jefferson.edu 
KLE Academy of Higher Education and Research, Jawaharlal Nehru Medical College (JNMC), Belagavi, 
Karnataka, India: 
Mrutyunjaya B. Bellad, MD 
Site Principal Investigator 
Professor of Obstetrics and Gynecology 
belladmb@gmail.com 
Roopa B. Bellad, MD 
Site Co-Principal Investigator 
Professor of Pediatrics 
belladroopa5@gmail.com  
Shivaprasad Goudar, MD 
Senior Foreign Investigator 
Professor, Department of Physiology 
sgoudar@jnmc.edu  
Yogesh Kumar, MD 
Project Coordinator 
Associate Professor of Community Medicine 
yogeshkumar23@gmail.com 
Umesh Charantimath, MD 
Project Coordinator 
Associate Professor, Community Medicine 
drumesh.charantimath@gmail.com  
Manjunath Somannavar, MD 
Laboratory Coordinator 
Associate Professor, Department of Biochemistry 
manjunathsomannavar@gmail.com  

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S. Nijalingappa Medical College (SNMC) Bagalkot, Karnataka, India: 
Ashalata Mallapur, MD 
Site Co-Principal Investigator 
Professor & Head, Department of Obstetrics & Gynecology 
drashalatamallapur@gmail.com  
 
Ramesh Pol, MD 
Co-Investigator 
Professor, Department of Pediatrics 
rameshpol@ymail.com 
 
Umesh Ramdurg, MD 
Project Coordinator 
Professor, Department of Community Medicine 
umeshramadurg79@gmail.com 
 
Raichur Institute of Medical Sciences (RIMS) Raichur and Model Rural Health Research Unit (MRHRU), 
Sirwar, Karnataka, India:  
Radha Sangavi, MD 
Site Co-Principal Investigator 
Professor & Head, Department of Obstetrics & Gynecology 
 
Basavaraj V. Peerapur 
Co-Investigator 
Dean cum Director 
peerapur_2003@yahoo.co.in  
 
Nasima Banu 
Co-Investigator 
Associate Professor, Department of Pediatrics 
nasimakazi@rediffmail.com 
 
Praveen S. Patil 
Project Coordinator 
Tutor, Department of Physiology 
praveenkmc@gmail.com 
 
             Subarna Roy 
In-Charge, MRHRU and Director,  
ICMR – National Institute of Traditional Medicine (ICMR/NITM), Belagavi, Karnataka, India 
 
Phaniraj Vastrad,  
Scientist C (Medical), MRHRU 
 
Other Co-Investigators: 
Michael K. Georgieff, MD 
Professor of Pediatrics and Developmental Psychology, Director of the Center for 
Neurobehavioral Development 
University of Minnesota, Minneapolis, MN 
georg001@umn.edu 

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Dennis Wallace, PhD 
Biostatistician Consultant 
Chief Quality and Safety Officer for RAPIDIRON Trial and RAPIDIRON-KIDS 
wallacejhawk@gmail.com 
Zubair Aghai, MD 
Director of Neonatal/Perinatal Fellowship Program, Associate Professor of Pediatrics 
Nemours Children’s Health, Philadelphia, PA 
zubair.aghai@nemours.org 
Michael Auerbach, MD 
Clinical Professor of Medicine 
Georgetown University School of Medicine, Academic Medical Practice (hematology and 
oncology), Baltimore, MD 
mauerbachmd@abhemonc.com 
 
 
 
 
 
 
 
 
 
 
 

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Version Tracking 
Version 
Date 
Author(s) 
Notes 
1.0 
April 7, 2022 
RAPIDIRON Working 
Group 
Original Version 
2.0 
April 7, 2022 
RAPIDIRON Working 
Group 
Minor amendments 
made to content and 
format 
2.1 
May 12, 2022 
RAPIDIRON Working 
Group 
Adjusted exclusion 
criteria; removed some 
assessments due to 
implementation 
feasibility issues; 
updated statistical plan 
2.2 
May 30, 2022 
RAPIDIRON Working 
Group 
Addition of Pediatrics 
site Investigators of 
Bagalkot and Raichur 
and Investigators of 
MRHRU, Sirwar, 
Raichur 
2.3 
September 20, 2022 
RAPIDIRON Working 
Group 
Minor amendments 
made to content, 
removed offspring 
blood draws at 24 and 
36M to reduce 
participant burden 
 
 
 

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Acronyms 
AE 
 
Adverse event 
ASHA  
Accredited Social Health Activist 
BSID 
 
Bayley Scales of Infant Development 
CBCL/1.5-5 
Child Behavior Checklist for Ages 1.5-5 
DQ 
 
Developmental quotient 
DSMB  
Data and Safety Monitoring Board 
Hb 
 
Hemoglobin 
HIF-1α  
Hypoxia induced growth factor 
ICFI 
 
Infant Child Feeding Index 
ID 
 
Iron deficiency 
IDA 
 
Iron deficiency anemia 
IGF-1  
Insulin-like growth factor-1 
ISAA 
 
Indian Scale for Assessment of Autism 
IV 
 
Intravenous 
JNMC  
Jawaharlal Nehru Medical College 
LBW 
 
Low birth weight 
MRHRU 
Model Rural Health Research Unit 
MRI 
 
Magnetic resonance imaging 
NICHD  
National Institute of Child Health and Human Development 
NFHS  
National Family Health Survey 
PI 
 
Principal Investigator 
PPH 
 
Postpartum hemorrhage 
RCT 
 
Randomized controlled trial 
RIMS 
 
Raichur Institute of Medical Sciences 
SAE 
 
Serious adverse event 
SNMC  
S. Nijalingappa Medical College 
TIBS 
 
Total iron binding saturations 
TSAT 
Serum transferrin saturation 
WHO 
World Health Organization 
WHODAS 
World Health Organization Disability Assessment Schedule 

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Abstract 
Background: Anemia is a worldwide problem with iron deficiency being the most common cause. When 
anemia occurs in pregnancy, it increases the risk of adverse maternal, fetal and postnatal outcomes. It 
induces preterm births and low birth weight (LBW) deliveries, long-term developmental sequelae and an 
increased risk of earlier onset of postnatal iron deficiency. Anemia rates are among the highest in South 
Asia, and India’s National Family Health Survey (NFHS-5) for 2019-21 indicated that over half of pregnant 
women, and more than 65% of children, in the country are classified as anemic. [1] In 2021, the 
RAPIDIRON Trial [2] was initiated in two states in India, with the goal of assessing if a dose of 
intravenous (IV) iron given to anemic women during pregnancy will result in a greater proportion of 
participants with normal hemoglobin concentrations in the third trimester and a lower proportion of 
participants with low birth weight deliveries compared to oral iron. As a follow-up to the RAPIDIRON Trial, 
the RAPIDIRON-KIDS study will follow the previously randomized mothers as well as their offspring after 
birth to assess neurodevelopmental, hematological, and health outcomes. This study’s overarching goal 
is to determine if the offspring born to RAPIDIRON Trial mothers in the IV iron groups, compared to the 
oral iron group, will achieve superior neurodevelopment, iron stores, and growth at specific time points 
during the first three years of life. 
 
 
 
 
 
 

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A. Specific Aims 
Hypotheses and Specific Aims 
This study has two primary hypotheses as follows: 
(1) Infants born to RAPIDIRON Trial maternal participants from the intravenous (IV) iron arms will 
have higher hemoglobin and ferritin levels at birth (determined by cord blood) and at 4 months of 
age compared to infants born to mothers in the oral arm; and 
(2) Offspring born to RAPIDIRON Trial participants who received IV iron treatment will have higher 
developmental quotients (DQs) on the cognitive domain of the Bayley Scales of Infant 
Development (BSID) at 2 years of age compared to offspring born to RAPIDIRON Trial 
participants given oral iron treatment. 
The RAPIDIRON-KIDS Study is designed to determine if offspring born to RAPIDIRON Trial maternal 
participants in the IV iron groups, compared to the oral iron group, will achieve superior neurodevelopment, 
iron stores, and growth at specific postnatal timepoints during the first 3 years of life. Differences will be 
assessed between offspring based on the iron deficiency anemia (IDA) treatment of the mother.  
 
The specific aims of this study are: 
 
To provide evidence that a single-dose of IV iron (either ferric carboxymaltose or iron isomaltoside, 
also known as ferric derisomaltose) given to pregnant women in the early second trimester of 
pregnancy during the RAPIDIRON Trial will prove more effective for prevention of neonatal and 
postnatal iron deficiency in the offspring than the oral iron given to pregnant women per the parent 
trial protocol; 
 
To assess if the offspring of women in the parent trial IV iron arms have better neurodevelopmental 
outcomes compared to the offspring of women treated with oral iron; and 
 
To determine longer-term hematologic effects in previously randomized mothers by obtaining 
ongoing hematologic indices, documented history of transfusion and hospitalization, and quality of 
life based on the use of a validated instrument. 
 
Long-Term Objective 
The long-term objectives of the RAPIDIRON-KIDS Study are to improve the iron status and health 
outcomes in both mothers and their offspring as well as to improve growth and neurodevelopment in 
children. Assuming that the primary hypotheses of both this follow-up study and the parent RAPIDIRON 
Trial prove true without raising safety concerns and the parent trial presents evidence of economic 
feasibility of single-dose IV iron for treatment of IDA in pregnancy, the researchers involved in these 
studies intend to aggressively disseminate the research results to increase the probability that the 
Government of India’s Ministry of Health and Family Welfare will endorse broader and appropriate scale-
up of a single-dose IV iron infusion as frontline treatment for IDA in pregnancy for women with moderate 
IDA. Further, dissemination of positive findings for these related studies can be globally promoted as 
supportive of goals to improve care and outcomes in women during pregnancy and the postpartum period 
and reduce the rates of anemia, iron deficiency, poor growth, and the occurrence of developmental 
disabilities in children.  
B. Background and Significance 
Based upon the National Family Health Survey (NFHS)-5 for 2019-21, over half of the pregnant women in 
India are anemic. [1] The major cause of anemia is iron deficiency, which is considered one of the most 
common micronutrient deficiencies in the world according to the World Health Organization (WHO). [3] 
Additionally, iron deficiency anemia (IDA) is a major contributing factor to the high rate, >65%, of children 
(6 – 59 months of age) who are classified as anemic in India (with hemoglobin level of <11g/dL). 
[1]Optimal fetal, neonatal, and childhood brain growth and development require adequate iron. However, 

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women with moderate to severe anemia during the late second and early third trimesters of pregnancy 
are often unable to make up their iron deficit. Thus, despite active transport via the placenta, insufficient 
iron may be transmitted to the developing fetus with consequent negative sequelae, including long-term 
neurodevelopmental impairment of the offspring.  
A study of Chinese pregnant women found that daily oral iron, initiated at or prior to 20 weeks gestation 
and continued until delivery, improved oral iron parameters; however, 45% of the babies born to these 
women were nevertheless iron deficient, suggesting that oral iron supplementation may not optimally 
reach the developing fetus. [4] Published evidence confirms that iron deficiency in infancy is associated 
with a statistically significant increase in cognitive and behavioral abnormalities which may persist for 
decades despite iron repletion. [5] While one of the primary outcomes for the parent RAPIDIRON Trial is 
the rate of low birth weight (LBW) delivery (a leading cause of under-5 mortality and an independent risk 
for poorer neurodevelopment), iron deficiency anemia in pregnancy is associated with other adverse 
pregnancy outcomes including increased preterm birth and perinatal and neonatal mortality as well as 
iron deficiency leading to poor neurodevelopment of the offspring. [6] The risks of fetal underloading 
include: (1) abnormal fetal brain development and function; (2) a higher incidence of significant 
psychopathologies later in childhood or adulthood including autism, schizophrenia, and neurocognitive 
disorders; and (3) an earlier onset of postnatal iron deficiency, which carries its own attendant 
neurodevelopmental consequences including anxiety, depression, and hyperactivity/attention disorders. 
[6] The long-term effects occur in spite of a diagnosis of iron deficiency in infancy and prompt treatment, 
implying that prevention of neonatal and postnatal iron deficiency through adequate fetal iron loading is 
the preferred course of action. Apart from the costs associated with treatment of acute illness, it is the 
long-term effects that are the true cost to society in terms of lost educational potential, remediation costs 
during childhood, and ultimately lost job potential. [6] 
We are unable to find published data on the duration of effect of IV iron administered to pregnant women. 
Further, additional research is required to determine if an increase in hemoglobin level over time 
translates into health-related consequences and overall life quality. Following women who were 
previously randomized for treatment of iron deficiency anemia with a single dose IV iron infusion or oral 
iron will allow us to detect differences in outcomes based on the treatment approach and determine if 
there is a time when differences among the arms no longer exist. This information is especially important 
if differences are found at the time of the next pregnancy. 
The Cochrane Collaboration reported a paucity of quality trials that assessed clinical maternal and 
neonatal effects of iron administration to anemic pregnant women. The Cochrane reviewers indicated the 
need for large, high-quality trials that assess clinical outcomes and treatment effects. [7] This need has 
not been adequately met, especially in terms of assessing the efficacy of newer treatment approaches, 
specifically the use of single-dose IV iron during pregnancy to reduce LBW rates and to adequately iron 
load the fetus to preserve hematologic and brain development. The ongoing RAPIDIRON Trial addresses 
the identified need from the maternal and newborn perspective through six weeks postpartum, but 
hematologic, iron status, and neurodevelopmental follow-up of the offspring is urgently needed to assess 
the intergenerational impact of maternal iron and anemia management and to ensure that the offspring 
achieve full developmental potential. Together, the parent RAPIDIRON Trial and the RAPIDIRON-KIDS 
Study will increase the likelihood that India’s anemia guidelines and recommendations of respected 
health organizations will continue to undergo refinement. We anticipate that new and improved anemia 
and iron deficiency treatment approaches that are convenient, cost-effective, and feasible can be more 
rapidly accepted, implemented, and scaled-up adequately to meet a great need. 
Description of Parent Trial 
The RAPIDIRON Trial 
Reducing Anemia in Pregnancy in India: the RAPIDIRON Trial [2] is a 3-arm, randomized-controlled trial 
designed to assess if a single dose of an intravenous (IV) iron formulation (ferric carboxymaltose in 
intervention arm 1 or iron isomaltoside, also known as ferric derisomaltose, in intervention arm 2), 

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administered early in the second trimester of pregnancy for treatment of moderate iron deficiency anemia 
(IDA), will result in a greater percentage of pregnant participants in the IV iron arms achieving a normal 
for pregnancy hemoglobin (Hb) concentration of ≥11 g/dL in the third trimester (at either a 30-34 week 
antenatal visit or based on blood collected prior to delivery) when compared to the percentage of 
participants randomized to an active, comparator arm (arm 3) provided oral arm. 
The second primary outcome for the parent trial is low birth weight (LBW) under 2500 grams, which is one 
of several adverse pregnancy outcomes associated with IDA. It is hypothesized that LBW delivery rates 
for participants randomized to the IV iron arms will be significantly lower when compared to the LBW 
delivery rate of participants randomly assigned to the oral iron arm. Additionally, numerous secondary 
outcomes will be explored. 
Although a much larger number of pregnant women will be screened for eligibility at two timepoints for 
RAPIDIRON, it is expected that 4,320 pregnant participants will be randomized and treated for IDA 
consistent with the plan for the study arm to which they are assigned. This sample size should be 
sufficient to assure availability of approximately 1,332 complete data sets per arm even if up to 7.5% of 
study participants fail to complete study milestones. 
An initial eligibility requirement for consent to participate in the RAPIDIRON Trial is hemoglobin 
concentration of 7 – 10.4 g/dL when measured in the first trimester or early in the second trimester of 
pregnancy. However, for randomization and treatment, requirements include: 
 
Iron deficiency anemia, defined for this study as moderate anemia with hemoglobin concentration 
level between 7 – 9.9 g/dL as well as test results indicating that serum transferrin saturation 
(TSAT) is <20% and/or ferritin is <30 ng/mL; and 
 
A live singleton, intrauterine fetus and dating ultrasound that indicates a pregnancy that would 
allow IDA treatment initiation between the beginning of week 14 and prior to 17 weeks 0 days 
gestation. 
Exclusion criteria for the parent trial include: 
 
Fetal anomaly, if detectable when an initial ultrasound is done to date the pregnancy (subsequent 
discovery of a fetal anomaly is not viewed as an exclusion criterion); 
 
Maternal history of cardiovascular disease, hemoglobinopathy, or other disease or condition 
considered a contraindication for treatment, including conditions recommended for exclusion by 
the manufacturers of oral or IV iron to be used in this study; and 
 
Any condition that, in the opinion of the consenting physician, warrants study exclusion. 
Pregnant participants randomly assigned to an IV iron arm receive a single-dose IV iron formulation 
having 1000mg of iron. However, participants weighing under 50kg receive a lower dose of iron as 
determined by a formula used by both manufacturers of the IV iron formulations employed in the trial. As 
indicated in the RAPIDIRON Trial protocol, the IV iron infusion should be given between 14 and 17 weeks 
fetal gestational age. 
C. Research Team and Preliminary Studies 
Since 2001, Dr. Richard Derman has been a Principal Investigator (PI) for the Global Network for 
Women’s and Children’s Health Research, Eunice Kennedy Shriver National Institute of Child Health and 
Human Development (NICHD), in partnership with Jawaharlal Nehru Medical College (JNMC), Belagavi, 
Karnataka, India. The first collaborative study, implemented in the Belagavi District of Karnataka, was a 
single-site, randomized, placebo controlled, community-based trial of oral misoprostol for prevention of 
postpartum hemorrhage (PPH). This study’s findings, published in the Lancet in 2006, indicated a 
reduction in PPH of nearly 50%; severe PPH showed an 80% reduction. [8] Importantly, the study 
resulted in worldwide scale-up of misoprostol use for PPH prevention. Since then, eleven additional 
Global Network studies have been completed. Additionally, Belagavi Women’s and Children’s Health 

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Research Unit (or Global Network Site 8) has also completed, or is participating in, more than 20 other 
studies funded by various private and public sources – e.g., the Bill & Melinda Gates Foundation, the 
Thrasher Research Fund, the Children’s Investment Fund Foundation, and the World Health Organization 
(WHO) – which recently named JNMC a WHO Collaborating Centre for Research in Maternal and 
Perinatal Health. 
RAPIDIRON-KIDS Study Co-Investigators at Thomas Jefferson University include Dr. Rupsa Boelig, Dr. 
Benjamin Leiby, and Dr. Vanessa Short. Dr. Rupsa Boelig is an Assistant Professor of Obstetrics and 
Gynecology with a dual appointment in the Division of Maternal Fetal Medicine, where she serves as the 
Director of Research, and the Department of Clinical Pharmacology and Experimental Therapeutics. Her 
clinical background includes four years of residency training in obstetrics and gynecology followed by an 
additional four years of subspecialty training in maternal fetal medicine, with a two year concurrent 
assignment facilitated by a US National Institutes of Health Clinical Pharmacology fellowship. Dr. Boelig 
has been involved in the design and implementation of the RAPIDIRON Trial and will serve in the same 
capacityfor the RAPIDIRON-KIDS Study. Dr. Benjamin Leiby is a Professor and head of the Biostatistics 
division, skilled in the analysis and interpretation of biomedical and epidemiological data. He has 
collaborated widely with basic scientists, clinicians, and epidemiologists in many specialties. Dr. Leiby 
helped develop the parent RAPIDIRON Trial and works closely with the JNMC research team on the Data 
Management System as well as the implementation of the statistical analysis plans. Dr. Vanessa Short is 
a perinatal epidemiologist and an Assistant Professor in the Department of Obstetrics and Gynecology at 
Sidney Kimmel Medical College/TJU. Dr. Short currently serves as Director of Research at TJU’s 
Maternal Addiction Treatment, Education and Research Program, where she leads her own federally and 
intramurally-funded research projects within the realm of maternal and child health, including the conduct 
of multi-year cluster randomized clinical trials. Dr. Short frequently collaborates with Dr. Derman and other 
faculty on maternal and child health-related trials in low-income and middle-income countries. She is 
currently providing services as a member of the RAPIDIRON research team, and she will work on the 
RAPIDIRON-KIDS Study providing input into the design and implementation of the trial and assisting with 
interpretation of study data and dissemination of findings. 
The RAPIDIRON-KIDS Study will involve oversight from a research support team at Thomas Jefferson 
University and collaboration with researchers from JNMC Women and Children’s Health Research Unit. 
Key personnel from JNMC include: Dr. Shivaprasad S. Goudar, a Professor of Physiology and JNMC’s 
Senior Foreign Investigator; Dr. Mrutyunjaya Bellad, a Professor of Obstetrics and Gynecology, and the 
Site Principal Investigator of the parent RAPIDIRON Trial, who has participated in numerous studies 
being implemented by the Women’s and Children’s Health Research Unit based on the JNMC campus 
since it was established twenty years ago; Dr. Roopa Bellad, a Professor of Pediatrics who will serve as 
Co-Site Principal Investigator; and Dr. Yogesh Kumar, Associate Professor of Community Medicine at 
JNMC, who is the current Project Coordinator of the RAPIDIRON Trial.  
Critical input into the design of the RAPIDIRON-KIDS Study has been provided by co-investigators who 
are recognized experts in neonatology, hematology, and pediatric neurodevelopment. Dr. Michael 
Georgieff, Executive Vice Chair and Professor, Department of Pediatrics at the University of Minnesota 
School of Medicine, is an internationally known researcher whose laboratory studies focus on the 
biological basis for the negative neurodevelopmental effects of fetal/neonatal iron deficiency and anemia. 
His work has contributed to an understanding of the neurological effects of iron deficiency prior to birth 
and the mechanisms by which long-term effects can be carried into adulthood. [9, 10] Dr. Michael 
Auerbach, Clinical Professor of Medicine, Georgetown University School of Medicine, is involved in an 
academic medical practice in hematology and oncology. He is recognized as a global leader with vast 
experience in the use of IV iron. He conducted the first US prospective study of IV iron use in pregnancy, 
the results of which indicated that IV iron is safe, less toxic, and more effective than oral iron. [11] Dr. 
Zubair H. Aghai, MD, FAAP is a Neonatologist in the Division of External Specialty Pediatrics, Department 
of Pediatrics at Alfred I. duPont Hospital for Children of the Nemours Foundation. He is also a Professor 
of Pediatrics and the Director of the Neonatology Fellowship Program at the Sidney Kimmel Medical 

RAPIDIRON-KIDS Study: Version 2.3 September 20, 2022 
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College of Thomas Jefferson University Hospital. Dr. Aghai has expertise in clinical and translational 
research in neonatal infection, inflammation, and nutrition, with a special interest in neurodevelopmental 
follow-up. He is active in several research projects including those funded by the National Institutes of 
Health. He is also collaborating with sites in India on an NIH R01 global health research project to 
improve maternal and child health in low- and lower-middle-income countries.  
D. Research Design and Methods 
Design of the RAPIDIRON-KIDS Study 
Table 1 below indicates the plan for assessing participating infants and post-delivered women for this 
study. The parent RAPIDIRON Trial will be the source of much of the information for the first two visits 
listed. The visits at 4 months, 12 months, 24 months, and 36 months of age will involve assessments 
done specifically for the RAPIDIRON-KIDS Study. The study outcomes are as follows: 
Offspring Primary Outcomes: 
(1) Iron status – indicated by hemoglobin and ferritin at birth and at 4 months 
(2) Developmental quotient (DQ) for cognitive domain of the Bayley Scales of Infant Development 
(BSID) at 24 months 
Secondary Outcomes (Offspring): 
 
Iron status – indicated by hemoglobin, and reticulocyte hemoglobin content at 12 months and 
other indices at birth and four months 
 
Performance on Preferential Looking assessment at 4 and 12 months 
 
Scoring of performance on motor and language domains of BSID IV at 24 months and scoring of 
performance on all domains and 36 months 
 
Performance on Behavioral Rating Scale at 24 months 
 
Performance on Ages & Stages Questionnaire -3 at 12 months 
 
Internalizing and Externalizing behaviors on the Child Behavior Checklist for Ages 1.5-5 
(CBCL/1.5-5) at  24 and 36 months 
 
Risk of autism diagnosis on Indian Scale for Assessment of Autism (ISAA) at 36 months 
 
Growth parameters (weight, length/height, and head circumference) at 4, 12, 24, and 36 months 
 
Hospitalizations and etiologies during the first three years of the child’s life 
 
Infant feeding practices measured via Infant and Child Feeding Index (ICFI) 
Secondary Outcomes (Mothers): 
 
Iron status (Hb, ferritin and TSAT) at 4, 12, and 241 months 
 
Quality of life indices at 4 and 12 months measured via the World Health Organization Disability 
Assessment Schedule 2.0 (WHODAS 2.0) 
 
Hospitalizations through three years post-delivery 
 
Documented blood transfusions 
 
Infant feeding practices measured via Infant and Child Feeding Index (ICFI) 
 
 
 
                                                     
1 If no difference in iron status between RAPIDIRON treatment arms is detected at 12 months (once 12 
month sample analysis is complete), we will no longer collect 24 month maternal blood samples. 

RAPIDIRON-KIDS Study: Version 2.3 September 20, 2022 
14 
 
Table 1. Schedule of RAPIDIRON-KIDS Study 
 
 
 
 
 
Assessment Visit # 
1 
2 
3 
4 
5 
6 
Age at Assessment 
Birth 
6 weeks 
4 months 
12 months 
24 months 
36 months 
Iron Status 
Hemoglobin 
X 
  
XX 
XX 
XXX** 
 
Reticulocyte Hgb content 
X 
 
XX 
XX 
XXX 
 
Ferritin 
X 
  
XX 
XXX 
XXX 
 
%TSAT 
X 
  
XX 
XXX 
XXX 
 
Neurobehavioral 
Preferential Looking 
  
  
X 
X 
  
  
Ages & Stages Questionnaire 3 
 
 
 
X 
 
 
Bayley Scales of Infant Development IV 
  
  
  
 
X 
 X 
Behavioral Rating Scale 
  
  
  
 
X 
  
India Scale for Assessment of Autism 
  
  
  
  
  
X 
Child Behavior Checklist for Ages 1.5-5 
(CBCL/1.5-5) 
  
  
  
X 
X 
General Growth & Breastfeeding Practices 
Weight 
X 
X 
X 
X 
X 
X 
Length 
X 
X 
X 
X 
X 
X 
Head circumference 
X 
X 
X 
X 
X 
X 
Breastfeeding 
XXX 
XXX 
XXX 
XXX 
XXX 
XXX 
Infant Child Feeding Index (ICFI) 
X 
X 
X 
X 
X 
 
  
  
  
  
  
  
  
Quality of life indices (WHODAS 2.0) 
  
XXX 
XXX 
XXX 
 
  
  
  
  
  
  
  
  
Rates of hospitalization (post-delivery) 
XX 
XX 
XX 
XX 
XX 
XX 
  
  
 
  
  
  
  
Rates of transfusions (post-delivery) 
XXX 
XXX 
XXX 
XXX 
XXX 
XXX 
 
 
 
 
 
 
 
X = offspring 
 
  
RAPIDIRON 
 
 
XX = offspring and mother 
 
  
RAPIDIRON-KIDS 
 
 
XXX = mother only 
 
  
 
 
 
*Note: The RAPIDIRON study will provide much of the information for the first two visits (as made clear by the different color boxes and 
key). Cord blood is the source of iron status for Visit 1.  
 
**See footnote on page 14. 
 
As previously noted, if a participating mother experiences an intercurrent pregnancy within 36 months post-delivery, a blood sample will 
be collected and analyzed to obtain a complete set of iron indices. 
 
 
 

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Domains and Timing of Assessments 
Primary Outcomes, Related Hypotheses, and Rationale: 
(1) Iron status as indicated by hemoglobin and ferritin at birth and at 4 months of age 
Hypothesis: Infants born to RAPIDIRON mothers from the IV iron arms will have higher hemoglobin 
and ferritin levels at birth and at 4 months of age compared to infants born to mothers in the oral iron 
arm. 
 
Rationale: Hemoglobin concentration is used to assess the presence of anemia and to calculate 
iron in the red cell mass. Serum ferritin concentration primarily assesses iron stores and is 
important for calculating total body iron along with other parameters. The sample obtained at birth 
– using cord blood – directly assesses fetal loading, which is the direct goal of maternal iron 
therapy. Iron status at 4 months allows assessment of whether fetal iron loading results in longer 
lasting improvements in hematologic and iron status during a period of rapid neurodevelopment. 
The timing of assessments is also based on prior studies that show improved hemoglobin 2-4 
months after birth. However, assessing iron status at 4 months of age has been recommended by 
many experts, including a noted neonatologist-researcher who has expertise related to the impact 
of iron deficiency on young children and serves as a Co-Investigator for both the parent 
RAPIDIRON Trial and this follow-up study. He noted that the World Health Organization 
recommends exclusive breastfeeding with no supplemental foods until an infant reaches six 
months of age. However, infants born at term who have delayed cord clamping, are breastfed 
and not supplemented with iron will start to “run out” of fetal iron stores beginning at four months 
of age, which puts their brain at risk for the effects of iron deficiency. But, if oral supplementation 
of an infant begins at about four months to address the deficiency, it can result in a shift toward a 
more pathogenic microbiome. Babies are relatively immunocompromised and such a shift can 
place them at greater risk for diarrheal illness earlier in life when they are less able to tolerate 
infection. If the above RAPIDIRON hypothesis is true, babies born to mothers in the IV iron arms 
will not have a need for postnatal oral iron therapy as early in life as the infants whose mothers 
received prenatal oral iron. The infants with mothers who received IV iron are expected to be 
loaded with prenatal IV iron, to be born with greater iron stores, and to have greater residual total 
body iron, as indexed by hemoglobin and ferritin concentrations, as well as greater storage iron, 
as indexed by ferritin concentration at birth and 4 months of age.  
 
(2) Developmental quotient (DQ) on the cognitive domain of the Bayley Scales of Infant Development 
(BSID) at 2 years of age (24 months) 
Hypothesis: Offspring born to RAPIDIRON participants who received IV iron treatment will have 
higher developmental quotients (DQs) on the cognitive domain of the Bayley Scales of Infant 
Development (BSID) at 2 years of age compared to offspring born to RAPIDIRON mothers given oral 
iron treatment. 
 
Rationale: Bayley Scales of Infant Development (BSID) is a validated tool for assessment of 
general developmental abilities routinely used in large-scale randomized controlled trials (RCTs) 
to detect deficits in the motor, language, and cognitive domains. The BSID will be applied at 24 
months as this is considered the optimal time for testing. The test scores are age normed to a 
median of 100 (similar to an IQ test) and components are referred to as developmental quotients 
(DQs). Iron deficiency anemia has been shown to reduce DQ at one year and beyond. 
 
Secondary Outcomes for the Offspring: 
Content experts that suggested the timing of assessment of iron status in infants also provided 
information utilized for determining the schedule associated with assessing secondary outcomes. From a 
neurodevelopmental perspective, four months is the first age at which reliable assessments of the 
integrity of the cortex are possible. Cortical maturation is critical for motor, cognitive, and social well-
being.  

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Prior to four months, infants’ behaviors are primarily subcortical and reflexic (as characterized by the 
presence of primitive reflexes). At four months, cortical control is evidenced by controlled motor 
movements, social smiling, and novel visual object discrimination memory. Based on the RAPIDIRON 
hypotheses, infants loaded with prenatal IV iron will be neurologically more mature with faster speed of 
processing and better discrimination memory performance at four months than infants whose mothers 
received prenatal oral iron.  
Neurobehavioral Development 
A secondary hypothesis of this study is that infants born to women randomized to the IV iron arms of the 
parent RAPIDIRON Trial will have superior neurobehavioral development compared to infants born to 
women randomized to the oral iron arm. To test this secondary hypothesis, standard generalized tests of 
development and brain region domain-specific tests that are expected to be influenced by iron status will 
be administered at 4, 12, 24, and 36 months of age. 
The tests and ages applied include: 
 
Preferential Looking Time – a behavioral test that assesses recognition learning and memory. 
This is a test of the integrity of the hippocampus brain region and will be applied when the child is 
4 and 12 months of age. The test leverages the brain development principle of novelty 
preference. The hippocampus is rapidly developing in the last trimester through the first 18 
postnatal months. Iron deficiency reduces hippocampal capacity. Infants are presented with an 
object (or picture of an object) and are expected to familiarize themselves with it. After a time 
delay, the familiar object is presented along with a novel object. The infant is expected to spend 
more time (>60% of total looking time) observing the novel object. 
o 
Hypothesis: Infants born to mothers randomized to IV iron arms will spend more time 
looking at the novel object compared to those in the oral iron arm. 
 
Ages & Stages Questionnaire®, 3rd Edition (ASQ-3) – a questionnaire designed to obtain parental 
input regarding a child’s general developmental milestones which can be used for children from 
one month to five and a half years of age. Mothers will be expected to provide answers for the 
age-appropriate questionnaire that will be used when the child is 12 months of age.  
o 
Hypothesis: Infants born to mothers randomized to IV iron arms will have more advanced 
developmental milestones compared to those in the oral iron arm. The age-equivalent 
stage will be positively correlated with iron status at birth. 
 
Bayley Scales of Infant Development Version 4 (BSID)– this is a validated assessment of general 
developmental abilities which is routinely used in large-scale randomized controlled trials to 
detect deficits in the motor, language, and cognitive domains. The BSID will be administered at 
24 and 36 months. The test scores are age normed to a median of 100 (like an IQ test), and 
components are referred to as developmental quotients (DQs). IDA has been shown to reduce 
DQs at one year of age. 
o 
Hypothesis: Infants born to mothers randomized to IV iron arms will have higher DQs on 
the subscales of these tests at 24 and 36 months compared to those in the oral iron arm.  
 
Behavioral Rating Scale – this was devised by the pioneer iron researcher Betsy Lozoff. [12] It is 
coded from a video recording of a child undergoing the BSID and it assesses engagement, 
tractability, exploration, hesitancy, and activity level. All domains that are dopamine-dependent 
and reported by Lozoff and others as perturbed by iron deficiency effects on dopamine 
metabolism will be administered during visits to children at 24 months of age.  
o 
Hypothesis: Infants born to mothers randomized to IV iron arms will have higher scores 
for exploration, tractability, and engagement, and less evidence of hesitancy and 
hyperactivity at 24 months of age compared to children in the oral iron arm when 
assessed at the same age. 
 
Child Behavior Checklist for Ages 1.5 to 5 years (CBCL/1.5-5) – this test will be administered to 
the parent/caretaker who spends the most time with the child and requires 20-30 minutes for 
completion. Parents rate items on a three-point Likert scale assessing internalizing and 

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17 
 
externalizing behaviors. Total scores, t-scores, and percentiles are available for each scale. A 
recent study reported that children with iron deficiency at birth had higher internalizing behavior 
problem scores at 5 years compared to those born iron sufficient {median (IQR) 9.0 (5.3, 12.0) 
compared with 5.0 (3.0, 10.0), p = 0.006; [adjusted estimate (95% CI): 2.8 (0.5, 5.1), p = 0.015] 
and total [24.5 (15.3, 40.8) compared with 1.0 (10.0, 30.0), p = 0.009; adjusted estimate (95% 
CI): 6.6 (0.1, 13.1), p = 0.047]}. [13] This will be administered at 24 and 36 months of age. 
 
India Scale of Assessment for Autism – this test is similar to the standard screening tool for 
autism (the Modified Checklist for Autism in Toddlers) and has been validated for the Indian 
population. Iron deficiency during early pregnancy is associated with a higher risk of autism in the 
offspring. This test will be applied at the 36 month visit. 
o 
Hypothesis: Infants born to mothers randomized to IV iron arms will have a lower risk of 
autism diagnosis compared to those in the oral iron arm. The risk will be related to lower 
initial maternal iron status at entry into the study and randomization to the oral iron arm. 
Iron Status at 12 months 
A secondary hypothesis of this study is that children born to women randomized to the two IV iron arms of 
the parent RAPIDIRON Trial will have better iron status at 12 months compared to children born to 
women randomized to the oral iron arm. To evaluate this hypothesis, a number of standard hematologic 
and iron indices will be assessed in all subjects. The trajectory of differences in iron status in children will 
be graphically presented from birth through 12 months. 
The biomarkers include: 
 
Hemoglobin concentration to assess presence of anemia and to calculate iron in the red cell 
mass; and 
 
Reticulocyte hemoglobin content to assess iron dependent erythropoiesis. 
The timing of the visits for iron assessments are: 
 
Birth – the sample to determine % TIBS will be obtained from cord blood (which will be available 
from the parent RAPIDIRON Trial). Assessment of indices related to fetal iron loading will also 
occur at birth since 88% of total body iron can be calculated from the cord blood hemoglobin and 
cord serum ferritin 
o 
Hypothesis: Infants born to mothers randomized to IV iron arms will have higher total 
body iron than infants born to mothers randomized to the oral iron arm. 
 
4 months – this is a critical time for an initial blood draw of infants to determine iron status 
 
12 months – this sample time is consistent with standard of care recommendations for infants 
from the World Health Organization and the American Academy of Pediatrics and coincides with 
the first global neurodevelopmental testing scheduled to occur during the study. 
o 
Hypothesis: The incidence of iron deficiency (ID) or iron deficiency anemia (IDA) will be 
lower in the infants born to mothers randomized to receive an IV iron infusion compared 
to mothers that receive oral iron. 
Following infant development through 12 months of age will provide critical data regarding associations of 
early iron deficiency with autism and schizophrenia. It will also enable a better understanding of growth 
and hemoglobin trajectories related to overall neurocognitive development. 
While there is a paucity of data on the longer-term impact of iron supplementation (especially the use of 
IV iron) given to women in pregnancy, this study will enable us to describe correlations between women 
recruited in the 3 arms of the RAPIDIRON Trial as well as their hematologic, clinical, and overall health 
impact. These results will be additive to the critical primary outcome data generated on the offspring of 
these women. 

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General Growth and Nutrition 
A secondary hypothesis of the RAPIDIRON Trial is that IV iron treatment early in pregnancy will reduce 
iron-deficiency-associated low birth weight (LBW) primarily due to premature delivery or intrauterine 
growth restriction. Both are risks for low iron stores at birth and poor postnatal growth in the offspring. 
Offspring born to mothers in the oral iron group, compared to the IV iron group, are more likely to be iron 
deficient during their fetal life, at birth, and during infancy. Prolonged iron deficiency can negatively impact 
linear growth. Chronic iron deficiency during the rapid phase of growth in infancy and early childhood 
results in vulnerability for growth impairment. Iron plays an important role in tissue oxygen delivery and 
transport of electrons within the cells, and iron acts as a co-factor for essential enzymatic reactions. Iron 
deficiency anemia likely delays growth by creating a hypoxemic condition and inhibiting insulin-like growth 
factor-I (IGF-1). [14, 15] Hypoxemia due to iron deficiency anemia can also lead to an increase in hypoxia-
induced growth factor (HIF-1α), which in utero has been shown to decrease human growth hormone 
mRNA levels. [16] In infants and young children with iron deficieny anemia, iron therapy increased serum 
IGF-1 levels and growth velocity. [17] Several other studies have reported lower body weight, height, 
head circumference, and growth velocity in anemic children and improvement in growth parameters with 
iron therapy. [18, 19, 20] 
The following general growth and nutrition assessments will be performed: 
 
Standard anthropometrics – weight, length (height), and head circumference will be measured at 
each of the six timepoints and plotted on the WHO standard pediatric growth curves. From these 
data, weight for age, length for age, and head circumference for age Z-scores will be generated 
and growth velocities will be calculated. 
 
Nutritional survey – the Infant and Child Feeding Index (ICFI) is a composite index used to 
measure feeding practices, which has been previously used in the Indian context and allocates 
scores based upon breastfeeding, dietary diversity, meal frequency, etc. [21] Data regarding iron-
supplementation and iron-fortified foods will also be collected. This survey will be conducted at 
the birth, 6 week, 4 month, 12 month, and 24 month visits.  
Maternal Secondary Outcomes 
While the RAPIDIRON-KIDS Study represents an opportunity to understand the impact of two different 
approaches for the treatment of IDA during pregnancy on offspring, it also enables continued contact with 
a sample of RAPIDIRON participants. The interaction with these mothers during this follow-up study is an 
important means of appreciating the longer-term effects of the treatments used during the parent trial. As 
indicated in Table 1, the maternal secondary outcomes to be explored include the following: iron status 
when the offspring is 4, 12, and 24 months old; breastfeeding practices; quality of life, as measured by 
the World Health Organization Disability Assessment Schedule 2.0 (WHODAS 2.0); and rates of 
hospitalization and transfusion subsequent to the conclusion of the RAPIDIRON Trial.  
 
E. Statistical Methods 
Sample Size 
We anticipate that at least 2,000 pregnant women will be recruited for the primary RAPIDIRON Trial 
across the three sites in Karnataka state and project that at least 600 complete data sets will be available 
for each of the three arms for analysis. Thus, an adequate number of infants per arm should be eligible 
for enrollment in the RAPIDIRON-KIDS Study. However, as demonstrated in the summary of the 
preliminary power analyses below, the aims of the RAPIDIRON-KIDS Study can be achieved by obtaining 
complete data sets for approximately 147 mother-offspring dyads for each of the three study arms (or a 
total of 441 dyads). Since RAPIDIRON Trial participants will be asked to consent to this follow-up study 
prior to delivery, we expect that some of them will experience a stillbirth or neonatal death and, thus, not 

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provide evaluable information on the infants. In addition, we expect that cord blood data will not be 
obtainable or analyzable for a certain percentage of live births resulting in missing data on two of the 
primary outcomes.  To account for this, we plan on initially consenting approximately 525 RAPIDIRON 
Trial participants (~175 per arm). Because Aims 1 and 2 answer different essential research questions, 
the study is powered with a type I error rate of 0.05 for each of those aims. 
We propose to conduct a total of four primary hypothesis tests for Aim 1. Two of those tests will be 
conducted on data collected at birth with separate tests comparing offspring born to mothers across both 
IV iron arms to those born to mothers in the oral arm with respect to hemoglobin level and log-
transformed ferritin levels at birth. If either of those tests at birth show differences between the two arms, 
the comparable tests will be conducted at 4 months post-delivery. In all cases, the null hypothesis is that 
the mean across the IV arms is the same as that in the oral iron arm. To control the type I error rate 
across these two tests, the alpha level for each individual comparison will be 0.025. Although information 
on the likely effect sizes for the proposed intervention are insufficient to provide explicit estimates of 
power for this study, limited comparisons of infant hemoglobin levels in south Asian and Africa suggest 
that the differences in both hemoglobin levels and log ferritin levels in infants born to mothers with and 
without IDA differ by at least 0.8 standard deviations. Because we want to assure that this intervention is 
powered to detect effects somewhat smaller than that observed in mothers with and without IDA, we want 
to assure that this study has sufficient power to detect an effect that is in the range of 0.3 to 0.4 standard 
deviations. We anticipate that approximately 85% of the enrolled sample will provide data for our primary 
iron outcomes at birth. Thus, we estimate a sample size of at least 441 infants (147 per arm) will provide 
information at birth with a slightly higher number at 4 months as this time is not subject to difficulties with 
obtaining data from the cord blood at delivery. When evaluated in the context of the proposed mixed 
effects model where we assume an intraclass correlation coefficient of 0.5, a sample size of 147 per arm 
(294 IV iron vs. 147 oral) gives more than 80% power to detect a difference in means at birth of 0.31 
standard deviations at the alpha=0.025 level. Since demonstrating the effects at 4 months requires that 
the hypothesis tests at birth reject the null hypothesis, the overall study power to detect a difference of 0.3 
standard deviations or greater at 4 months is greater than 80%. 
The primary outcome for Aim 2 is the Bayley Developmental Quotient (DQ) measured at 2 years of age. 
The average score across the two IV iron arms will be compared to the oral arm at the alpha=0.05 level. 
We expect to obtain data for approximately 85% of consented participants at this time point for a sample of 
at least 147 per arm. This sample size gives more than 90% power to detect a difference in means of 5 
units on the BSID mean score assuming a standard deviation of 15 at the alpha=0.05 level. Because effect 
sizes in the range of 5 to 7.5 are generally clinically meaningful for the BSID, the study is well powered to 
detect clinically meaningful differences. 
Primary Data Analysis 
Statistical analyses involve both formal hypotheses testing for specified primary outcome measures and 
supportive descriptive analyses of secondary outcome measures. 
Hemoglobin and log transformed ferritin will be separately modeled using mixed effects linear regression. 
Both outcomes will be measured at birth and 4 months, and hemoglobin will also be measured at 12 months. 
The mixed effects models will treat time as a categorical value and include fixed effects for time, treatment, 
and time by treatment interaction. Correlation among repeated measurements for each subject will be 
modeled through structuring the variance-covariance matrix of the residuals. Inclusion of the time by 
treatment interaction will allow for estimation of mean iron indices at each measurement time for each 
group. Within these models, the primary hypothesis tests for aim 1 will be performed by comparing the 
average across the two IV iron groups to the oral iron group with respect to the mean values at birth and at 
4 months using a significance level of 0.025. 

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A similar mixed-effects model will be used for modeling Bayley DQ cognitive domain in participating children 
at 2 and 3 years of age. The primary test for aim 2 will be performed by comparing the average across the 
two IV arms to the oral iron arm at 2 years using a significance level of 0.05. 
Secondary Analyses 
Secondary analyses will examine the effect of the three treatment regimens on each of the secondary 
outcome measures including iron measures (hemoglobin, ferritin, reticulocyte hemoglobin content and % 
total iron binding saturation), neurodevelopmental measures (total score and scores for individual domains 
such as communication, fine motor, gross motor, problem solving ability, and personal-social functioning), 
preferential looking time (log transformed), scores for the four factors associated with the Behavioral Rating 
Scale (object orientation, motor quality, negative affect, and positive social responsiveness), T-scores for 
the CBCL/1.5-5 syndrome scales (Emotionally Reactive, Anxious/Depressed, Somatic Complaints, 
Withdrawn, Attention Problems, Aggressive Behavior, and Sleep Problems), the India Scale of Assessment 
for Autism, and the standard growth metrics (weight for age, length for age, and head circumference for 
age Z-scores). As noted previously, data from a nutritional survey will be collected; these data will only be 
included in analyses as potential effect modifiers or moderators, not as outcome measures. Maternal 
outcomes that include hematologic indices, a history of transfusion and/or hospitalization, and assessment 
of life quality are incorporated into this study as secondary analyses.  
Each of the outcome measures outlined above are continuous, and most are collected longitudinally on 
each of the infants enrolled in this follow-up study. Outcomes measured at only one time will be analyzed 
using two-sample t-tests.  The secondary analyses based on these outcome measures will generally 
examine the trajectories over time on each of these measures using linear mixed models. A first set of 
models will be designed to describe how the trajectory for each outcome differs as a function of treatment 
arm and will include the outcome measure (or an appropriate log transformation) as the independent 
variable, fixed effects of treatment, time, and the treatment by time interaction, with infant as a random 
effect. Analogous models will be generated for the secondary analysis of maternal iron levels with the 
inclusion of the woman as a random effect. Model results will be used to generate point and interval 
estimates of treatment-specific mean levels of the outcome measures at each developmental time. For 
each of these outcome measures, there will be an omnibus hypothesis test that assesses evidence for a 
difference in outcome by treatment at any time point with step-down tests of time-specific differences in 
treatment. All hypothesis tests are considered to be descriptive with p-values used as indicators of strength 
of evidence of treatment differences with no plans for formal statistical inference for the secondary analyses. 
As such, no control for multiple comparisons is planned. 
A second set of descriptive secondary analyses will examine the relationship between each of the 
neurodevelopmental outcomes and growth parameters and infant iron status at delivery measured as 
delivery total body iron. These analyses will utilize linear mixed models that evaluate the regression 
relationship between baseline iron levels and outcome measures over time, controlling for treatment arm. 
These models will also evaluate whether the relationship between baseline iron and neurodevelopmental 
outcomes are mediated by specific post-delivery nutritional measures.  
Interim Analysis 
An analysis of maternal hematologic indices will be completed when all women have passed the 12 
month collection period.  This analysis will include all available maternal blood data through 12 months.  
Based on the results of this analysis, the research team may decide to suspend collection of maternal 
blood at 24 months.  Other extensive interim analysis is not planned. However, typical study monitoring 
activities could trigger such an analysis for one or more of the following reasons: recruitment proves 
problematic, making it difficult to perform an adequate number of tests and obtain measurements at 
desired time points; study withdrawals, investigator-recommended discontinuations, or ‘loss-to-follow-up’ 
among children enrolled in the study are significant; and/or there is a poor ratio for data obtained vs. data 
expected for important study variables. If undertaken, the interim analysis would consider if continuation 

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of the study, despite existence of challenges such as those mentioned, could nevertheless provide 
enough valid evidence to judge whether the primary study hypotheses remain valid. If the answer is no, 
the study could be prematurely stopped. 
Safety Analyses 
All safety analyses will be descriptive based on the safety cohort with no planned formal inference for 
safety. All adverse events will be summarized by system organ class by arm with point and interval 
estimates of the risk of each of these events estimated by either exact binomial methods or normal 
approximations to the binomial as appropriate for the incidence of the event. 
Descriptive statistics will also be generated for maternal and infant mortality and for incidence of serious 
adverse events (SAEs). 
Analysis Cohorts 
Intent-to-treat (ITT) cohort: study participants (mothers or infants, depending on the particular analysis) 
will be included using group assignments for the mother as randomized. 
Delivery iron status cohort: Analyses that examine outcomes as a function of delivery iron status will 
include enrolled maternal participants with sufficient delivery data to determine iron status of the infant at 
delivery. 
Assignment of participants into the various cohorts will be determined based on a masked data review 
prior to initiation of analysis. 
F. Gender/Minority/Pediatric Inclusion for Research 
The parent study, the RAPIDIRON Trial, was initiated in mid-March 2021 in two states of India; thus, its 
participants are classified as adult, female Asians and their offspring will likewise be considered Asian. 
Pregnant women utilizing facilities classified in the Indian public health system as primary or community 
health centers will have the opportunity to participate if they meet specified eligibility criteria. No pregnant 
woman will be excluded because of socioeconomic status or other personal characteristic that could be 
considered to qualify such women as minorities. Similarly, the offspring for whom consent will be sought – 
from the RAPIDIRON Trial participant – will not be excluded due to minority or family economic status. 
Additionally, there will be no gender bias in the inclusion of the participants for this follow-up study. By 
definition, newborn infant participants are children (thus, there is pediatric inclusion).  
G. Human Subjects 
1. Study Population 
Participants of this study are mother-infant dyads consisting of women, residing in Karnataka state, who 
were randomized and treated for IDA during the RAPIDIRON Trial and their newborns. 
Inclusion Criteria: 
 
Informed consent of the RAPIDIRON Trial participant for their study inclusion and that of their 
offspring for the RAPIDIRON-KIDS Study 
 
Expressed intent of the RAPIDIRON Trial participant to remain in the designated Karnataka 
research area for delivery and during the follow-up period for the RAPIDIRON-KIDS Study to 
enable participation in study visits 
 
For the offspring – live-born singleton infants of Karnataka maternal participants randomized and 
treated in the RAPIDIRON Trial, if consent is provided by the mother 

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Exclusion Criteria: 
 
Unwillingness of maternal participant to provide RAPIDIRON-KIDS Study consent for herself and her 
offspring 
2. Sources of Research Material 
Results of blood tests will be a source of data for this research. The RAPIDIRON Trial will provide the 
hemoglobin (Hb) for the last blood sample taken for a participating woman prior to delivery, as well as iron 
indices at 6 weeks postpartum. The RAPIDIRON Trial protocol also calls for the collection of cord blood 
subsequent to delivery to assess Hb, transferrin saturation, ferritin, and reticulocyte indices of the 
newborns. For the purposes of the RAPIDIRON-KIDS Study, blood samples of approximately half a 
teaspoon of blood (2-3 mL) will be collected from the mother at the 4, 12, and 24 month study visits, and 
0.5 – 0.8 mL collected via heel stick from the offspring at the 4 and 12 month study visits to determine 
hemoglobin and iron indices.  
The visits for the RAPIDIRON-KIDS Study also include performing assessments as listed in Table 1 
(some of which overlap with the parent study). As part of the RAPIDIRON Trial, infant weight and length 
will be collected at birth, and maternal reports of labor and delivery practices and complications will be 
recorded. For the RAPIDIRON-KIDS Study, we will collect child head circumference at birth and 
administer the ICFI to the mother/parent to collect information regarding breastfeeding practices, general 
nutritional intake, and iron supplementation. Additionally, we will collect child height and head 
circumference at 6 weeks, 4, 12, 24, and 36 months of age. We will administer the ICFI at 6 weeks, 4, 12, 
and 24 months of age. We will also collect child weight at 4, 12, 24, and 36 months of age (as it will be 
collected via the RAPIDIRON Trial at the 6-week visit). The World Health Organization Disability 
Assessment Schedule 2.0 (WHODAS 2.0) will be utilized to assess the mother’s quality of life when the 
offspring has reached 6 weeks (as part of the RAPIDIRON Trial), 4 months, and 12 months of age. Data 
will also be collected to monitor rates of transfusion and hospitalization among the maternal participants 
through the end of the study. 
As part of the RAPIDIRON-KIDS Study, we will conduct a variety of neurobehavioral assessments with 
the offspring using validated tools, as follows: 
 
The Bayley Scales of Infant Development (BSID) is a validated tool for assessment of general 
developmental abilities which is routinely used in large-scale randomized controlled trials (RCTs) 
to detect deficits in the motor, language, and cognitive domains. This will be conducted at the 24 
and 36 month visit.  
 
The Ages & Stages Questionnaire, 3rd Edition (ASQ-3) is designed to obtain parental input 
regarding a child’s general developmental milestones. For the ASQ-3, mothers provide answers 
for the questionnaires, and this will be conducted at the 12 month visit.  
 
The Preferential Looking Time test assess recognition learning and memory by presenting an 
infant with an object (or a picture of an object) which they are expected to familiarize themselves 
with. After a time delay, the familiar object is presented along with a novel object, and the time 
spent observing each object will be measured (with the expectation that they will spend more time 
observing the novel object to demonstrate recognition memory). This test will be conducted at the 
4 and 12 month visits.  
 
The Behavioral Rating Scale is coded from a video recording of the offspring undergoing the 
BSID and assesses engagement, tractability, exploration, hesitancy, and activity level. At the 24-
month visit, the child will be recorded undergoing the BSID. Trained staff will code the recordings, 
with a random sub-sample sent to the Masonic Institute of the Developing Brain to be coded 
again for quality assurance. 
 
The Child Behavior Checklist for Ages 1.5 to 5 years (CBCL/1.5-5) is completed by the 
parent/caretaker who spends the most time with the child, and involves rating items on a three-

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point Likert scale assessing internalizing and externalizing behaviors. This requires 10-20 
minutes for completion and will be conducted at the 24 and 36 month visits.  
 
The India Scale of Assessment for Autism is similar to the standard screening tool for autism 
(Modified Checklist for Autism in Toddlers) and has been validated for the Indian population. This 
will be conducted at the 36 month visit.  
Personnel will be trained prior to initiating assessments and will be required to achieve competence in 
scoring performance, recording results, and completing and submitting data forms for entry into the data 
management system. Arrangements will be made for the use of suitable space for assessments and 
quality monitoring will ensure appropriate conduct of the research. Specific data forms will be used for 
recording data for participating mothers and offspring, and these forms will be available at locations 
where needed.  
3. Recruitment and Consent Procedures 
Community sensitization occurred in communities served by the primary and community health centers 
participating in the RAPIDIRON Trial and additional sensitization will occur in the Karnataka research 
area to promote awareness of plans for implementation of the RAPIDIRON-KIDS study. Sensitization 
generally consists of research staff holding meetings with community leaders, health officials, and other 
relevant parties. Accredited Social Health Activities (ASHAs) who live within the communities that 
constitute the research are may be asked to participate in the study (with authorization of the supervising 
health official), for example, by accompanying a mother and child to a center of engagement in the 
screening and consent process, or by ensuring that a participating child and mother keep an appointment 
for a blood draw or for developmental testing. ASHAs receive incentive-based fees for their work 
associated with government-defined responsibilities which include transporting mothers and children to 
health facilities for needed care. In prior situations when approval has been granted for their involvement 
in research designed to improve women’s and children’s health, their assistance has been a key factor in 
the success of the research. 
ASHAs who have received education relevant to anemia and iron deficiency can be an additional source 
of information about this study. However, consent will be obtained by a trained facility health care provider 
or research staff member who is a physician or other certified health care provider having adequate 
health-related training to provide information about the nature and purpose of the study and is able to 
answer questions during the consent process.  
Current RAPIDIRON participants will be given information about the study and an opportunity to consent 
as early as the 6th RAPIDIRON visit (26-30 weeks of pregnancy) and will be able to provide consent up 
until delivery, as long as they are not yet in active labor.  
4. Description of Risks, Assessment of Likelihood and Seriousness 
This RAPIDIRON-KIDS Study involves no intervention, and thus the risks associated with study 
participation can be considered minimal but include the possibility that drawing blood for analysis could 
cause bruising and, rarely, infection. No serious risks are associated with the various assessments 
indicated in Table 1, although some discomfort or anxiety on the part of the child or a mother cannot be 
ruled out but can often be ameliorated by individuals appropriately trained to conduct assessments. 
5. Procedures for Protecting Against or Minimizing Potential Risks 
Care will be taken to reduce the risk of bruising. Sterile technique will be utilized during blood draw to 
reduce risk of infection. Personnel conducting sociodemographic, neurodevelopmental, and other 
assessments will have adequate background and relevant training to ensure competency.  

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6. Potential Benefits, Importance to Participants and Others 
The possible benefits to participants may be earlier diagnosis of iron deficiency due to a screening 
schedule which encompasses more monitoring opportunities than is associated with usual practice. Both 
mothers and infants whose hemoglobin values fall below a pre-designated cutoff will be appropriately 
referred as per standard of care. Further, if neurodevelopmental testing indicate possible disorder, 
parents will be told about resources for confirming a diagnosis and for seeking supportive services (if they 
exist locally).   
In combination with the parent study, the RAPIDIRON Trial, it is hoped that this follow-up study will result 
in the expanded use of a more effective approach for the treatment of IDA and decrease the risk that 
offspring will suffer the consequences of an ineffective IDA treatment approach. Further, if the IV iron 
treatment approach is determined feasible and adopted widely as a frontline treatment for IDA in 
pregnancy, the number of future beneficiaries of these studies can be multiplied.  
7. Discussion of Why Risks are Reasonable in Relation to Benefits 
The RAPIDIRON-KIDS Study can be considered an observational study with minimal risk. However, if the 
study results in earlier diagnosis and earlier treatment of iron deficiency, and delivery of services to 
address developmental disorders, then participating children and mothers may receive greater benefit 
than would be the case if such problems were recognized later in life. 
H. Data and Safety Monitoring Plan 
1. Data and Safety Monitoring Plan (DSMP) 
For the RAPIDIRON-KIDS Study, a serious adverse event (SAE) involves mothers (and their infants) who 
were treated for iron deficiency anemia as participants in the RAPIDIRON Trial and who provide consent 
for themselves and their offspring to participate in the RAPIDIRON-KIDS Study. To qualify as an SAE, the 
event must fulfill at least one of the criteria below: 
 
Results in a maternal or neonatal death, or fetal death >20 weeks gestation; 
 
Is life-threatening; 
 
Requires an unanticipated hospitalization or prolongs an existing hospitalization 
 
Results in persistent or significant disability or incapacity; or 
 
Represents other serious or unexpected adverse events that a study investigator feels should be 
reported, including need for resuscitation. 
Specific forms will be utilized to report an SAE. For an SAE, the reporting form should be sent to the 
person designated to receive it within 7 days of the notification of the event. Reportable adverse and 
serious adverse events will be shared with the DSMB. If there is an unanticipated problem recognized as 
posing a risk to participants or others and it is likely to reoccur, a designated researcher should obtain 
additional information on the unanticipated event and compile a report to forward to the study Principal 
Investigator, Chief Quality and Safety Officer, and the Chair of the DSMB. The Chair will review the report 
and determine significance of the event to the study and the need for action to avoid harm to participants. 
The Chair may put the report on the agenda of the next DSMB meeting or communicate with members to 
address the unanticipated problem sooner. 
At each scheduled DSMB meeting after completion of the primary randomized trial, SAE data and a 
report summarizing retention and participant status will be prepared for review and discussion. Following 
its deliberations, the DSMB will issue any recommendations it may have and specify that the study should 
continue based upon absence of safety concerns or concerns about data quality or excessive loss-to-
follow-up. 

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2. Data and Safety Monitoring Board (DSMB) 
It is envisioned that the independent data and safety monitoring for this RAPIDIRON-KIDS Study will be 
provided by the same Data and Safety Monitoring Board (DSMB) that oversees the primary randomized 
controlled RAPIDIRON Trial. The Board Chair has expertise in high-risk obstetrics and global health 
research. The other members have expertise that spans obstetrics and gynecology, 
neonatology/pediatrics, biostatistics, clinical trials design and implementation, and clinical management of 
anemia. All members of the DSMB are free of significant conflicts of interest (i.e., financial, intellectual, 
professional, or regulatory) to enable performance of their duties in an unbiased manner. 
The duties to be performed by the DSMB specific to this study include: 
 
Review all serious adverse events (SAEs) and unanticipated problems that infants and mothers 
incur to determine if they pose ongoing risk for continuation in the RAPIDIRON-KIDS Study; 
 
Assure that the RAPIDIRON-KIDS Study activities are conducted in a manner to minimize risk to 
infants and mothers and assess maternal and infant burden of participation to confirm that the 
study is conducted in a safe and ethical manner; 
 
Assess data quality and participant retention to assure that the benefit of study continuation is 
consistent with participant burden; and 
 
Prepare reports that outline DSMB recommendations and provide them to the study Principal 
Investigator, the Senior Foreign Investigator, and the Chief Quality and Safety Officer. 
After data from the primary RCT have been collected for outcomes through 42 days post-delivery (which 
constitutes completion of the clinical component of the RAPIDIRON Trial), the DSMB will continue to meet 
approximately annually to review the data from this follow-up study. The DSMB may revise the annual 
meeting schedule if the workload justifies. If a scheduled meeting is considered too remote to review an 
SAE and determine relevant action, information may be distributed to DSMB members via email and input 
sent to the DSMB Chair who can communicate a recommendation representing consensus among DSMB 
members to senior key personnel of the study. The Chief Quality and Safety Officer will be the 
intermediary in supplying appropriate data to the DSMB for review. 
 
 

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I. 
Literature Cited 
 
[1]  International Institute for Population Sciences (IIPS), "National Family Health Survey-5, 2019-21, 
India Fact Sheet," [Online]. Available: http://rchiips.org/nfhs/NFHS-5_FCTS/India.pdf. [Accessed 8 
February 2022]. 
[2]  R. J. Derman, S. S. Goudar, S. Thind, S. Bhandari, Z. Aghai, M. Auerbach, R. Boelig, U. S. 
Charantimath, R. Frasso, M. S. Ganachari, K. L. Gaur, M. K. Georgieff, F. Jaeger, S. Yogeshkumar 
and La, "RAPIDIRON: Reducing Anaemia in Pregnancy in India - a 3-arm, randomized-controlled 
trial comparing the effectiveness of oral iron with single-dose intravenous iron in the treatment of 
iron deficiency anaemia in pregnant women and reducing low birth weight," Trials, vol. 22, p. 649, 
2021.  
[3]  World Health Organization, "Micronutrients," [Online]. Available: https://www.who.int/health-
topics/micronutrients#tab=tab_1. [Accessed 8 February 2022]. 
[4]  G. Zhao, G. Xu, M. Zhou, Y. Jiang, B. Richards, K. M. Clark, N. Kaciroti, M. K. Georgieff, Z. Zhang, 
T. Tardif, M. Li and B. Lozoff, "Prenatal iron supplementation reduces maternal anemia, iron 
deficiency, and iron deficiency anemia in a randomized clinical trial in rural China, but iron deficiency 
remains widespread in mothers and neonates," Journal of Nutrition, vol. 145, no. 8, pp. 1916-1923, 
2015.  
[5]  B. Lozoff, E. Jimenez and J. B. Smith, "Double burden of iron deficiency in infancy and low 
socioeconomic status: a longitudinal analysis of cognitive test scores to age 19 years," Archives of 
Pediatrics & Adolescent Medicine, vol. 160, no. 11, pp. 1108-13, 2006.  
[6]  M. K. Georgieff, "Iron deficiency in pregnancy," American Journal of Obstetrics and Gynecology, vol. 
223, no. 4, pp. 516-524, October 2020.  
[7]  L. Reveiz, G. M. Gyte, L. G. Cuervo and A. Casabuenas, "Treatments for iron-deficiency anaemia in 
pregnancy," Cochrane Database of Systematic Reviews, vol. 10, no. CD003094, 5 October 2011.  
[8]  R. J. Derman, B. S. Kodkany, S. S. Goudar, S. E. Geller, V. A. Naik, M. B. Bellad, S. S. Patted, A. 
Patel, S. A. Edlavitch, T. Hartwell, H. Chakraborty and N. Moss, "Oral misoprostol in preventing 
postpartum haemorrhage in resource-poor communities: a randomised controlled trial," Lancet, vol. 
368, no. 9543, pp. 1248-53, 7 October 2006.  
[9]  M. K. Georgieff, P. V. Tran and E. S. Carlson, "Atypical fetal development: Fetal alcohol syndrome, 
nutritional deprivation, teratogens and risk for neurodevelopmental disorders and psychopathology," 
Developmental Psychopathology, vol. 30, no. 3, pp. 1063-1086, August 2018.  
[10] P. V. Tran, S. J. Fretham, E. S. Carlson and M. K. Georgieff, "Long-term reduction of hippocampal 
brain-derived neurotrophic factor activity after fetal-neonatal iron deficiency in adult rats," Pediatric 
Research, vol. 65, no. 5, pp. 493-8, May 2009.  
[11] M. Auerbach, S. E. James, M. Nicoletti, S. Lenowitz, N. London, H. F. Bahrain, R. Derman and S. 
Smith, "Results of the first American prospective study of intravenous iron in oral iron-intolerant iron-
deficient gravidas," American Journal of Medicine, vol. 130, no. 12, pp. 1402-1407, 2017.  

RAPIDIRON-KIDS Study: Version 2.3 September 20, 2022 
27 
 
[12] B. Lozoff, N. K. Klein, E. C. Nelson, D. K. McClish, M. Manuel and M. E. Chacon, "Behavior of 
infants with iron-deficiency anemia," Child Development, vol. 69, no. 1, pp. 24-36, February 1998.  
[13] E. K. McCarthy, D. M. Murray, J. O'B Hourihane, L. C. Kenny, A. D. Irvine and M. E. Kiely, 
"Behavioral consequences at 5 y of neonatal iron deficiency in a low-risk maternal-infant cohort," 
American Journal of Clinical Nutrition, vol. 113, no. 4, pp. 1032-1041, April 2021.  
[14] T. Tsunawaki, K. Sakai, M. Momomura, Y. Wachi, Y. Matsuzawa and M. Iwashita, "Hypoxia alters 
phosphorylation status of insulin-like growth factor (IGF)-binding protein-1 and attenuates biological 
activities of IGF-1 and HepG2 cell cultures," Journal of Obstetrics and Gyenaecology Research, vol. 
39, no. 9, pp. 1367-1373, 2013.  
[15] S. Kajimura, K. Aida and C. Duan, "Insulin-like growth factor-binding protein-1 (IGFBP-1) mediates 
hypoxia-induced embryonic growth and developmental retardation," Proceedings of the National 
Academy of Sciences, vol. 102, no. 4, 2005.  
[16] H. Vakili, Y. Jin and P. A. Cattini, "Negative regulation of human growth hormone gene expression 
by insulin is dependent on hypoxia-inducible factor binding in primary non-tumor pituitary cells," 
Metabolism, vol. 287, no. 40, pp. P33282-33292, 2012.  
[17] A. Soliman, M. Eldabbagh, A. Adel and A. Sabt, "765 Linear growth and circulating IGF-1 
concentrations in children with iron deficiency anemia after treatment," Archives of Disease in 
Childhood, vol. 97, no. Suppl 2, 2012.  
[18] I. T. Angeles, W. J. Schultink, P. Matulessi, R. Gross and S. Sastroamidjojo, "Decreased rate of 
stunting among anemic Indonesian preschool children through iron supplementation," American 
Journal of Clinical Nutrition, vol. 58, no. 3, pp. 339-342, September 1993.  
[19] D. Bhatia and S. Seshadri, "Growth performance in anemia and following iron supplementation," 
Indian Pediatrics, vol. 30, no. 2, pp. 195-200, February 1993.  
[20] R. Bandhu, N. Shankar and O. P. Tandon, "Effect of iron on growth in iron deficient anemic school 
going children," Indian Journal of Physiology and Pharmacology, vol. 47, no. 1, pp. 59-66, January 
2003.  
[21] S. R. Chaudhary, S. Govil, M. K. Lala and H. B. Yagnik, "Infant and Young Child Feeding Index and 
its association with nutritional status: A cross-sectional study of urban slums of Ahmedabad," 
Journal of Family and Community Medicine, vol. 25, no. 2, pp. 88-94, 2018.