Feasibility or Oral Lactoferrin to Prevent Iron Deficiency Anemia in Obese Pregnancy
Phase: NA
Conditions: Iron-deficiency, Pregnancy Anemia, Obesity
Sponsor: University of Illinois at Chicago

 
Feasibility of oral lactoferrin to prevent iron deficiency anemia in obese 
pregnancy 
 
 
Principal Investigator: 
 
Mary Dawn Koenig, PhD, RN, CNM, University of Illinois at Chicago, marydh@uic.edu 
 
 
Co-Investigators: 
 
Lisa Tussing-Humphreys, PhD, RD, MS, University of Illinois at Chicago, 
ltussing@uic.edu 
 
Gloria Elam, MD, University of Illinois at Chicago, gelam@uic.edu 
 
Alana Steffen, PhD, University of Illinois at Chicago, steffena@uic.edu 
Kylea Liese, PhD, CNM, University of Illinois at Chicago, kylea3@uic.edu 
Consultant: 
Piera Valenti, PhD, University of Rome “La Sapienza”, piera.valenti@uniroma1.it 
 
 
 
 
Study Location(s): University of Illinois Hospital and Health Sciences System, 
Outpatient Care Center, Center for Women’s Health, 1801 W Taylor St. Chicago, IL 
60612 
 
 
Sponsor: 
 
National Institutes of Health, National Heart, Lung, and Blood Institute 
 
 
Version: 24 
 
Date: 09/17/2024 

Feasibility of oral lactoferrin to prevent iron deficiency anemia in obese pregnancy 
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TABLE OF CONTENTS 
Page 
Table of Contents ........................................................................................................ 2 
List of Abbreviations .................................................................................................... 3 
1.0 Project Summary/Abstract ................................................................................ 4 
2.0 Background/Scientific Rationale ....................................................................... 5 
3.0 Objectives/Aims................................................................................................. 8 
4.0 Eligibility ............................................................................................................ 8 
4.1 
Inclusion Criteria ..................................................................................... 9 
4.2 
Exclusion Criteria .................................................................................... 9 
4.3 Excluded or Vulnerable Populations ......................................................... 10 
5.0 Subject Enrollment .......................................................................................... 10 
6.0 Study Design and Procedures......................................................................... 11 
7.0 Expected Risks/Benefits.................................................................................. 16 
8.0 Data Collection and Management Procedures ................................................ 17 
9.0 Data Analysis ................................................................................................... 21 
10.0 Quality Control and Quality Assurance .......................................................... 21 
11.0 Data and Safety Monitoring ............................................................................. 21 
12.0 Statistical Considerations ............................................................................... 22 
13.0 Regulatory Requirements................................................................................ 22 
13.1 Informed Consent .................................................................................. 22 
13.2 Subject Confidentiality .......................................................................... 22 
13.3 Unanticipated Problems ........................................................................ 23 
14.0 References ....................................................................................................... 24 

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LIST OF ABBREVIATIONS 
 
ABR 
Auditory Brainstem Response  
Lf 
Oral Lactoferrin 
BMI 
Body Mass Index 
COI 
Conflict of Interest 
CRP 
C-reactive Protein 
CV 
Coefficient of Variation 
DHA 
Docosahexaenoic Acid 
DHHS 
Department of Health and Human Services 
DMC 
Data Monitoring Committee 
DSMB 
Data and Safety Monitoring Board 
DSMP 
Data and Safety Monitoring Plan 
EPO 
Erythropoietin 
EMR 
Electronic Medical Record 
Fe 
Iron 
FERPA 
Family Educational Rights and Privacy Act 
FDA 
Food and Drug Administration 
GCP 
Good Clinical Practice 
Hg 
Hemoglobin 
HIPAA 
Health Insurance Portability and Accountability Act 
IBC 
Institutional Biosafety Committee 
ICD 
Informed Consent Document 
ICH 
International Conference of Harmonization 
ID 
Iron Deficiency 
IDA 
Iron Deficiency Anemia 
IDE 
Investigational Device Exemption 
IDS 
Investigational Drug Service 
IL-6 
Interleukin-6 
IND 
Investigational New Drug 
IRB 
Institutional Review Board 
LAR 
Legally Authorized Representative 
LD 
Labor and Delivery 
OHRP 
Office of Human Research Protections 
OPRS 
Office for the Protection of Research Subjects 
PHI 
Protected Health Information 
PI 
Principal Investigator 
PNV 
Prenatal Vitamin 
PPRA 
Protection of Pupil Rights Amendment 
QA/QI 
Quality Assurance/Quality Improvement 
SAE 
Serious Adverse Event 
SOP 
Standard Operating Procedure 
UIC 
University of Illinois at Chicago 
WG 
Weeks Gestation 

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1.0 
Project Summary/Abstract 
 
Maternal iron deficiency anemia (IDA) increases the risk of adverse pregnancy 
outcomes and can negatively impact the iron endowment of the neonate that may  
cause irreversible deleterious effects on neurodevelopment. To avert IDA in pregnancy, 
women are universally prescribed a daily oral supplement containing ~27 mg of iron. 
However, there is growing concern regarding the efficacy of this one-size-fits-all 
approach for women with systemic inflammation, including women with obesity. Obese 
women experience disruption to the hepcidin-ferroportin complex deeming iron from 
diet, including supplemental iron, and body stores less bioavailable. This indicates an 
urgent need to develop and test new methods to prevent maternal IDA among women 
with obesity. In this planning grant application, we will develop the clinical and research 
infrastructure to test the preliminary efficacy of oral lactoferrin (Lf) supplementation for 
the prevention of maternal IDA among obese women. Lactoferrin is a natural  
compound found in secretions from mammals, including milk, and is an important 
regulator of body iron balance given its effects on inflammation and the hepcidin- 
ferroportin complex. Daily oral Lf has been shown to be superior to oral iron 
supplementation for treating IDA in pregnant women with inflammation but has not  
been studied for its ability to prevent IDA in pregnancy. The specific aims of this clinical 
trial planning grant include: 1) Conduct provider facing one-on-one interviews to inform 
and optimize a clinical research intervention into the clinical infrastructure at the Center 
for Women’s Health at UIC and, 2) Pilot test a randomized controlled trial of oral Lf 
supplementation from early second trimester up through delivery among 40 pregnant 
women with obesity at risk of IDA. In this aim we seek to understand recruitment, 
adherence, and retention rates and intervention acceptability to determine feasibility  
and preliminary efficacy on maternal inflammation and maternal and neonatal iron and 
hematological-related markers. This study will provide first of its kind data, necessary 
and sufficient to inform and develop a large-scale efficacy trial of oral Lf as a safe and 
scalable natural compound for the prevention of IDA among obese women. Ultimately if 
successful, this project is an important first step toward improving prenatal care for 
obese women who represent more than a third of reproductive-age women in the  
United States. 

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2.0 Background/Scientific Rationale 
 
Iron is essential for all cells and is particularly vital in pregnancy for the growth and 
maintenance of the placenta, expansion of the maternal red blood cell mass, fetal 
growth and neurocognitive development, and the neonatal iron endowment that 
supports ex utero brain development. 1 Conversely, decreases in maternal iron status 
that lead to IDA increase the risk of maternal and infant mortality, spontaneous preterm 
birth, and enduring neurocognitive defects in infants. 2 Recent animal and human data 
indicate that maternal IDA and reduced maternal iron bioavailability is associated with 
poor infant iron status at birth,3,4 posing a significant threat to neurocognitive 
development. In an effort to optimize iron status and avert IDA in pregnancy, U.S. 
women are universally prescribed a daily oral prenatal supplement containing ~27 mg of 
iron as a ferric or ferrous iron salt. 5 However, there is growing concern regarding the 
efficacy of this one-size-fits-all approach given the discovery of the hepcidin-ferroportin 
complex and its role in systemic iron regulation.5,6 
 
In pregnancy, a woman’s dietary iron absorption efficiency is enhanced, a 
compensatory mechanism to fulfill maternal iron needs and increased placental iron 
uptake and iron flux to the fetus as the pregnancy progresses1. It is believed this 
mechanism is largely supported by the suppression of maternal hepcidin, a liver-derived 
peptide hormone and master regulator of systemic iron homeostasis.7,8 Hepcidin 
controls systemic iron metabolism through its regulation of the ferroportin iron exporter.9 
Hepcidin promotes ferroportin’s degradation thereby reducing the efflux of iron from 
intestinal enterocytes, reticuloendothelial macrophages, and hepatocytes. Hepcidin is 
simultaneously regulated by iron stores, erythropoiesis and systemic inflammation.10 In 
persons with systemic inflammation, hepcidin production is enhanced, resulting in 
diminished ferroportin expression and reduced iron export into circulation from diet and 
stores.11 This phenotype, commonly referred to as the anemia of inflammation, is 
associated with iron delocalization specifically normal to elevated ferritin (i.e., iron 
trapping), low hematological parameters [e.g., hemoglobin (Hb)], and systemic 
inflammation [e.g., elevated interleukin-6 (IL-6)].12 Importantly, supplementing with oral 
iron salts (e.g., ferrous sulfate) in persons with underlying systemic inflammation has 
limited efficacy for preventing or treating IDA because hepcidin is a negative regulator of 
dietary iron absorption.13 
 
There is increasing evidence that hepcidin can be overexpressed in pregnant women 
with obesity. Pre-pregnancy obesity, a condition affecting 36% of reproductive age 
women in the U.S.,14 is often accompanied by chronic systemic inflammation and is a 
risk factor for neonatal ID.15,16 In 2014, a small case control study reported that women 
with pre-pregnancy obesity (n = 20) had higher systemic inflammation and hepcidin in 
the second trimester and at the time of delivery compared to lean women (n = 20, BMI 
18.5 - 24.9 kg/m2).17 Moreover, the neonates birthed by the obese women had lower 
cord iron status compared to the neonates born to the lean women. In a separate case- 
control study, maternal obesity was associated with impaired neonatal cord iron status 
at delivery compared to neonates birthed by lean women.15 Specifically, neonatal tissue 
iron stores were compromised to support Hb, a partitioning that may have a profound 

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effect on growth and neurodevelopment. Several other research teams have examined 
links between maternal obesity and maternal-fetal iron regulation in large observational 
cohorts, with findings garnering mixed results.16,18–20 The lack of consistency among the 
various studies may be related to the degree of adiposity of the women investigated.19 
For example, one study stratified by extreme obesity (BMI ≥ 35.0 kg/m2) found that 
serum hepcidin was 50% higher in the 2nd trimester and at the time of delivery among 
severely obese women (but not among overweight or class I obese women) compared 
to women that were lean, indicating that a certain BMI threshold may be necessary to 
skew iron homeostasis in pregnancy.19 In our preliminary data, described in detail 
below, pregnant women with pre-pregnancy obesity (mean BMI 35.3 ± 5.0 kg/m2) had 
greater systemic inflammation, overexpressed hepcidin, lower dietary iron uptake and 
less iron transferred to their fetus. Our findings are important given we used a stable 
iron isotope to objectively trace iron from maternal uptake to neonatal cord blood – no 
other studies to date have used this rigorous approach to examine iron homeostasis in 
obese pregnancy. Our findings indicate that the flow of iron from mother to fetus is 
impaired in women with pre-pregnancy obesity. Moreover, our preliminary data suggest 
that the current universal approach to prevent IDA in pregnant women with obesity, a 
daily oral prenatal supplement containing iron, is suboptimal given their underlying 
inflammatory status. This is a call to action given that disturbances to maternal iron 
homeostasis can result in irreversible, lifelong adverse health effects in infants,21 
coupled with the high rate of obesity among reproductive age women in the U.S.14 
Clearly, alternatives to preventing maternal IDA are needed for pregnancies 
complicated by obesity. 
 
Lactoferrin (Lf) is an iron binding glycoprotein secreted by exocrine glands and 
neutrophils that is present in mammalian biological fluids including milk, saliva, tears, 
and cervical mucus.22 Lf is involved in both iron homeostasis and inflammation and is 
closely related to the iron transport protein transferrin although it can retain iron at a 
lower pH23. Of relevance to the proposed trial is evidence that Lf can suppress the 
production and release of pro-inflammatory cytokines including IL-6.24 This mechanism 
of action is important given IL-6 modulates hepcidin (i.e., promotes its upregulation) 
which leads to reduced ferroportin activity.9 In a trial conducted in pregnant women with 
concurrent hereditary thrombophilia, IDA, and underlying systemic inflammation, led by 
our team member Valenti,22 oral supplementation with bovine-derived Lf (Lf) (which has 
similar structure to human Lf and can bind to human Lf receptors to exert biological 
activity)23 led to large reductions in circulating IL-6 and clinically significant improvement 
in iron and hematological parameters, compared to standard oral iron salt 
supplementation (330mg ferrous sulfate). Importantly this effect was observed in the 
absence of additional oral supplemental iron. Oral Lf is thought to improve body iron 
balance by enhancing ferroportin-mediated iron export by limiting inflammation and 
decreasing blood levels of both IL-6 and hepcidin — ultimately increasing iron 
bioavailability in the absence of additional direct iron supplementation.22 Moreover, in 
pregnant women with IDA, daily oral Lf when tested against standard oral ferrous 
sulfate and ferrous fumarate supplementation was superior for improving Hb and was 
associated with significantly fewer adverse side-effects (i.e., gastrointestinal 
disturbances) and enhanced acceptability.25 To our knowledge, there exists no clinical 

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data to show the effectiveness of oral Lf supplementation for preventing IDA among 
pregnant women with obesity, despite a biological pretense for why oral Lf may be 
beneficial in this context. In summary, oral Lf is a safe compound to counteract 
inflammation, regulate the hepcidin-ferroportin axis and rebalance iron homeostasis in 
the absence of additional oral iron in a way that may be uniquely beneficial for 
preventing IDA among pregnant women with obesity given their underlying inflammatory 
status. 
 
Effects of oral Lf vs. oral iron on iron homeostasis among women with hematologic 
disorders and complicated pregnancies. Our team member, professor Piera Valenti 
recently published a study examining the efficacy of twice daily (100mg/dose) oral Lf vs. 
once daily oral iron supplementation (330mg ferrous sulfate) for the treatment of 
concurrent anemia of inflammation and IDA in pregnant women with the hereditary 
hematologic disorder thrombophilia.22 Women with anemia of inflammation/IDA have a 
similar phenotype, albeit more extreme, to what is observed in obesity, i.e., systemic 
inflammation, overexpressed hepcidin, elevated body iron stores (ferritin) and 
decreased Hb. Valenti found that after 30 days of supplementation and at the time of 
delivery, oral Lf was associated with a significant reduction in IL-6 and hepcidin and 
statistically and clinically significant improvements in iron and hematological parameters 
(Table 1). Notably there were no changes in the women receiving oral ferrous sulfate. 
Also noteworthy is the fact that there were no adverse effects on the fetus/neonate 
(e.g., amniotic fluid index or birth outcomes) with either intervention. An exploratory 
analysis examining the impact of daily oral Lf on iron and hematological parameters in 
pregnant women with insulin resistance (n = 3) showed that after 30 days of 
supplementation, there was an increase from baseline in Hb (10.4 ± 0.3 to 11.4 ± 0.3 
g/dL) and serum iron (33 ± 2 to 56 ± 11 µg/dL). This data is particularly intriguing given 
pregnant women with insulin resistance most closely represent the metabolic phenotype 
observed in obesity. Together, the data suggest that oral Lf supplementation safely 
elicits an anti-inflammatory effect that modulates the hepcidin-ferroportin complex to 
improve maternal iron homeostasis. This trial provides strong evidence that oral Lf 
supplementation, in the absence of additional oral iron, could be used to safely optimize 
iron homeostasis and prevent IDA in women with obesity. 

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Table 1. Hematological, iron, IL-6 and hepcidin mean values ± SD of pregnant women with hereditary 
thrombophilia and anemia of inflammation treated with oral bLf or ferrous sulfate baseline, after 30 
days of treatment, and at delivery 
Oral bLf (n=40) 
Oral ferrous sulfate (n=25) 
Baseline 
After 30 days 
Delivery 
Baseline 
After 30 days 
Delivery 
Red blood cells x 103 
3,750 ± 127 
4,354 ± 198* 
4,482 ± 147* 
3,650 ± 163 
3,960 ± 230 
4,029 ± 287 
Hb, g/dL 
10.6 ± 0.4 
12.2 ± 0.4* 
12.7 ± 0.5* 
10.8 ± 0.8 
11.3 ± 1.1 
11.2 ± 0.9 
Serum iron, µg/dL 
36 ± 11 
63 ± 9* 
85 ± 10* 
40 ± 15 
40 ± 6 
38 ± 11 
Serum ferritin, ng/mL 
11 ± 6 
18 ± 2 
31 ± 3* 
17 ± 14 
14 ± 9 
12 ± 10 
Serum IL-6, pg/mL 
89 ± 8 
58 ± 6* 
50 ± 5* 
85 ± 12 
108 ± 7 
113 ± 15 
Serum hepcidin, 
115 ± 23 
65 ± 10* 
54 ± 13* 
107 ± 29 
112 ± 32 
116 ± 26 
ng/mL 
*significantly different from baseline, p < 0.001 
 
 
3.0 Objectives/Aims 
 
Aim 1: Conduct provider interviews to gauge best practices for integrating clinical 
research and patient recruitment into the Center for Women’s Health clinical 
workflow. We will recruit 9-12 providers from the Physician attending/Physician resident, 
nurse-midwife and nurse positions to complete a 30-60-minute one-on-one interview and 
5-minute electronic demographics survey. 
 
Aim 2: Pilot test a randomized controlled trial of oral Lf supplementation among 
pregnant women with pre-pregnancy obesity at risk of IDA. We will randomize 40 
women to once-daily oral Lf (250mg) or usual care from early second trimester through 
delivery to: a) Understand recruitment, adherence, and retention rates and intervention 
acceptability to determine feasibility; and b) Determine preliminary efficacy on maternal 
inflammation and maternal and neonatal iron and hematological markers. 
 
 
4.0 
Eligibility 
 
Aim 1: We will recruit physician attendings, physician residents, nurse-midwives and 
nurses from the Center for Women’s Health at UIC (9-12 individuals in total). The only 
eligibility is to be a current provider at the Center for Women’s Health. 
Aim 2: We will recruit 60 women to enroll 40 women and obtain a final sample of 32 
women (16/arm). Women will be referred by their provider to the study team and 
recruited following their initial new OB visit either in-person at an upcoming clinical visit 
or by phone to determine interest and confirm eligibility. Women will also be identified 
and pre-screened by study staff through provider scheduling records and patient EMR 
to assess provider referral rates. We have found in our previous studies that waiting 

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until after the new OB visit is completed streamlines the recruitment process 
substantially, since the initial visit yields a detailed History and Physical, estimated 
dating of gestational age, and new OB clinical labs. Interested and eligible women will 
be invited to schedule a baseline research visit. 
 
4.1 Inclusion Criteria 
Aim 1: 
• current physician, nurse-midwife or nurse in the Center for Women’s Health at 
UIC 
Aim 2: 
• single 
• naturally conceived pregnancy 
• at risk of IDA [Hb 11.0 – 12.9 g/dL (first trimester)/10.5 – 12.5 g/dL (second 
trimester)] based on new OB complete blood count (CBC) results obtained from 
the EMR 
• 18 – 45 years old 
• pre-conception BMI ≥ 28.0 kg/m2 [based on measured height in EMR and recent 
pre-conception weight (within 3 months of pregnancy) from EMR if available or 
self-reported]; < 24 WG 
• fluency in English to provide consent and complete study procedures 
• ability to provide consent 
• and ownership of a smartphone (currently more than 90% of our patient 
population at the Center for Women’s Health) 
• For ABR testing, the infant must have a normal hearing screen recorded in the 
electronic medical record (EMR). This is a requirement for normal ABR testing 
protocol. 
 
 
4.2 Exclusion Criteria 
Aim 1: 
• 
None 
 
Aim 2 
• IDA requiring high dose supplemental iron 
• allergy to milk proteins or wheat 
• vegan (due to content of the supplements) 
• recent blood transfusion 
• previously diagnosed type 1 or type 2 diabetes 
• autoimmune disorder (e.g., rheumatoid arthritis) 
• inflammatory bowel disease 

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• current bacterial or viral infection 
• history of bariatric surgery 
• malabsorptive disease 
• current hyperemesis 
• current eating disorder 
• hematologic disorder or trait carrier (e.g., hemochromatosis, β-thalassemia) 
• current tobacco, alcohol or illicit drug use (not including marijuana) 
• and regular use of medications that may interfere with nutrient absorption 
• We have also found in our previous studies a recent traumatic event (e.g., death 
of a significant other or parent) may make it difficult to comply with the 
interventions, hence these women will also be excluded. 
• First trimester Patient Health Questionnaire depression screener (PHQ-8) >10 is 
indicative of moderate to severe depressive symptoms and in our previous 
studies women with a score PHQ-8 >10 had difficulty complying with study 
measures. Women with a score >10 from the initial prenatal visit in the EMR, or if 
one is not available in the EMR, at screening will be excluded for study 
participation. The participant’s provider will be notified via EPIC in basket 
message flagged as high priority and a member of the research team will monitor 
whether the provider read the message from the study team. If the participant is 
found to have a score >10 in the preliminary screening of the EMR, they will not 
be approached for recruitment. 
 
 
4.3 Excluded or Vulnerable Populations 
Sufficient fluency in English is required to complete consent, study forms, and 
questionnaires. During screening, a member of the research team will abstract from the 
medical record (if available) whether the participant is English speaking. A member of 
the research team will ask the participant whether they have sufficient fluency in English 
to complete the consent study forms and questionnaires. 
Pregnant women will be enrolled and are considered a vulnerable population. 
 
5.0 
Subject Enrollment 
Aim 1: We will recruit 9-12 (3-4 from each group) physician attending/resident, nurse- 
midwife and nurse in the Center for Women’s Health at UIC. 
 
Aim 2: We will recruit 60 pregnant women to enroll 40 and obtain a final sample of 32 
women (16/arm). Women will be referred by their provider to the study team and 
recruited following their initial new OB visit either in-person at an upcoming clinical visit, 
by phone, by text or via MyChart messaging to determine interest and confirm eligibility. 
Only one MyChart message will be sent to gauge individual interest. Potential 
participants can decline participation in the study through MyChart messaging. A 
member of the research staff will indicate through MyChart that the participant’s 
response will be directed to a member of the research staff only and not to the 
practitioner. Women will also be identified and pre-screened by study staff through 
provider scheduling records and patient EMR to assess provider referral rates. We have 
found in our previous studies that waiting until after the new OB visit is completed 
streamlines the recruitment process substantially, since the initial visit yields a detailed 

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History and Physical, estimated dating of gestational age, and new OB clinical labs. 
Interested and eligible women will be invited to schedule a baseline research visit. 
 
 
 
 
6.0 Study Design and Procedures 
Aim 1. 
Center for Women’s Health providers agreeing to participate will engage in a one-on- 
one interview with study staff. The interview consists of prepared questions as well as 
organic dialogue and subsequent questions stemming from the interview responses. 
The interview will take place in person at UIC in a private office in the College of 
Nursing or College of Medicine (with COVID distancing and masking in place) or via 
phone or UIC hosted video conferencing platform, depending on provider preference. 
The one-time conversation will be audio recorded and will last approximately 30-60 
minutes. The provider/participant will also complete a brief 5-minute demographics 
questions on a study provided tablet via REDCap. Provider/subjects will be 
compensated with a $25 Starbuck’s gift card upon completion of the interview. 
 
Aim 2. 
Study Design. The feasibility and acceptability of daily oral Lf supplementation taken 
from early second trimester (15 – 24 WG) up through delivery will be assessed in a 
parallel 2-arm randomized controlled pilot trial conducted at the University of Illinois 
Hospital and Health Sciences System (i.e., Center for Women’s Health and 

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Department of Family Medicine at University Village and Pilsen).

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 among 40 pregnant women with pre-pregnancy obesity at risk of IDA (Fig. 1). Women 
will be asked to complete the PHQ-8 to determine eligibility. This will only be completed if 
there is not an available score in the EMR from the patient’s initial prenatal visit. If the 
woman has a PHQ-8 score <10 she will be eligible to participate. Women will be 
randomized (3:1) to one of two arms in the early second trimester (15 - 24 WG): once 
daily oral Lf + prenatal vitamin/mineral supplement without iron or once daily standard 
prenatal vitamin/mineral supplement with iron (usual care). We will examine feasibility, 
acceptability, and preliminary efficacy as main study outcomes. 
 
Randomization. Dr. Steffen, the study biostatistician, will develop the treatment arm 
allocation schedule and ensure it is concealed from staff responsible for recruitment and 
enrollment until after written informed consent via electronic signature is obtained. We 
will use stratified, permuted block randomization considering pre-pregnancy BMI (28-
34.9 kg/m2 and 35+ kg/m2), Hb (10.5 – 11.4 g/dL and 11.5 – 12.5 g/dL) and CRP (3.0 – 
6.49 mg/L and 6.5 – 9.99 mg/L) to ensure balance across treatment arms. Forty women 
will be randomized to either once daily oral Lf + prenatal vitamin/mineral supplement 
without iron or daily prenatal vitamin/mineral supplement with iron (usual care) using the 
REDCap® randomization module developed to obtain a final sample of 32 women 
(16/arm assuming 20% attrition). 
 
Study Arms. Women will be randomly assigned (3:1) to one of the two study arms 
described below. Women will not be blinded to their treatment assignment. Women will 
receive a month’s worth of supplements at a time. Refills will occur at monthly research 
visits that are coordinated with clinical care. Based on our current probiotic 
supplementation trial, women receive between 5-6 refills over the course of their 
pregnancy depending on the timing of their study entry. We have opted to test once 
daily Lf (250mg) versus a lower dose (100mg) taken twice daily (as was used by our 
team member Valenti in her previous trial)22 due to the common availability of Lf 
formulations in the U.S. commercial market, as well as the logistics around once vs. 
twice daily supplementation in our patient population. The supplements will be 
dispensed in  bottles by the UIC Investigational Drug Service. 
 
Once daily Oral Lf (250mg). Women assigned to this group will be instructed to 
consume an oral Lf capsule one hour prior to their afternoon meal and two prenatal 
vitamin/mineral supplement gummies without iron with omega-3 fatty acids before bed 
from early second trimester (15 - 24 WG) up through delivery. Women are advised to 
consume the Lf prior to meals, given our team member Valenti’s unpublished work 
shows its superior efficacy for improving iron and hematological parameters among 
pregnant women with hereditary thrombophilia versus when consumed with meals. The 
250mg Lf capsule will be sourced from Jarrow Formulas (Los Angeles, CA. The 
supplement (Fig. 2), available commercially, has been previously tested in pregnancy25 
and includes 250mg Lf in addition to cellulose, magnesium stearate (vegetable source), 
silicon dioxide, and gelatin capsule material. Jarrow sources their Lf from Glanbia 
Nutritionals (Chicago, Illinois). Per the manufacturer, the Lf is approximately 14.3% 
saturated with iron or contains 17.6 mg/100g; we will confirm this with our own purity 
testing as described below. The prenatal vitamin/mineral gummies will be a 
commercially available product (OLLY The Essential Prenatal Multi). The nutritional 
content of the prenatal gummies is presented in Fig. 3. Women in both groups will be 
advised to consume an iron-rich diet and provided a handout detailing foods rich in 
heme and non-heme iron. 

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Figure 2. Jarrow Formulas Oral Lactoferrin supplement Facts Label 
 
 
 
 
 
Figure 3. OLLY The Essential Prenatal Multi, Supplement Facts 
 
 
Usual Care (Control). Women assigned to this group will be instructed to consume a 
commercially available prenatal vitamin/mineral supplement with iron and omega-3 fatty 
acids (Prenatal 1, Bayer Healthcare, Whippany, NJ) before bed from early second 
trimester (15-24 WG) through delivery. To minimize variability in prenatal 
vitamin/supplement use across the participants, we have opted to standardize the 
prenatal vitamin/mineral supplement by providing women in the usual care arm a 
supplement that is nutritionally like what is prescribed by the Center for Women’s Health 
providers. The nutritional content of the study-provided prenatal vitamin/mineral 
supplement is presented in Fig. 4. Women will be advised to consume an iron-rich diet 
and provided a handout describing foods rich in heme and non-heme iron.

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Figure 4. Prenatal-1, Bayer Healthcare, Supplement Facts 
 
 
Purity Testing of Oral Lf Capsules. Purity testing of the Lf capsules will be conducted 
using SDS-PAGE and silver nitrate staining, concentration via UV spectroscopy, iron 
saturation via spectroscopy and lipopolysaccharide contamination by Limulus 
Amebocyte Lysate assay (Sigma Aldrich, St. Louis, MO). Lf purchased from Sigma 
Aldrich will serve as the reference standard (St. Louis, MO). All purity testing will be 
conducted by Avomeen Analytical Services (Ann Arbor, MI). 
 
Study Feasibility. We will collect detailed records of the number of women eligible and 
then referred by Center for Women’s Health clinical staff to the research team. We will 
also track the number of referred women approached (by phone & in person) by our 
research team for enrollment and the number of women who decline and their reasons 
for non-enrollment. Once a woman is enrolled in the study, attendance at study visits, 
completeness of data, and overall and treatment specific loss to follow-up/withdrawal 
will be closely monitored. To track progress of subjects through the trial, we will adhere 

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to and update weekly the Consolidated Standards of Reporting Trials guidelines subject 
flow diagram. Women who voluntarily withdraw will be asked to provide reasons for 
revoking their enrollment. To further supplement these process measures, the study 
coordinator will take detailed notes during weekly research clinic meetings to document 
barriers to and facilitators of subject enrollment and retainment in the study, which will 
be used in our implementation analysis. Rates of enrollment and retainment are key 
feasibility outcomes. To facilitate recruitment and retention, transportation and 
participation monetary incentives will be provided. Together these activities will allow for 
the assessment of success and failure points to guide future work. Expected results: 
The study will be deemed feasible if clinicians refer ≥ 75% of women that are eligible, 
we recruit ≥ 50% of those referred at a minimum rate of 3-4 women/month, women 
complete ≥ 80% of planned study visits, and we retain ≥ 80% of women in both 
treatment arms through delivery. 
 
Optimizing Adherence.   . Hand pill counts will be performed by UIC Investigational Drug 
Service. Expected result: Women will be deemed adherent to the interventions if ≥ 
80% of the supplements provided are consumed. 
 
Health Status Monitoring. Health status will be assessed weekly through text messaging 
via texting service SimpleTexting (subject will be contacted by phone if additional 
information is needed beyond what is captured by text) and at monthly research visits. 
SimpleTexting is a HIPPA compliant online texting service. Subjects will receive this 
message on their smartphone. The SimpleTexting account is password protected and 
only accessible by key research personnel. Reported health changes will be reviewed 
and coded using a standardized approach.26This information will be stored in REDCap. 
Other maternal (e.g., preeclampsia, gestational diabetes mellitus, infections), pregnancy 
(e.g., spontaneous pre-term birth), fetal (e.g., amniotic fluid index measured clinically in 
the second and third trimesters) and neonatal (e.g., birth weight, hypoglycemia) health 
outcomes will be collected from the subject EMR. We anticipate the most frequently 
reported health changes will be gastrointestinal (GI) in nature (e.g., constipation, loose 
stools). Therefore, a GI symptoms survey27 will be administered at each monthly in-
person research encounter. Expected result: We expect that the type and rate of 
health events will be similar between study arms. 
 
The research participants will complete 7-9 study visits depending on their gestational 
age when they enter the study. T1 (Screening/Baseline) will occur at 15-24 weeks 
gestation (WG), T2 will occur at 19-24 WG,T3 will occur at 23-28 WG, T4 will occur at 
27-32 WG, T5 will occur at 31-36WG, T6 will occur at 35-40 WG, T7 will occur at 39- 
41WG, T8 will occur at delivery, and T9 will occur 2-3 days after delivery or at 38-42 
weeks postmenstrual age for infants born preterm. 

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Participants will receive cash for each completed study visit. If they screen out at 
baseline due to their PHQ-8 score (if applicable), they will receive $10 and be 
withdrawn. If they do not finish the study, they will be compensated only for the visits 
they have completed. Participants will receive $50 at baseline, $20 at each monthly 
visit, and $29 at delivery. If a participant completes all parts of the study, they will 
receive a total of 
$179-$199 in cash depending on when they entered the study, compensation for UIC 
parking and/or public transportation costs, and a baby-related gift with a cash value of 
approximately $101 (car seat, highchair or bassinet). Participants will receive another 
baby-related gift with a cash value of approximately $101 (car seat, highchair or 
bassinet) if they agree and complete ABR testing. Participants will choose a gift from 
the gift catalog provided by a member of the research team. Participants who screen 
out for PHQ-8 (if applicable) will receive $10 and compensation for UIC parking or 
public transportation costs. 
 
We will monitor hemoglobin and iron status monthly at study visits and review results 
weekly with Dr. Gloria Elam, the medical oversight physician for the study. If needed, 
Dr. Gloria Elam will coordinate care with the participant’s primary practitioner. 
 
 
7.0 Expected Risks/Benefits 
 
Aim 1: 
• We do not anticipate risks related to the one-on-one provider interviews. The only 
identified risk is related to loss of confidentiality. However, every precaution will be 
taken to keep information safe. A unique study ID number will be assigned to 
protect identity. Any electronic data will be stored in a password protected, restricted 
access electronic database (REDCap). The audio data will be stored on a password 
protected UIC Box folder. Only authorized research personnel will have access to 
this data. 
 
Aim 2: 
Side effects, risks, and/or discomforts from participation in this study include: 
• Blood draws. Some people may be uncomfortable with having their blood drawn and 
may feel lightheaded, dizzy or even faint. Other risks and discomforts associated 
with drawing blood from a vein include pain or bruising at the site of the blood draw 
and rarely, infection at this site. Care will be taken to reduce these risks by having 
the phlebotomist conduct the blood draw.  
• Loss of confidentiality. Because participants are sharing personal information, there 
is always the possibility that confidentiality will be violated. However, every 
precaution will be taken to keep information safe. A unique study ID number will be 
assigned to protect identity. Any electronic data will be stored in a password 
protected, restricted access electronic database. The blood and urine samples will 
be stored in a locked laboratory at UIC in a locked freezer. The survey data will be 
directly entered into a password protected research database. Only authorized 
research personnel will have access to this data. 
• Questionnaires and interviews. Responding to study questionnaires and interview 

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questions is not expected to but may provoke uncomfortable feelings or cause mild 
distress. If participants experience any of these symptoms, they may choose not to 

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answer any question that causes discomfort or may choose to withdraw from the 
study altogether. If their responses to the questionnaires indicate they may have 
depression or anxiety, it is our standard practice to refer them back to their clinical 
caregiver for further screening and evaluation. We will also screen using the PHQ-8, 
and the participant must complete this questionnaire to remain eligible. 
• Adverse effects of consuming a prenatal supplement that lacks iron. Based on 
existing data, it is highly unlikely to see a significant decrease in Hb or iron related 
parameters with oral Lf supplementation is the subject is adherent with the regimen. 
Nonetheless, we will monitor Hb and iron status monthly and review results with Dr. 
Gloria Elam who is the medical oversight for the study and medical director of the 
clinic where all the research subjects are receiving prenatal care. 
• Adverse effects from oral Lf use. We will monitor daily changes to the subject’s 
health reported through text messaging via SimpleTexting throughout the study and 
follow up with a phone call for additional information if needed. Additionally, the 
research coordinator will call participants to evaluate reported symptoms and advise 
administration for improved tolerability during the first week of Lf or usual care 
treatment. If in the unlikely situation that symptoms due to the intervention are 
intolerable or significant (unable to maintain intake by mouth or dehydration from 
diarrhea), the intervention will be immediately discontinued. To safeguard the risk of 
significant adverse effects, women with contraindications or history of bariatric 
surgery, inflammatory bowel disease, chronic diarrhea, or with hyperemesis 
gravidarum or eating disorder with the current pregnancy are excluded from the 
study. All adverse health effects will be coded and reviewed by the study team. 
Decision regarding continuing in the study rests solely with Drs. Koenig and Tussing- 
Humphreys. 
• Anthropometric assessments. To minimize discomfort related to body 
measurements, assessments will be conducted by a trained and experienced 
research team member in a private area. 
• ABR testing. If you agree to ABR testing on your baby, it is possible that your 
baby may experience minor skin irritation at the site of probe placement from 
the adhesive pads after testing is done, and this is expected to resolve in one 
day. The adhesive pads used in the testing are the same used for the clinical 
hearing testing performed in the university hospital. Care will be taken to 
ensure that the adhesive does not stay on your baby for longer than 
necessary for testing to reduce any possibility of irritation. 
 
8.0 Data Collection and Management Procedures 
 
Aim 1: Providers will be involved in one interview and will complete one on-line survey 
(REDCap). The interview will be audio recorded or UIC hosted video conferencing 
platform. 
 
Aim 2: Women will be involved in 7-9 research visits (depending on timing of study 
entry) that will take place at the Clinical Research Center and the Hospital LD unit. 

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Antenatal visits will be coordinated with clinical care to minimize subject burden. 
Measures to be obtained at each study visit are detailed in Table 2. Follow-up visit 7 will 
likely only be required for women entering the trial at 15-16 WG. A brief description and 
rationale for each measure is described below. 
Maternal Iron Status 
& Hematological 
Parameters. Maternal 
iron status and 
hematological 
parameters will be 
assessed from 
antecubital non- 
fasting blood at 
baseline, monthly, 
and at admittance for 
delivery. Expected 
results: The primary 
maternal outcome is 
Hb. Compared to the 
control group, we 
expect (based on our 
preliminary data in 
pregnant women with 
insulin resistance) a 
clinically significant improvement in Hb (+ 0.5 – 1.0 g/dL) at timepoint (T) 2 (~ 30 days 
after starting the supplement) for women randomized to daily oral Lf that is maintained 
through delivery. We also expect in the women randomized to oral Lf significantly higher 
serum ferritin, iron, hematocrit and red blood cells and significantly lower soluble 
transferrin receptor (sTFR) at T2 that is maintained through delivery compared with the 
control group. 
• 
Ferritin & Iron. Maternal ferritin and iron will be measured in serum by 
immunoassay and spectrophotometry by a local commercial lab (Quest Diagnostics, 
Wood Dale, IL). 
• 
sTFR. Maternal sTFR will be measured in serum using a commercially available 
immunoassay (ELISA) (R&D Systems, Minneapolis, MN). Our lab’s intra-assay 
coefficient of variation (CV) for this kit is 5.3%. 
• 
Calculated Total Body Iron (TBI). Maternal TBI will be calculated from sTFR and 
serum ferritin using an equation developed by Cook and colleagues.28,29 
• 
Complete Blood Count (CBC). Maternal CBC with differential will be measured in 
whole blood by electronic cell sizing/counting/cytometry/microscopy by a local 
commercial lab (Quest Diagnostics, Wood Dale, IL). Hb, obtained from the CBC, will be 
used to define trimester-specific IDA with a downward correction of 0.8 g/dL for Black 
women.30 Hb is the primary clinical outcome for the trial given it is the most commonly 
assessed iron/hematological status marker in pregnancy. 

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Correcting Maternal Ferritin, sTFR & TBI for Systemic Inflammation. The Biomarkers 
Reflecting Inflammation and Nutritional Determinants of Anemia (BRINDA) project 
inflammation-related correction factors will be used to correct serum ferritin, sTFR and 
TBI values for C-reactive protein (CRP).31 CRP will be measured via nephelometry by a 
local commercial lab (Quest Diagnostics, Wood Dale, IL). 
 
Maternal Hepcidin. Maternal hepcidin will be measured in serum at baseline, T3, T5, 
and at admittance for delivery with a commercially available ELISA (Intrinsic 
LifeSciences, La Jolla, CA).32 The minimum level of detection for the assay is 2.5 
ng/mL. Expected results: We expect significantly lower hepcidin at T2 that is maintained 
through delivery in women randomized to oral Lf compared with control. 
 
Maternal IL-6. Inflammation, specifically the pro-inflammatory cytokine IL-6, is a positive 
regulator of hepcidin.12 Maternal IL-6 will be measured in serum at baseline, T3, T5, and 
at admittance for labor and delivery with a commercially-available ELISA (R&D 
Systems, Minneapolis, MN). Our lab’s intra-assay CV is 5.7%. Expected results: We 
expect significantly lower IL-6 at T2 that is maintained through delivery in women 
randomized to oral Lf compared with control. 
 
Maternal Erythropoietin. Erythropoiesis is a negative regulator of hepcidin.9 Maternal 
erythropoietin will be measured in serum at baseline, T3, T5, and at admittance for labor 
and delivery with a commercially available ELISA (R&D Systems, Minneapolis, MN). 
Our lab’s intra-assay CV is 2.6%. 
 
Maternal Dietary Intake. Dietary macro- and micro-nutrient intake will be assessed using 
the web-based Diet History Questionnaire III (DHQ-III) food frequency questionnaire 
(baseline only).28 The DHQ-III will be self-administered. Recent dietary 
intake/supplement use will be assessed at baseline, T3, and T5, via Nutrition Data 
System for Research interviewer-assisted 24-hour diet recall software.33 Mean total, 
food, and supplemental iron intake can be estimated from both dietary measures. 
 
Maternal Anthropometrics & Gestational Weight Gain. Maternal height will be measured 
using a fixed stadiometer (baseline only) and weight at baseline, monthly, and at 
admittance for labor and delivery with a calibrated digital scale. BMI will be calculated 
as kg/m2. Pre-pregnancy weight will be self-reported (confirmed by EMR, if available) 
and used to derive gestational weight gain using published methods.29 Rationale for 
determining gestational weight gain is the reported evidence that excessive gestational 
weight gain can negatively affect maternal and neonatal iron status and regulation.15–18 
 
Neonatal Cord Iron Status & Hematological Parameters. Neonatal cord iron status and 
hematologic parameters will be assessed from venous umbilical cord blood obtained 
bedside following delivery of the placenta. Even with delayed umbilical cord-clamping, 
we can successfully obtain 40-50 milliliters of umbilical venous cord blood. We will 
employ our effective 24/7 call schedule (including holidays) with two team members on 
call daily to cover LD collections. We have an 87% (40 of 46 women) success rate of 
collecting samples bedside at delivery as indicated in our preliminary studies section. 

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Expected results: Compared with control, we expect the neonates born to women in the 
oral Lf group will have superior cord markers of iron status, cord TBI and cord Hb at 
delivery. 
• 
Cord Ferritin & Iron. Cord ferritin and iron will be measured in serum by 
immunoassay and spectrophotometry at a local commercial lab (Quest Diagnostics, 
Wood Dale, IL). 
• 
Cord sTFR. Cord sTFR will be measured with an immunoassay (ELISA) (R&D 
Systems, Minneapolis, MN). Our lab’s intra-assay coefficient of variation (CV) for this kit 
is 5.3%. 
• 
Cord TBI. Cord TBI will be calculated from cord sTFR and serum ferritin using a 
published equation.28,29 
• 
Cord CBC. Cord CBC with differential, which includes Hb, will be measured by 
electronic cell sizing/counting/cytometry/microscopy at a local commercial lab (Quest 
Diagnostics, Wood Dale, IL). 
 
Maternal Urinary Metabolite. Urine metabolite analysis related to oxysterols will be 
assessed from spot urine collection at T1, T3 and T5. Pregnant individuals will provide 
a spot urine sample in a sterile cup. 0.5ml of urine will be aliquoted and frozen at -80C 
until further analysis. Urine oxysterols and chemicals, such as neonicotinoids, present 
in the urine will be quantified using mass spectrometry at the UIC metabolomics core. 
 
Neonatal Characteristics. There is evidence that fetal sex (considering sex as a 
biological variable), neonatal weight at delivery and gestational age at delivery and can 
affect maternal and neonatal iron status and hematological parameters.15,34 We will 
obtain these variables from the patient EMR. 
 
Neonatal Auditory Brainstem Response (ABR) Testing. Cognitive development in 
neonates will be assessed with ABR testing at T9. ABR testing in hearing infants 
measures the conduction speed of neurons along different levels of the auditory 
pathway.35 Longer latencies indicate delayed myelination in the brainstem and the 
brain more broadly.36 Maternal and infant ID have been associated with poorer ABR 
performance in neonates and later in infancy, which is hypothesized to due to iron’s 
role in synthesizing lipid required for myelination.37,38 An experienced audiologist will 
conduct the ABR testing at a target age of 2-3 days postpartum in neonates of 
individuals enrolled on the trial. Infants born preterm will be tested at 38-42 weeks 
postmenstrual age. The audiologist will be blinded to the mother’s assigned treatment 
group, and the ABR data will be analyzed by an audiologist who is blind to iron status 
and treatment group. The test in noninvasive and performed while the infant is lying 
supine in quiet sleep. Testing will be conducted either in the hospital or in the UIC 
audiology clinic. ABRs will be elicited with click stimuli at 75-85 dB (Biologic version 
5.70 model 317, Bio-logic Navigator Pro-Natus Medical Incorporated, San Carlos, CA).  
Bilateral monaural ABR testing will produce latency and amplitude measures for waves 
I, III and V and inter-peak latencies between I and III, III and V, and I and V in each 
ear. Latencies, amplitudes and inter-peak latencies for each wave will be analyzed 
separately in relation to treatment group and maternal and neonatal iron status 
measures to investigate whether, to what extent and in what location(s) brainstem 
myelination differs by iron status or treatment in the study participants. Recordings will 
be obtained from both ears. At least two recordings will be obtained for each intensity 

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and data from the better ear (shorter interpeak latency I-V) will be used for the final 
analyses of interpeak latencies. 
 
Additional Surveys & Questionnaires. Subjects will complete surveys pertaining to 
socio-demographics, reproductive/mental health, physical activity39 and 
medication/supplement use. If the participant is interested in completing questionnaires 
remotely in advance of the subsequent monthly visits after the baseline visit, they will 
have the option to receive a link via REDCap to the GAD 7, PSS 10, and PPAQ 
questionnaires which should be completed 1-3 days before their research visit. We will 
also obtain data at follow-up and supplement refill visits pertaining to health or 
medication/supplement changes since the last research-related visit that we will verify 
with the EMR. 
 
Satisfaction Survey. Subjects will complete the Treatment Satisfaction Questionnaire for 
Medication (TSQM)40 to determine satisfaction with the interventions. The TSQM will 
assess participants’ satisfaction with the effects and ease of taking the intervention 
supplements. There are 14 items in the TSQM that cover four domains: effectiveness (3 
items), side effects (5 items), convenience (3 items), and global satisfaction (3 items). 
Expected results: Subject satisfaction with the oral Lf intervention will be determined by 
achieving a mean score ≥ 70. 
 
Overall trial acceptability. Semi-structured interviews using questions adapted from 
DasMahaptra et al.41 and Pflugeisen et al.42 will address multiple domains of 
participation in the trial including satisfaction with study visits, study staff, 
reimbursement, and intentions for participation in the trial. The interviews will be 
conducted one day after delivery in the patient’s hospital room. We have used this 
technique in our other studies with success. Responses will inform refinement and 
implementation procedures for the larger efficacy trial. 

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9.0 Data Analysis 
 
Aim 1. Interview data will be transferred to and stored on UIC Box. The interviews will 
be de-identified and transcribed verbatim. Survey questions will be summarized using 
descriptive statistics. Interview transcripts will be analyzed using NVivo, a qualitative 
analysis software. The PI and co-investigators will review transcripts to sort the 
discussion topics into codes and categories. An initial code list will be created. Data will 
be analyzed using the constant comparative method to identify recurrent themes until 
theoretical saturation is achieved. The research team will discuss the codes, categories, 
and themes generated to resolve discrepancies through interpretive discussions, 
consensus building, and refinement as needed. Results will be presented as quotes and 
vignettes as supportive evidence. Measures to maintain trustworthiness will include 
audit trail, peer debriefing, and reflexivity notes. Data will be used to inform and refine 
Aim 2. 
 
Aim 2. We will summarize feasibility metrics and preliminary efficacy using descriptive 
statistics such as frequencies, percentages, means, standard deviations, and effect 
sizes, and corresponding confidence intervals, to estimate a plausible range of values 
for planning a larger efficacy trial. We will examine both intent-to-treat and as-treated 
effects and seek to understand possible differences. Missing data and attrition will be 
monitored continuously and investigated to refine our methods to prevent their 
occurrence. We will seek to identify predictors of missingness to support the missing at 
random assumption for future analyses of a fully powered efficacy trial.43,44 These pilot 
data will also be valuable for identifying possible subgroups for whom oral Lf may be 
more or less effective (e.g., pre-pregnancy BMI ≥ 35.0 kg/m2). We will examine the 
trend plot of each participant and summarize trends by treatment arm to understand the 
pattern of change (linear, quadratic) in inflammation, maternal iron status, and 
hematologic parameters collected monthly over the course of gestation. Mixed effect 
linear regression will be used to explore the relationships among these intensive 
longitudinal measures.45 We will examine sex of the fetus as a biological variable that 
may influence our intervention effects or the relationships among inflammation and 
hematologic parameters.  
 
10.0 Quality Control and Quality Assurance 
The PI will monitor the following items: data quality, completeness, and timeliness; 
adequacy of compliance with goals for recruitment and retention, including those 
related to participation of minorities; adherence to the protocol; and adverse events 
reported by subjects at study and adherence visits. Co-I Liese will evaluate the 
qualitative interview data for quality. For Aim 2, If an adverse event is reported during 
the study, subjects will be requested to report the event to their physician and the PI.  
All adverse events will be documented and reported to the UIC IRB in a timely manner 
based on the type of adverse event (adverse event vs. serious adverse event). We will 
also record and track reasons for dropout. All authorized study personnel will be 
required to notify the PI of any unanticipated problems/adverse events immediately 
upon discovery. The PIs will immediately notify the UIC IRB. The study will be subject 
to annual review and recertification by the OPRS. 
 
11.0 Data and Safety Monitoring 

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The PI will establish a data safety monitoring plan and follow all procedures for the 
protection of human subjects as required by UIC. The purpose of this data and 
safety monitoring plan is: (1) to ensure the safety of study subjects and the validity 
of data; and (2) to produce high quality research while considering both risks and 
benefits. The PI and research assistants will meet weekly to monitor the 
components of the research study. Any instances of adverse events or 
unanticipated problems will be reported according to the standard forms and/or 
procedures that have been established by the IRB. 
 
12.0 Statistical Considerations 
This clinical trial pilot study is not powered to detect significant effects for improvement 
in maternal inflammation or maternal/cord Hb/iron status. This sample size will be 
sufficient for calculating confidence intervals for feasibility and effect size components.46 
 
13.0 Regulatory Requirements 
 
13.1 Informed Consent 
• Trained key personnel will explain the purpose of the study risks and 
benefits and answer any questions that arise from participants in-person 
or via zoom. Prior to engaging participants in any part of this research, an 
electronic informed consent document that contains appropriate human 
subject protection information relevant to the study in plain language will 
be presented to the participants via REDCap. The electronic informed 
consent document will be presented in-person at the baseline visit or prior 
to the baseline visit remotely over Zoom. If consented over Zoom, the 
consent form will still be signed electronically by the research participant 
and key research personnel but be presented remotely. If consented 
remotely, participants will be consented by key research personnel, and it 
will be documented in REDCap with the date and time, how it occurred, 
the name of the witness and the participant, and confirmation that the 
subject received the informed consent document. The same REDCap 
electronic informed consent document will be presented to the participant 
via a survey invitation tool for the participant to sign and date. The survey 
invitation tool will be sent via participant email and verification of receipt 
will be obtained by the participant’s response to the email and their ability 
to complete the form via Zoom. The participant and key research 
personnel will sign and date the form together utilizing the methods listed 
below. Participants will be consented by project staff who have completed 
human subjects training and project- specific training, whether consenting 
is taking place in person or remotely. The participants are advised that 
their participation is completely voluntary and participation/non-
participation does not affect their health care at the University of Illinois 
Hospital and Health Sciences System. Upon agreeance, subjects will 
grant consent to participate in the study by clicking “Yes” in the eConsent 
instrument in REDCap. Both the participant and key research personnel 
will e-sign the consent from in REDCap. All participants will be provided 
with a hard copy of the informed consent form. Whether consented in-

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person or via Zoom, the hard copy of the informed consent form will be 
printed at the Clinical Research Center. Remotely consented participants 
will also receive a copy of the signed consent form via email. Participants 
are free to not answer any questions that they find uncomfortable and 
they are free to withdraw at any time. All electronically signed eConsents 
will be stored on the secure REDCap server. Only key research 
personnel will have access to these documents. 
 
13.2 Subject Confidentiality 
• Participants will be given a unique study ID number to protect their 
identity. Any electronic data will be stored in a password protected, 
restricted access electronic database. Blood and hair samples will be 
stored in a locked laboratory with subject ID. Only authorized research 
personnel will have access to the data. 

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13.3 Unanticipated Problems 
• All authorized study personnel will be required to notify the PI of any 
unanticipated problems adverse events immediately upon discovery. The 
PIs will immediately notify the UIC IRB. The study will be subject to annual 
review and recertification by the OPRS. 
• First trimester Patient Health Questionnaire depression screener (PHQ-8) 
>10 is indicative of moderate to severe depressive symptoms and in our 
previous studies women with a score PHQ-8 >10 had difficulty complying 
with study measures. Women with a score >10 at screening will be 
excluded for study participation. If the participant has a score >10 at 
baseline screening, their provider will be notified via EPIC in basket 
message flagged as high priority and a member of the research team will 
monitor whether the provider read the message from the study team. 

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14.0 References 
 
1. 
Bothwell TH. Iron requirements in pregnancy and strategies to meet them. In: 
American Journal of Clinical Nutrition. Vol 72. ; 2000. doi:10.1093/ajcn/72.1.257s 
2. 
Allen LH. Anemia and iron deficiency: Effects on pregnancy outcome. In: 
American Journal of Clinical Nutrition. Vol 71. ; 2000. doi:10.1093/ajcn/71.5.1280s 
3. 
Rao R, Georgieff MK. Iron in fetal and neonatal nutrition. Semin Fetal Neonatal 
Med. 2007;12(1):54-63. doi:10.1016/j.siny.2006.10.007 
4. 
Shao J, Lou J, Rao R, et al. Maternal serum ferritin concentration is positively 
associated with newborn iron stores in women with low ferritin status in Late 
Pregnancy1-3. J Nutr. 2012;142(11):2004-2009. doi:10.3945/jn.112.162362 
5. 
Friedrisch JR, Friedrisch BK. Prophylactic Iron Supplementation in Pregnancy: A 
Controversial Issue. Biochem Insights. 2017;10:117862641773773. 
doi:10.1177/1178626417737738 
6. 
Brannon PM, Taylor CL. Iron supplementation during pregnancy and infancy: 
Uncertainties and implications for research and policy. Nutrients. 2017;9(12). 
doi:10.3390/nu9121327 
7. 
Koenig MD, Tussing-Humphreys L, Day J, Cadwell B, Nemeth E. Hepcidin and 
iron homeostasis during pregnancy. Nutrients. 2014;6(8):3062-3083. 
doi:10.3390/nu6083062 
8. 
Bah A, Pasricha SR, Jallow MW, et al. Serum hepcidin concentrations decline 
during pregnancy and may identify iron deficiency: Analysis of a longitudinal 
pregnancy cohort in the Gambia. J Nutr. 2017;147(6):1131-1137. 
doi:10.3945/jn.116.245373 
9. 
Nemeth E, Ganz T. Regulation of iron metabolism by hepcidin. Annu Rev Nutr. 
2006;26:323-342. doi:10.1146/annurev.nutr.26.061505.111303 
10. 
Tussing-Humphreys LM, Liang H, Nemeth E, Freels S, Braunschweig CA. Excess 
adiposity, inflammation, and iron-deficiency in female adolescents. J Am Diet 
Assoc. 2009;109(2):297-302. doi:10.1016/j.jada.2008.10.044 
11. 
Darshan D, Anderson GJ. Interacting signals in the control of hepcidin expression. 
In: BioMetals. Vol 22. Biometals; 2009:77-87. doi:10.1007/s10534-008-9187-y 
12. 
Nemeth E, Rivera S, Gabayan V, et al. IL-6 mediates hypoferremia of 
inflammation by inducing the synthesis of the iron regulatory hormone hepcidin. J 
Clin Invest. 2004;113(9):1271-1276. doi:10.1172/jci20945 
13. 
Nemeth E, Valore E V., Territo M, Schiller G, Lichtenstein A, Ganz T. Hepcidin, a 
putative mediator of anemia of inflammation, is a type II acute-phase protein. 
Blood. 2003;101(7):2461-2463. doi:10.1182/blood-2002-10-3235 
14. 
Hales CM, Fryar CD, Carroll MD, Freedman DS, Ogden CL. Trends in obesity 
and severe obesity prevalence in us youth and adults by sex and age, 2007-2008 
to 2015-2016. JAMA - J Am Med Assoc. 2018;319(16):1723-1725. 
doi:10.1001/jama.2018.3060 
15. 
Phillips AK, Roy SC, Lundberg R, et al. Neonatal iron status is impaired by 
maternal obesity and excessive weight gain during pregnancy. J Perinatol. 
2014;34(7):513-518. doi:10.1038/jp.2014.42 
16. 
Jones AD, Zhao G, Jiang YP, et al. Maternal obesity during pregnancy is 
negatively associated with maternal and neonatal iron status. Eur J Clin Nutr. 

Feasibility of oral lactoferrin to prevent iron deficiency anemia in obese pregnancy 
Page 29 of 27 
Version 24 
09/17/2024 
 
 
2016;70(8):918-924. doi:10.1038/ejcn.2015.229 
17. 
Dao MC, Sen S, Iyer C, Klebenov D, Meydani SN. Obesity during pregnancy and 
fetal iron status: Is hepcidin the link? J Perinatol. 2013;33(3):177-181. 
doi:10.1038/jp.2012.81 
18. 
Garcia-Valdes L, Campoy C, Hayes H, et al. The impact of maternal obesity on 
iron status, placental transferrin receptor expression and hepcidin expression in 
human pregnancy. Int J Obes. 2015;39(4):571-578. doi:10.1038/ijo.2015.3 
19. 
Cao C, Pressman EK, Cooper EM, Guillet R, Westerman M, O’Brien KO. 
Prepregnancy Body Mass Index and Gestational Weight Gain Have No Negative 
Impact on Maternal or Neonatal Iron Status. Reprod Sci. 2016;23(5):613-622. 
doi:10.1177/1933719115607976 
20. 
Flynn AC, Begum S, White SL, et al. Relationships between maternal obesity and 
maternal and neonatal iron status. Nutrients. 2018;10(8). 
doi:10.3390/nu10081000 
21. 
Lozoff B. Perinatal iron deficiency and the developing brain. Pediatr Res. 
2000;48(2):137-139. doi:10.1203/00006450-200008000-00003 
22. 
Lepanto MS, Rosa L, Cutone A, Conte MP, Paesano R, Valenti P. Efficacy of 
lactoferrin oral administration in the treatment of anemia and anemia of 
inflammation in pregnant and non-pregnant women: An interventional study. Front 
Immunol. 2018;9(SEP):2123. doi:10.3389/fimmu.2018.02123 
23. 
Rosa L, Cutone A, Lepanto MS, Paesano R, Valenti P. Lactoferrin: A natural 
glycoprotein involved in iron and inflammatory homeostasis. Int J Mol Sci. 
2017;18(9). doi:10.3390/ijms18091985 
24. 
Frioni A, Conte MP, Cutone A, et al. Lactoferrin differently modulates the 
inflammatory response in epithelial models mimicking human inflammatory and 
infectious diseases. BioMetals. 2014;27(5):843-856. doi:10.1007/s10534-014- 
9740-9 
25. 
Rezk M, Dawood R, Abo-Elnasr M, Al Halaby A, Marawan H. Lactoferrin versus 
ferrous sulphate for the treatment of iron deficiency anemia during pregnancy: A 
randomized clinical trial. J Matern Neonatal Med. 2016;29(9):1387-1390. 
doi:10.3109/14767058.2015.1049149 
26. 
Medical Dictionary for Regulatory Activities. https://www.meddra.org. 
27. 
Dimenäs E, Glise H, Hallerbäck B, Hernqvist H, Svedlund J, Wiklund I. Quality of 
life in patients with upper gastrointestinal symptoms: An improved evaluation of 
treatment regimens? Scand J Gastroenterol. 1993;28(8):681-687. 
doi:10.3109/00365529309098272 
28. 
Slattery ML, Caan BJ, Duncan D, Dennis Berry T, Coates A, Kerber R. A 
computerized diet history questionnaire for epidemiologic studies. J Am Diet 
Assoc. 1994;94(7):761-766. doi:10.1016/0002-8223(94)91944-5 
29. 
Kleinman KP, Oken E, Radesky JS, Rich-Edwards JW, Peterson KE, Gillman 
MW. How Should Gestational Weight Gain Be Assessed? A Comparison of 
Existing Methods and a Novel Method, Area under the Weight Gain Curve. 
30. 
Institute of Medicine (US) Committee on the Prevention D and M of IDAAUSC and 
W of CA, Earl R, Woteki CE. Iron Deficiency Anemia: Recommended Guidelines 
for the Prevention, Detection, and Management Among U.S. Children and 
Women of Childbearing Age. 1993. 

Feasibility of oral lactoferrin to prevent iron deficiency anemia in obese pregnancy 
Page 30 of 27 
Version 24 
09/17/2024 
 
 
31. 
Mei Z, Namaste SM, Serdula M, et al. Adjusting total body iron for inflammation: 
Biomarkers Reflecting Inflammation and Nutritional Determinants of Anemia 
(BRINDA) project. Am J Clin Nutr. 2017;106(Suppl 1):383S-389S. 
doi:10.3945/ajcn.116.142307 
32. 
Ganz T, Olbina G, Girelli D, Nemeth E, Westerman M. Immunoassay for human 
serum hepcidin. Blood. 2008;112(10):4292-4297. doi:10.1182/blood-2008-02- 
139915 
33. 
Forero R, Nahidi S, De Costa J, et al. Application of four-dimension criteria to 
assess rigour of qualitative research in emergency medicine. BMC Health Serv 
Res. 2018;18(1). doi:10.1186/s12913-018-2915-2 
34. 
Viteri FE. Iron endowment at birth: maternal iron status and other influences. Nutr 
Rev. 2011;69(SUPPL. 1):S3-S16. doi:10.1111/j.1753-4887.2011.00449.x 
35. 
Jewett Don, L., Romano Michael, N. & Williston John, S. Human Auditory Evoked 
Potentials: Possible Brain Stem Components Detected on the Scalp. Science 
167, 1517-1518, doi:10.1126/science.167.3924.1517 (1970). 
36. 
Amin, S. B., Orlando, M. & Wang, H. Latent iron deficiency in utero is associated 
with abnormal auditory neural myelination in ≥ 35 weeks gestational age infants. 
J Pediatr 163, 1267-1271, doi:10.1016/j.jpeds.2013.06.020 (2013). 
37. 
Larkin, E. C. & Rao, G. A. Importance of Fetal and Neonatal Iron: Adequacy for 
Normal Development of Central Nervous System in Brain, behaviour, and iron in 
the infant diet 43-57 (Springer, 1990) 
38. 
Roncagliolo, M., Garrido, M., Walter, T., Peirano, P. & Lozoff, B. Evidence of 
altered central nervous system development in infants with iron deficiency 
anemia at 6 mo: delayed maturation of auditory brainstem responses. The 
American Journal of Clinical Nutrition 68, 683-690, doi:10.1093/ajcn/68.3.683 
(1998). 
39. 
Chasan-Taber L, Schmidt MD. Pregnancy Physical Activity Questionnaire. Can J 
Public Health. 2016;106(8):e563. doi:10.17269/cjph.106.5397 
40. 
Atkinson MJ, Sinha A, Hass SL, et al. Validation of a general measure of 
treatment satisfaction, the Treatment Satisfaction Questionnaire for Medication 
(TSQM), using a national panel study of chronic disease. Health Qual Life 
Outcomes. 2004;2. doi:10.1186/1477-7525-2-12 
41. 
DasMahapatra P, Raja P, Gilbert J, Wicks P. Clinical trials from the patient 
perspective: survey in an online patient community. BMC Health Serv Res. 
2017;17(1). doi:10.1186/s12913-017-2090-x 
42. 
Pflugeisen BM, Rebar S, Reedy A, Pierce R, Amoroso PJ. Assessment of clinical 
trial participant patient satisfaction: A call to action. Trials. 2016;17(1):483-483. 
doi:10.1186/s13063-016-1616-6 
43. 
Graham JW, Hofer SM, Piccinin AM. Analysis with missing data in drug 
prevention research. NIDA Res Monogr. 1994;142:13-63. 
44. 
Graham JW. Missing Data Analysis: Making It Work in the Real World. Annu Rev 
Psychol. 2009;60(1):549-576. doi:10.1146/annurev.psych.58.110405.085530 
45. 
Stadler G, Bolger N, Ochsner S, et al. A good starting point: Sources of variance 
in dyadic longitudinal data and reliability of change. Psychol Heal. 2013;28:44-44. 
46. 
Moore CG, Carter RE, Nietert PJ, Stewart PW. Recommendations for planning 
pilot studies in clinical and translational research. Clin Transl Sci. 2011;4(5):332- 
337. doi:10.1111/j.1752-8062.2011.00347. 

Feasibility of oral lactoferrin to prevent iron deficiency anemia in obese pregnancy 
Page 31 of 27 
Version 24 
09/17/2024