Periconceptional iron supplementation does not reduce anemia or
improve iron status among pregnant women in rural Bangladesh1–4
Amina Z Khambalia, Deborah L O’Connor, Colin Macarthur, Annie Dupuis, and Stanley H Zlotkin
ABSTRACT
Background: There is a growing interest in periconceptional iron
supplementation in developing countries by researchers and policy
makers; however, there are no randomized controlled trials that
examine the effectiveness of this strategy in decreasing anemia
during pregnancy.
Objective: The aim was to determine whether periconceptional iron
supplementation reduces anemia during pregnancy.
Design: A randomized, double-blind, controlled trial was conducted
in rural Bangladesh. Married, nulliparous women were randomly
assigned to receive daily iron and folic acid (IFA; 60 mg ferrous
fumarate and 400 lg folic acid) (n = 134) or folic acid (FA; 400 lg)
(n = 138) in the form of a powdered supplement added to food.
Women were followed until pregnancy or the end of 9 mo. Primary
outcomes included hemoglobin, plasma ferritin, and plasma trans-
ferrin receptor concentrations.
Results: Among 88 pregnant women, periconceptional IFA in com-
parison with FA did not affect anemia or iron status at 15 wk
gestation. However, each 1% increase in adherence was associated
with a 10-g/L increase in change in hemoglobin from baseline (P =
0.03), and those who initiated supplementation at a mean (6SD)
time of 72.9 6 57.8 d before conception showed a 7.3-g/L increase
in change in hemoglobin from baseline compared with those who
initiated supplementation at 26.3 6 12.3 d after conception (P =
0.01). Among 146 nonpregnant women, IFA decreased anemia
(odds ratio: 0.19; 95% CI: 0.04, 0.95) and improved iron stores
(P = 0.001) more than did FA.
Conclusion: Good adherence and initiation of supplementation be-
fore conception are needed to reduce anemia during early preg-
nancy. This trial was registered at www.clinicaltrials.gov as
NCT00953134. Am J Clin Nutr 2009;90:1295–302.
INTRODUCTION
The global figures for the prevalence of anemia among non-
pregnant and pregnant women are 35% and 51%, respectively (1).
The major cause of nutritional anemia is iron deficiency (2). Iron
intervention programs during pregnancy have traditionally been
initiated between 10 and 15 wk of gestation when a pregnant
woman attends her first prenatal visit (3, 4). However, ’50% of
women in developing world settings enter pregnancy with in-
adequate iron stores to meet pregnancy requirements (3, 5).
Recent evidence suggests that anemia before conception may be
associated with an increased risk of low-birth-weight deliveries
and fetal growth restriction (6). Maternal iron deficiency in the
first trimester of pregnancy has been associated with significant
Am J Clin Nutr 2009;90:1295–302. Printed in USA. Ó 2009 American Society for Nutrition
decrements in infant size at birth (7, 8), and well-designed
randomized controlled trials of iron supplementation starting
early in pregnancy have shown significant reductions in the
frequency of low-birth-weight infants (7, 9, 10).
Periconceptional iron and folic acid (IFA) supplementation has
been suggested as a complementary strategy to traditional iron
supplementation that commences after pregnancy is confirmed
and/or a woman knows she is pregnant (11, 12). The rationale for
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IFA supplementation in nonpregnant women in developing
countries is to treat preexisting anemia, increase body iron stores,
and reduce the risk of neural tube defects. Iron supplementation
programs during pregnancy have shown limited effectiveness
because of low adherence, inefficient health care service delivery,
and high rates of preexisting anemia, infection, and adolescent
childbearing (3, 13, 14). In young infants, poor adherence to
standard iron drops led to the development of a powdered
micronutrient supplement as an alternative delivery method.
Powdered micronutrient supplements are a home-fortification
method whereby micronutrients are added to foods prepared in
the home. The use of a powdered micronutrient supplement
among adolescent and adult women has not been well studied. A
single study found that a powdered micronutrient supplement was
as efficacious as a tablet supplement in eliciting a hemoglobin
response among pregnant women in Bangladesh (15). Further
1
From the Department of Nutritional Sciences (AZK, DLO, and SHZ),
the Department of Pediatrics (SHZ and CM) and the Dalla Lana School of
Public Health (SHZ), University of Toronto, Toronto, Canada; the Depart-
ment of Pediatrics and Research Institute, Hospital for Sick Children, Tor-
onto, Canada (AZK, DLO, AD, and SHZ); and the Bloorview Research
Institute, Toronto, Canada (CM and AD).
2
The sources of funding for the research (Foundation of the Hospital for
Sick Children and HJ Heinz Company Foundation) had no input into the
design, analysis or interpretation of the data.
3
Supported by the Foundation of the Hospital for Sick Children and the
HJ Heinz Company Foundation for studies on micronutrient deficiencies and
the use of the micronutrient powder, Sprinkles, to treat and prevent micro-
nutrient deficiencies (to SHZ). AZK was supported by the Enid Walker
Award and the Helen Marion Walker Award from the Women’s College
Research Institute and the Danone Doctoral Award in Education and Com-
munication from the Danone Institute.
4
Address correspondence and requests for reprints to SH Zlotkin. Divi-
sion of Gastroenterology, Hepatology, and Nutrition, Hospital for Sick Chil-
dren, 555 University Avenue, Suite 8260 Toronto, ON, Canada M5G1X8.
E-mail: stanley.zlotkin@sickkids.ca.
Received July 7, 2009. Accepted for publication August 29, 2009.
First published online September 30, 2009; doi: 10.3945/ajcn.2009.28350.
1295
1296 KHAMBALIA ET AL
studies are needed to examine alternative methods of supple-
mentation among other high-risk age groups.
In Bangladesh, anemia affects 43% of adolescents, 45% of
nonpregnant women, and 49% of pregnant women (16). In 2007
the National Strategy for Anemia Prevention and Control in
Bangladesh included adolescents and newly married women as
important target groups for IFA supplementation programs (17).
However, no study to date has examined the effect of peri-
conceptional IFA supplementation on iron status during preg-
nancy in Bangladesh. The primary aim of our study was to
examine the effect of daily periconceptional IFA compared with
folic acid (FA) in a powdered supplement on anemia, hemoglobin
concentrations, and other iron indicators during pregnancy. The
periconceptional period was defined as the period before the
ascertainment of a pregnancy during the study period. Outcomes
were also measured for women who did not become pregnant
during the study period.
SUBJECTS AND METHODS
Study setting and design
The study site was a 22-square-kilometer area in Kaliganj, 1 of
the 5 administrative divisions in the Gazipur district in central
Bangladesh. This rural area has a high population density, relies
primarily on agricultural labor, and is a predominantly Muslim
population. We conducted a randomized controlled trial from
March 2007 to the end of February 2008. Participants were
randomly assigned to receive IFA or FA and then followed up
monthly for 9 mo or until they became pregnant. The primary
outcome was hemoglobin concentration among women who
became pregnant (IFA compared with FA). On the basis of our
sample size calculations, we estimated that 30 pregnancies per
group would be an adequate sample size to detect a clinically and
statistically significant difference of 8 g/L in hemoglobin con-
centration between experimental and control groups (on the basis
of 80% power and an SD of 11 g/L) (18). On the basis of a pilot
study, we assumed that 20% of women would become pregnant;
thus, we aimed to recruit 152 women per group. To identify
eligible women, field interviewers made house-to-house visits
starting with households located nearest to the medical clinic
where blood samples would be collected and moved outward
from this location in a circular fashion. Women were ineligible if
they met at least one of the following criteria: not married, had
previously given birth, age 40 y old, not a permanent house-
hold member (living in household ,6 mo), not living in same
household as their husband, using an implant form of birth
control, previous surgery to prevent pregnancy, had used iron
supplements within the previous 3 mo, were known to be
pregnant at enrollment, or were identified as severely anemic at
baseline (hemoglobin concentration of ,70 g/L) (1). Severely
anemic women (n = 1) were excluded from the study and pro-
vided with iron supplements. At recruitment, women who were
otherwise deemed eligible but had not menstruated for .45 d
were administered a urine pregnancy test. Only women with
a negative urine pregnancy test were included in the study.
Intervention
Women were randomly assigned to receive daily iron (60 mg
as ferrous fumarate) and FA (400 lg) or FA alone (400 lg). The
iron dose corresponds to recommendations for areas in which
the prevalence of anemia is .40% (19). The FA dose is based on
the amount of synthetic FA recommended for women who plan
a pregnancy (or are capable of becoming pregnant) for pre-
vention of a neural tube defect (20). FA was used as the control
because the protective effect of folate against the development
of neural tube defects, specifically anencephaly and spina bifida,
is well established (21, 22). In keeping with standard care in
Bangladesh, once a pregnancy was confirmed, women were
removed from the study and advised to take IFA supplements
(17). Nutrients were delivered in a powdered form in individual
sachets. Sachets were identical in appearance, except for a small
embossed letter on the back of the sachet to identify the treat-
ment group (“A” or “B”). The use of these sachets, which
contained powdered micronutrients, is a home-fortification
strategy that allows micronutrients to be added to semisolid
foods for consumption (Sprinkles; Ped-Med Ltd, Toronto,
Canada). Powdered micronutrient supplements have success-
fully been evaluated for efficacy in infants and young children
and for acceptability and safety in diverse settings (23, 24). Each
woman was given 35 sachets at enrollment and at each moni-
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toring visit (30 sachets for a 1-mo daily supply and 5 extra sa-
chets). Participants were instructed to pour the entire contents of
the package into any semiliquid food after the food had been
cooked and at a temperature acceptable to eat and then to mix it
into the food. Participants were told to use one full package per
day at any mealtime.
Data Collection
Study participants were administered a standardized baseline
interview on socioeconomic and reproductive health factors.
Anthropometric measurements included weight and height. Field
interviewers visited participants once a month to replenish
supplies of sachets, to count the number of empty sachet
packages, to monitor side effects, and to encourage women to
consume the contents of the sachets. For each participant, ad-
herence was calculated as percentage of the total eligible doses
consumed during the study period. During monthly visits, par-
ticipants were asked if they had menstruated in the past 45 d.
After 2 missed menstruations, a urine-based human gonadotropin
pregnancy test was administered. Venous blood samples were
collected at baseline and after a pregnancy was ascertained or
after 9 mo among nonpregnant women. Interviewers read an
approved assent form to the study participants and obtained oral
and thumbprint consent. Ethical approval was obtained from the
Medical Research Council in Bangladesh and the Hospital for
Sick Children in Toronto, Canada.
Laboratory Analysis
Hemoglobin was assessed using a hemoglobinometer
(HemoCue, Angelholm, Sweden). Hemocue machines were
checked daily against a standard. Venous blood was centrifuged
at 1500 · g for 10 min, and plasma was separated from cells.
Aliquots of plasma were stored at 220°C and shipped in a fro-
zen state on dry ice to the International Centre for Diarrheal
Disease Research, Bangladesh (ICDDR,B) for plasma ferritin
and transferrin receptor (TfR) analyses and to the Hospital for
 PERICONCEPTIONAL IRON SUPPLEMENTATION
Sick Children (Toronto, Canada) for plasma folate analyses.
Aliquots of plasma for folate analyses included sodium ascor-
bate (1%, wt:vol) to prevent the oxidation of folate. Plasma
ferritin was measured by using an enzyme-linked immunosor-
bent assay method with a commercial kit (Roche Diagnostics,
Indianapolis, IN) with an interassay CV of ,4.0%. Plasma TfR
was measured by using an enzyme-linked immunosorbent assay
method with a commercial kit (BioVendor, Modrice, Czech
Republic) with an interassay CV of ,3.3%. C-reactive protein
was measured at baseline by using a latex agglutination method
and the automated method on the Roche Integra 400 (Roche
Diagnostics) with an analytic goal of 610%. Plasma folate
concentrations were measured by using a microbiologic assay
that uses the test organism Lactobacillus rhamnosus (ATCC
7649; American Type Tissue Culture Collection, Manassas, VA)
as described by Molloy and Scott (25). The accuracy and re-
producibility of these assays were assessed by using a whole-
blood control standard with a certified value (29.5 nmol/L)
(Whole Blood 95/528; National Institute of Biological Standards
and Control, Hertfordshire, United Kingdom). Analysis of the
whole-blood standard in our laboratory yielded a folate content
of 31.0 6 2.4 nmol/L with an interassay CV ,9.4%.
Statistical analysis
An intent-to-treat analysis was performed. Analyses were
conducted separately for pregnant women (IFA compared with
FA) and nonpregnant women (IFA compared with FA). The main
outcomes were anemia and changes in hemoglobin, plasma
ferritin, and plasma TfR concentrations from baseline. Anemia
was defined as hemoglobin ,120 g/L among nonpregnant
women and hemoglobin ,110 g/L among pregnant women (26,
27). Plasma ferritin concentrations were log-transformed. As
a secondary analysis, change in plasma folate concentrations
from baseline were examined in both treatment groups. Plasma
folate concentrations were also examined by using linear re-
gression analysis with the independent variable adherence to
examine the accuracy of self-reported intake of supplements.
Univariate analyses of hematologic indexes were performed by
using t tests for continuous variables and chi-square tests for
categorical variables. Multivariate analyses were performed by
using linear regression models for continuous response variables
and logistic regression for the binary response variable for
anemia.
Multivariate analyses for pregnant women were adjusted for
treatment group, literacy, adherence, gestational age, and timing
of supplementation. Gestational age was the number of days since
last menstruation reported by women who were identified as
pregnant after 2 missed menstruations and a positive urine
pregnancy test. Timing of supplementation is the number of days
between commencement of supplementation and conception
(days of last menses minus first day of supplementation). Timing
of supplementation was examined as a continuous variable and as
a categorical variable by categorizing pregnant women into those
who initiated supplementation before and after conception, re-
spectively. Multivariate analyses for nonpregnant women were
adjusted for literacy and adherence. A 2-sided P value of 0.05
indicated statistical significance. Statistical analyses were con-
ducted by using SAS (version 9.1; SAS Institute, Cary, NC).
1297
RESULTS
Of the 15,357 women who were initially assessed for in-
clusion, 303 were eligible to participate (Figure 1). A total of
15,085 women were excluded because they did not meet in-
clusion criteria [eg, not married (n = 7407)], had already given
birth (n = 6387), no longer married (n = 26), age . 40 y old (n =
976), not living with their husband (n = 158), not a permanent
household member (n = 47), using a permanent form of con-
traception (n = 19), use of an iron supplement in the past 3 mo
(n = 2), and severely anemic (n = 1). A small number refused to
participate (n = 25) or were unreachable (n = 6). A total of 272
women were randomized to supplement groups, giving an ac-
ceptance rate of 89.8%.
Baseline characteristics have been described elsewhere (28).
No differences existed between groups, except in literacy (see
Table S1 under “Supplemental data” in the online issue).
Women who were randomly assigned to the IFA group had
a higher literacy rate (able to read and write a letter) than those
in the FA group (P = 0.02). A separate comparison of baseline
and follow-up measures for pregnant and nonpregnant women
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showed no significant differences by supplement group, except
for a significantly higher proportion of literate pregnant women
in the IFA group (Table 1). Literacy was adjusted for in all
multivariate analyses. There were no statistically significant
differences in baseline characteristics or in biochemical indexes
for iron and folate between women who completed the study and
those lost to follow-up (data not shown).
During the 9 mo trial, 88 pregnancies were confirmed by urine
testing: 43 in the IFA group and 45 in the FA group. The median
(range: 0–100) time since last menstruation was 15 wk (6–21 wk)
and was not statistically different between the IFA and FA groups
(P = 0.69). There was an unexpectedly long delay between
recruitment and the first monthly follow-up visit. Due to this
delay, some women who initially reported that they were
menstruating at recruitment (and thus were eligible for the
study) had stopped menstruating (had become pregnant) by the
first monthly visit. For this reason, a small number of women
(n = 31) had an unidentified pregnancy between recruitment and
the first monthly follow-up visit. These 31 women received
supplements for a mean (6SD) time of 26.3 6 12.3 d after
conception. The majority of women (n = 57) started supple-
mentation before conception (72.9 657.8 d before estimated
conception). The mean (6SD) number of days between re-
cruitment and the first monthly follow-up visit was 40.3 6 9.9
d for 229 participants. Five participants were not reached until
the next visit, ’1 mo later. The number of days between initi-
ation of supplementation and conception did not differ by sup-
plement group (P = 0.27). The median number of days from
when supplementation was commenced and conception oc-
curred, as indicated by last menstruation was 25.5 d and ranged
(first–third quartiles) from 216.5 to 82.5 d.
The results of multivariate analyses showed that daily peri-
conceptional IFA supplementation compared with FA supple-
mentation did not reduce anemia or improve iron status among
pregnant women (Table 2). Among pregnant women, overall
percentage adherence and timing of supplementation in relation
to conception were found to be significantly associated with
change in hemoglobin concentration from baseline (Figure 2).
Each 1% increase in percentage adherence was associated with
1298 KHAMBALIA ET AL
FIGURE 1. Participant flowchart according to the Consolidated Standards of Reporting Trials (CONSORT). *Nonpregnant women who, by error, had their
final venous blood measurement taken before 9 mo of supplementation were excluded from analyses.
a 10-g/L improvement in change in hemoglobin from baseline
(P = 0.03). Women who initiated supplementation before con-
ception showed a 7.3 g/L greater change in maternal hemoglo-
bin from baseline compared with those who initiated
supplementation after conception (P = 0.01). Timing of sup-
plementation as a continuous variable was not significantly as-
sociated with change in hemoglobin (P = 0.37).
Among nonpregnant women, daily periconceptional IFA
supplementation for 9 mo compared with FA supplementation
significantly improved iron stores (P = 0.001) (Table 3). There
were no significant differences between IFA and FA groups in
changes in hemoglobin from baseline. However, with the use of
the cutoff of a hemoglobin concentration 120 g/L to define
anemia among nonpregnant women, IFA supplementation sig-
nificantly reduced anemia compared with FA supplementation
(odds ratio: 0.19; 95% CI: 0.04, 0.95). There were no significant
differences between IFA and FA groups in changes in plasma
TfR concentrations from baseline.
At baseline, the median (first–third quartile) plasma folate
concentration was 15.9 nmol/L (12.1–20.5 nmol/L) for all
women. The median (first–third quartile) change in plasma folate
concentration from baseline when women in both treatment
groups were pooled for statistical analysis was 11.7 nmol/L (2.5–
26.5 nmol/L) in pregnant women and 11.4 nmol/L (0.5–24.6
nmol/L) in nonpregnant women. The prevalence of inadequate
folate status (,10 nmol/L) decreased in both groups from 8.8%
to 7.5% (P = 0.71) among pregnant women and from 16.3% to
7.8% (P = 0.03) among nonpregnant women. Self-reported ad-
herence was significantly associated with change in plasma
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folate concentrations from baseline among nonpregnant women
(P , 0.0001) but not among pregnant women (P = 0.20), re-
gardless of whether the pregnant women initiated supplemen-
tation before or after conception (P = 0.18). Each 1% increase in
overall percentage adherence was associated with a 0.42-nmol/L
improvement in change in plasma folate from baseline (P ,
0.0001) among nonpregnant women compared with only a 0.14-
nmol/L improvement in change in plasma folate from baseline
(P = 0.20) among pregnant women.
DISCUSSION
The design of the current study was unique in 2 ways: the
intervention (IFA or FA) commenced before or very early in the
first trimester of pregnancy rather than later during pregnancy,
and the supplement was provided as a powder added to food
rather than a more traditional tablet or capsule. Among pregnant
women in the current study, periconceptional IFA supplemen-
tation compared with FA supplementation did not significantly
decrease anemia at 15 wk of gestation. Results indicate that
at least a moderate level of adherence to iron supplementation well
before conception is necessary to prevent anemia during early
pregnancy. Among nonpregnant women, 9 mo iron supplemen-
tation significantly decreased anemia and improved iron stores.
Our final sample size of .30 women per treatment group
provided us with 80% power to detect a difference of 8 g/L
(611 g/L) in hemoglobin concentration at a 0.05 significance
level (18). Our study showed that the actual difference in he-
moglobin concentration between the treatment groups was much
 PERICONCEPTIONAL IRON SUPPLEMENTATION 1299
TABLE 1
Selected characteristics of study participants by treatment group and pregnancy status at follow-up1
Pregnant Not pregnant

Iron and folic acid Folic acid Iron and folic acid Folic acid
(n = 43) (n = 45) P value (n = 75) (n = 71) P value

Age (y) 19.1 6 3.12 18.9 6 2.6 0.76 21.8 6 5.6 22.9 6 6.4 0.27
Age at menarche (y) 12.9 6 0.7 12.8 6 0.8 0.70 13.0 6 0.8 12.9 6 0.7 0.59
Age at marriage (y) 17.7 6 3.1 17.6 6 2.8 0.88 17.8 6 3.5 18.3 6 4.5 0.44
Can read and write a letter (%) 100 86.7 0.01 89.3 81.7 0.18
Had a previous miscarriage (%) 4.7 8.9 0.68 13.3 7.0 0.21
Uses a contraceptive device (%) 25.6 33.3 0.43 17.3 28.2 0.12
Weight (kg) 46.3 6 7.0 46.7 6 6.6 0.75 49.8 6 9.9 49.9 6 8.5 0.93
Height (cm) 150.7 6 5.5 150.2 6 5.4 0.66 149.4 6 5.5 149.8 6 5.3 0.65
BMI (kg/m2) 20.4 6 2.6 20.7 6 2.8 0.52 22.3 6 4.2 22.2 6 3.5 0.93
Hemoglobin (g/L) 123.0 6 8.8 122.6 6 11.6 0.85 122.4 6 9.9 121.0 6 11.5 0.43
Plasma ferritin (lg/L) 37.1 (24.3–54.9)3 38.6 (25.7–60.3) 0.86 34.6 (26.2–53.6) 42.3 (25.0–61.3) 0.81
Plasma transferrin receptor (mg/L) 3.2 (2.5–3.7) 3.4 (2.3–4.0) 0.19 3.0 (2.5–3.7) 2.0 (2.5–3.8) 0.97
C-reactive protein (mg/L) 1.3 6 1.7 1.4 6 1.7 0.79 2.4 6 3.3 2.2 6 3.0 0.82
Plasma folate (nmol/L) 17.6 6 7.5 18.1 6 6.5 0.76 15.4 6 6.8 17.2 6 6.5 0.10
Anemia (%)4 44.2 31.1 0.21 37.3 35.2 0.70
Iron deficiency (%)5 2.3 8.9 0.18 4.0 7.0 0.42

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Iron deficiency and anemia (%)6 2.3 8.9 0.18 4.0 7.0 0.42
Total no. of sachets used 74.3 6 55.6 88.6 6 53.7 0.22 175.5 6 52.6 176.8 6 56.5 0.88
Overall percentage adherence 53.8 6 27.8 61.5 6 25.7 0.18 65.0 6 19.5 65.5 6 20.9 0.88
No. of times reported side effects
Loose motion 0.3 6 0.6 0.3 6 0.7 0.92 1.2 6 1.4 0.9 6 1.3 0.34
Constipation 1.8 6 1.7 1.9 6 1.6 0.93 2.8 6 2.5 2.6 6 2.3 0.62
Nausea 2.7 6 1.7 2.9 6 1.4 0.43 3.3 6 2.4 2.4 6 2.4 0.04
Dark stools 1.9 6 2.0 0.8 6 1.3 0.004 5.1 6 2.3 1.6 6 1.7 ,0.0001
Length of supplementation (d) 134.5 6 62.0 147.1 6 67.8 0.37 277.5 6 5.3 276.9 6 4.5 0.48
1
n = 272. Values were calculated by using univariate analyses.
2
Mean 6 SD (all such values).
3
Median; range (first–third quartile) in parentheses (all such values).
4
Defined as hemoglobin ,110 and ,120 g/L in pregnant and nonpregnant women, respectively.
5
Defined as plasma ferritin concentration ,12 lg/L.
6
Defined as hemoglobin concentration ,120 g/L and plasma ferritin concentration ,12 lg/L.

smaller (mean: 1.00 g/L; 95% CI: 24.1, 6.1) and had a larger change in maternal hemoglobin among pregnant women despite
degree of individual variability. Whereas a larger sample size iron supplementation.
would have been needed to detect a statistically significant A possible explanation for why maternal hemoglobin and iron
difference, results of our study found no clinically significant stores were not significantly affected by IFA supplementation in

TABLE 2
Change in hemoglobin and iron indicators by treatment group among rural Bangladeshi women who became pregnant1
Change
n Baseline Follow-up (follow-up 2 baseline) P value

Hemoglobin (g/L)
FA 45 125.0 (116.0 to 129.0) 114.0 (108.0 to 123.0) 210.0 (213.0 to 25.0)
IFA 43 122.0 (116.0 to 130.0) 112.0 (105.0 to 124.0) 28.0 (218.0 to 0.0) 0.76
Plasma ferritin (lg/L)
FA 38 37.3 (24.9 to 61.4) 48.5 (24.5 to 70.5) 8.0 (23.2 to 22.2)
IFA 39 36.8 (24.3 to 54.9) 47.2 (31.4 to 60.8) 5.7 (23.2 to 24.4) 0.92
Plasma transferrin receptor (mg/L)
FA 41 3.4 (2.3 to 4.0) 2.4 (1.9 to 3.0) 20.8 (21.4 to 20.4)
IFA 40 3.2 (2.5 to 3.7) 2.2 (1.9 to 2.7) 20.7 (21.4 to 20.3) 0.66
Anemia (%)2
FA 43 31.1 64.4 +33.3
IFA 45 44.2 65.1 +20.9 0.93
1
Values are medians or percentages, where indicated; ranges (first–third quartiles) in parentheses. FA, folic acid; IFA, iron and folic acid. Multivariate
linear regression analyses were performed for change in hematologic indexes controlled for treatment group, literacy, and initiation of supplementation (before
or after pregnancy) (n = 88).
2
Defined as hemoglobin ,110 g/L among pregnant women.
1300 KHAMBALIA ET AL

for a much larger randomized controlled trial in Nepal that

FIGURE 2. Change in hemoglobin concentrations (g/L) from baseline by
overall percentage adherence among pregnant women who initiated
supplementation before pregnancy or in early pregnancy. Values on the x
axis represent quartiles for overall percentage adherence (minimum, first,
second, third, and maximum). Error bars represent upper and lower 95% CIs
for adjusted mean estimates of the change in hemoglobin from baseline
found that iron supplementation in early pregnancy to women
with initially poor nutritional status increased mean birth weight
by 37 g (95% CI, 216, 90) compared with control (7). A dis-
tinguishing feature of both of those studies (7, 9) and the present
study is that iron supplementation started earlier in pregnancy as
compared with previous iron supplementation trials that have
been recently reviewed (4, 30). Unfortunately, the current study
was not powered to detect differences in infant birth weight;
therefore, we were unable to determine whether periconcep-
tional iron supplementation led to significantly heavier infants
compared with a control group. On the basis of calculations by
Rasmussen (4), we would have needed 250 women per treat-
ment group to detect a 100-g difference in birth weight. The
effect of periconceptional iron supplementation on functional
outcomes in both the mother and infant is an important area of
future research and would benefit from investigation of the bi-
ological pathways by which maternal iron status affects fetal
metabolism and growth (29).
It is also possible that IFA supplementation had no effect
among pregnant women because of low adherence (57.7% 6

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performed by using multivariate analysis (n = 88). To clearly see the size
of the error bars, upper error bars are shown for women supplemented before
pregnancy and lower error bars are shown for women supplemented in early
pregnancy. P values for timing of supplementation and overall percentage
adherence are adjusted for treatment group, literacy, and gestational age.
pregnant women is that iron status was already fairly good at
baseline. Among pregnant women, the mean (6SD) ferritin
concentration at baseline was 54.9 6 38.5, and 6% of these
women were iron deficient (ferritin ,12 lg/L). It has been
suggested that the placenta and fetus may be able to compete
effectively for iron among mothers with good iron status (29). A
study in the United States found that iron supplementation ini-
tiated in early pregnancy to initially iron-replete women did not
improve maternal iron status and yet led to a surprisingly large
increase in mean (6SD) birth weight (206 6 565 g; P = 0.01)
among infants whose mothers received iron compared with
a placebo (9). As stated by Rasmussen and Stotlzfus (29), those
results (9) may have been interpreted as spurious had it not been
26.9%) and a relatively short period of supplementation. Preg-
nant women were in the study for an average of 136.3 6 36.6
fewer days and therefore consumed the contents of 94.5 6 54.5
fewer sachets compared with nonpregnant women. On the as-
sumption that plasma folate concentrations would reflect ad-
herence, we attempted to verify self-reported adherence by
examining the association between adherence and plasma folate
concentrations. We found that the association was statistically
significant among nonpregnant but not pregnant women. Preg-
nant women may have reported their adherence inaccurately or
were at different stages of hemodilution at the time of blood
sampling, which affected the relation between self-reported in-
take and plasma folate concentration.
The level of adherence in the current study was similar to
percentage adherence (61%) reported in a study in Bangladesh
that used a pill bottle with an electronic counting device to
compare the effectiveness of a daily versus weekly regimen of
iron supplementation during pregnancy (31). However, adher-
ence among pregnant women in this study (57.7%) was much

TABLE 3
Change in hemoglobin and iron indicators by treatment group among rural Bangladeshi women who did not become pregnant1
Change
n Baseline Follow-up (follow-up 2 baseline) P value

Hemoglobin (g/L)
FA 71 123.0 (113.0 to 128.0) 124.0 (115.0 to 130.0) 2.0 (24.0 to 8.0)
IFA 75 122.0 (117.0 to 129.0) 126.0 (119.0 to 133.0) 2.0 (24.0 to 8.0) 0.30
Plasma ferritin (lg/L)
FA 58 45.6 (25.0 to 61.3) 45.7 (27.5 to 64.8) 3.1 (25.6 to 11.3)
IFA 59 34.0 (25.6 to 50.5) 60.8 (35.5 to 89.0) 18.8 (3.2 to 41.8) 0.0001
Plasma transferrin receptor (mg/L)
FA 64 2.9 (2.5 to 3.8) 2.8 (2.3 to 3.6) 20.2 (20.8 to 0.2)
IFA 68 3.0 (2.5 to 3.7) 2.7 (2.2 to 3.2) 20.4 (20.8 to 0.0) 0.48
Anemia (%)2
FA 75 35.2 12.7 222.5
IFA 71 37.3 2.7 234.7 0.05
1
Values are medians or percentages, where indicated; ranges (first–third quartiles) in parentheses. FA, folic acid; IFA, iron and folic acid. Multivariate
linear regression analyses were performed for change in hematologic indexes controlled for treatment group and literacy (n = 116).
2
Defined as hemoglobin ,120 g/L.
 PERICONCEPTIONAL IRON SUPPLEMENTATION
lower than in Nepal (86–88%), where fieldworkers also con-
ducted monthly house-to-house visits to deliver and monitor iron
supplements (7). One possible reason is the timing of supple-
mentation. In the Nepal study, supplementation commenced after
a woman knew she was pregnant. It is possible that women in our
study were less compliant because they perceived less benefit
from supplement use before pregnancy. In a subsequent article,
investigators of the Nepal study found that adherence was greater
among women who were older and who had more children,
possibly due to more exposure to antenatal messages (32).
Woman in our study may have been less adherent than women in
the Nepal study because they were nulliparous and considerably
younger (50% were adolescents).
The current study is the first trial to examine the effect of
periconceptional supplementation by using a powdered micro-
nutrient supplement. Recently, a study was conducted to de-
termine the efficacy of 60 mg elemental iron and 400 lg folic
acid delivered as a powdered micronutrient supplement com-
pared with tablets among pregnant women in rural Bangladesh
(15). At 32 wk, mean (6SD) hemoglobin was significantly higher
among women who consumed the tablet (110.2 6 11.1 g/L) than in
those who consumed the powdered supplement (105.6 6 11.1 g/L)
(P , 0.05). Mean percentage adherence was higher in the tablet
group than in the powdered supplement group (76% compared
with 58%; P , 0.05). After adherence was controlled for,
hemoglobin response did not differ between the groups. The
study also found that the powdered supplement was commonly
mixed with rice, curry, banana, and dry biscuits with a small
amount of water (15). In the present study, both treatment
groups received supplements in the form of a powdered mi-
cronutrient supplement, and we did not collect information on
acceptability or on how women consumed the contents of the
sachets. However, because our study was conducted in the
same geographical region as the previous study, it is likely
that the 2 study populations share similar food habits and
practices. Reduced iron absorption from the powdered mixture
may have occurred because of high amounts of phytate in rice
or from mixing the powdered micronutrients with liquids,
causing some of the particles to be lost in the process because of
sticking to a glass or bowl. Although women in Bangladesh
traditionally consume their meal after their husband and chil-
dren have been fed, it is uncertain whether adding a powdered
form of micronutrients to their food was difficult in household or
social situations in which food is shared. Further studies on
adherence in this age group need to examine preferences for
modality of supplements in relation to socioeconomic and cul-
tural factors.
In conclusion, results show that 9 mo of daily IFA supplied as
a powder supplement to nonpregnant women in rural Bangladesh
decreases anemia and increases iron stores. We did not find
a positive effect among pregnant women. Before large-scale
periconceptional IFA supplementation programs are imple-
mented, there is a need for effectiveness studies and research that
examines the effect of periconceptional IFA supplementation on
maternal and infant outcomes. The current study examined
plasma folate, which is an indication of recent folate intake (33),
as a subsidiary outcome. Future studies would benefit from
determining red blood cell folate concentrations, which are long-
term indicators of folate status and can be related to reduction
in risk of neural tube defects (27, 34). The present study was
1301
a small-scale research trial with sufficient support to theoretically
ensure that the IFA supplements were distributed and consumed,
and yet adherence was only moderate. It has yet to be determined
whether periconceptional IFA supplementation is a cost-effective
strategy for preventing and controlling anemia during pregnancy
and whether it can be effectively implemented at the operational
level.
The authors’ responsibilities were as follows—AZK: designed the study,
developed and implemented the study protocol, supervised field operations,
ensured data quality, analyzed the data, and wrote the manuscript; DLO: su-
pervised laboratory analysis of folate, interpreted the findings, and reviewed
the manuscript; CM: contributed to the analytic strategy, interpreted the find-
ings, and reviewed the manuscript; AD: contributed to the analytic strategy
and interpreted the findings; and SHZ: contributed to various phases of the
project from review and implementation of the study protocol to interpretation
of the findings and review of the manuscript. No conflicts of interest were
declared.
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