Am. J. Trop. Med. Hyg., 69(6), 2003, pp. 574–581
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene
MALARIA-ASSOCIATED CYTOKINE CHANGES IN THE PLACENTA OF WOMEN
WITH PRE-TERM DELIVERIES IN YAOUNDE, CAMEROON
AMORSOLO L. SUGUITAN JR.,* TIMOTHY J. CADIGAN,* THU A. NGUYEN,* AINONG ZHOU, ROBERT J. I. LEKE,
SIMON METENOU, LUCY THUITA, ROSETTE MEGNEKOU, JOSEPHINE FOGAKO, ROSE G. F. LEKE, AND
DIANE WALLACE TAYLOR
Department of Biology, Georgetown University, Washington, District of Columbia; Department of Biology, University of Scranton,
Scranton, Pennsylvania; Mayo Medical Center, Rochester, Minnesota; AZ Data Clinic, Inc., Rockville, Maryland; Faculty of Medicine
and Biomedical Sciences, Biotechnology Center, University of Yaounde I, Yaounde, Cameroon
Abstract. The prevalence of pre-term deliveries (PTDs) is increased in women who become infected with Plasmodium falciparum during pregnancy. Because prematurity is a risk factor for newborns, it is important to identify
conditions that contribute to malaria-associated PTDs. Plasmodium falciparum−infected erythrocytes sequester in the
placenta and attract activated mononuclear cells that secrete pro-inflammatory cytokines. Increased inflammatory
cytokine levels in other microbial infections are associated with PTDs. To determine if such is the case in women with
placental malaria, concentrations of interferon-␥ (IFN-␥), tumor necrosis factor-␣ (TNF-␣), interleukin-4 (IL-4), and
IL-10 were measured in placental plasma of 391 malaria-infected and -uninfected Cameroonian women with premature
and full-term deliveries. Risk factors for malaria-associated PTDs included peripheral and placental parasitemias greater
than 1%, maternal anemia, elevated IL-10 levels, and low TNF-␣:IL-10 ratios due to over-expression of IL-10. Alterations in cytokine levels may contribute to PTDs through the induction of anemia and/or altering cellular immune
responses required for eliminating placental parasites.
in fetal death and abortion.18 The influence of TNF-␣ and
other pro-inflammatory cytokines on pregnancy outcomes in
humans with malaria is less clear. Significant increases in
TNF-␣ levels have been reported in pregnant women as a
result of malaria, yet the majority of women had full-term
deliveries (FTDs).15–17 Moorman and others found no association between increased TNF-␣ mRNA expression in the
placenta and PTDs among malaria-infected women in
Malawi.19 The investigators, however, noted that the sample
size was small (n ⳱ 44, with eight cases of PTD), making the
conclusion provisional. Therefore, the relationship between
cytokine production at the maternal-fetal interface and pregnancy outcome, specifically PTD, requires elucidation.
The current study sought to determine whether malariarelated cytokine changes in the placenta of women with PTDs
were significantly different from those in women who had
FTDs. Based on previous studies, we hypothesized that in
PTDs high placental parasitemias during the third trimester
would lead to elevated levels of TNF-␣ without the production of compensatory levels of IL-10. Results from the current
study showed that high placental parasitemia and maternal
anemia were important risk factors for malaria-related PTD,
but contrary to predictions, both TNF-␣ and IL-10 were significantly elevated in malaria-infected women with PTDs. An
association between parasitemia, anemia, and IL-10 was
found, suggesting that high IL-10 levels are associated with
pathologies contributing to PTD.
INTRODUCTION
Women who become infected with Plasmodium falciparum
during pregnancy have an increased risk of delivering low
birth weight babies, due either to pre-term delivery (PTD) or
intra-uterine growth retardation.1–3 Malaria in pregnant
women also contributes significantly to infant mortality. In
sub-Saharan Africa, it is estimated that up to 200,000 infants
die annually as a result of maternal infection during pregnancy.4 Accordingly, there is significant interest in understanding how malaria influences fetal development and pregnancy outcome.
PTD is a leading cause of neonatal mortality.5 Neurologic
and respiratory systems of premature infants may not have
completed development at birth, causing infants to have a
variety of morbidities.5 Most premature infants are also low
birth weight, thereby increasing their risk of dying during
their first year of life.6 At least 40% of all pre-term births in
developed countries occur in mothers with intrauterine infections, mainly of bacterial etiology.7 In tropical countries, malaria is a significant risk factor for premature deliveries.8–10
During successful pregnancies, fetal trophoblasts and maternal leukocytes secrete predominantly Th2-type cytokines,
e.g., interleukin-10 (IL-10), to prevent initiation of inflammatory and cytolytic-type responses that might damage the integrity of the materno-fetal placental barrier.11,12 In response
to invading pathogens, however, Th1-type cytokines may be
produced that reverse the Th2-type bias within the placenta.
Excessive secretion of inflammatory cytokines against microbial pathogens along with insufficient quantities of counterregulatory IL-10 are reported to be important factors associated with PTD.13,14 The sequestration of P. falciparum parasites in the intervillous space of the placenta also stimulates
the production of Th1-type cytokines, including interferon-␥
(IFN-␥) and tumor necrosis factor-␣ (TNF-␣).15–17 Early
studies by Clark and Chaudhri demonstrated that elevated
TNF-␣ levels in pregnant mice infected with P. vinckei result
MATERIALS AND METHODS
Subjects. The study was reviewed and approved by the
Institutional Review Board of Georgetown University, the
National Ethical Committee, Ministry of Health, Cameroon,
and is covered by Single Project Assurance #S-9601-01.
Women residing in the capital city of Yaounde and nearby
surrounding areas who delivered at the Biyem Assi Hospital
between August 1996 and August 1997 and Central Hospital
from September 1997 to August 2001 were consecutively recruited. A member of the hospital staff explained the purpose
* These authors contributed equally to this work.
574
575
PLACENTAL MALARIA, CYTOKINES, AND PREMATURITY
of the study to each woman and, only after obtaining verbal
informed consent, was the woman formally enrolled. A questionnaire was used to record relevant clinical information,
including maternal age, gravidity, use of anti-malarial drugs
during pregnancy, and date of last menstrual period. After
delivery, information about the newborn was also recorded.
Infants born between the 28th and 37th weeks of gestation
were considered premature.
Study design. Women were excluded if they had abortions,
stillbirths, caesarian sections, or multiple births, i.e., only
women with singleton, vaginal deliveries were included. A
detailed description of the 391 women included in the study is
provided in Table 1. Among the women recruited, 83 women
had PTDs. To compare women with PTDs with those who
had FTDs, a representative group of 308 women with FTDs
was selected by frequency matching with respect to placental
malaria infection.
Sample collection. Immediately after delivery, ∼ 5 mL of
maternal placental blood was collected using the biopsy-pool
method for cytokine analysis. Briefly, a block of tissue (5 cm
× 5 cm × 5 cm) was excised from the basal side of the placenta,
resulting in the formation of a large pool of intervillous blood
at the excision site. Heparinized blood was quickly withdrawn
and placed on ice until processed in the laboratory. A piece of
placental tissue (2 cm × 2 cm × 2 cm) was also collected, and
a portion was placed in 10% buffered formalin and processed
for histology using routine methods. The remainder of the
tissue was used to prepare impression smears for parasitologic
analysis. In addition, ∼ 5 mL of heparinized, maternal venous
blood was collected for hematologic and parasitologic analysis.
Parasitemias and anemia. Thick and thin blood films were
prepared using maternal peripheral and placental blood. Impression smears of placental tissue were also prepared. The
slides were stained with Diff-Quick solution (Baxter Scientific
Products, Miami, FL) and examined for the presence of P.
falciparum parasites. Parasitemias were calculated by counting the number of infected erythrocytes per 2,000 erythrocytes and expressing the value as a percentage. Based on the
presence/absence of parasites in placental impression smears,
smears of placental blood, and histologic sections, women
were classified as either positive or negative for placental
malaria.
An aliquot of maternal peripheral blood was used to determine the packed cell volume (PCV). Using the definition
of the World Health Organization, a woman was considered
anemic if the PCV was less than 30%.
Measuring cytokine levels in placental plasma by an enzyme-linked immunosorbent assay (ELISA). Plasma samples
collected prior to August 1997 (n ⳱ 175) were assayed
for IFN-␥, TNF-␣, IL-4, and IL-10 using the DuoSet ELISA
Development System (Genzyme Diagnostics, Cambridge,
MA), while samples collected thereafter were assayed for
the same cytokines using ELISA kits from BD Pharmingen
(San Diego, CA). The sensitivity of the assays was comparable: 2 pg/ml for IFN-␥ and IL-10 and 5 pg/ml for TNF-␣ and
IL-4.
Statistical analyses. The Wilcoxon rank sum test was used
to determine significant differences in the amounts of cytokines and other continuous variables between malariainfected and non-infected women who delivered prematurely
or at term. The Pearson’s chi-square test was used for uni-
TABLE 1
Comparison of women with pre-term deliveries and full-term deliveries*
Women with pre-term deliveries (PTD)
All women
Malaria +
Malaria −
Women with full-term deliveries (FTD)
P
All women
Malaria +
Malaria −
P
P†
Number studied
83
38
45
308
110
198
Maternal age (years)‡
25.0 ± 6.4
24.0 ± 5.0
25.9 ± 7.4
0.20
24.8 ± 5.4
24.5 ± 5.3
24.9 ± 5.5
0.45
0.68
Primigravidae
41.5%
36.8%
44.4%
0.48
39.9%
40.4%
39.1%
0.85
0.81
Anti-malarial drug
86.7
84.2%
88.9%
0.53
89.0%
90.9%
87.9%
0.10
0.58
usage
Placental parasitemias
2.1%
0
0.7%
0
(%)§
(0.8%, 23.1%)
(0.2%, 2.6%)
Peripheral parasitemias
0.12%¶
0
0.03%
0
(%)§
(0%, 0.8%)
(0%, 0.3%)
Packed cell volume‡
31.8 ± 6.4%
28.4 ± 5.9%
34.4 ± 5.4% <0.001
34.4 ± 6.1%
33.8 ± 6.0%
34.8 ± 6.2% 0.05
0.003
% Anemia
46.7%
75.8%
23.8%
<0.001
28.2%
33.3%
25.1%
0.12
0.003
Gestational age
33.1 ± 3.2
33.4 ± 2.8
32.9 ± 3.5
0.47
40.1 ± 1.7
40.1 ± 1.6
40.1 ± 1.8
0.90
<0.0001
(weeks)‡
Infant birth weight
2,159 ± 651
2,150 ± 607
2,166 ± 693
0.91
3,322 ± 441
3,235 ± 410
3,370 ± 451
0.01
<0.0001
(grams)†
Cytokines (pg/mL)#
IFN-␥
13.2 ± 10.4
13.5 ± 10.5
12.8 ± 10.6
0.43
13.9 ± 27.1
20.4 ± 40.4
9.5 ± 10.5 <0.0001 0.07
(11.4)
(12.5)
(9.7)
(8.2)
(10.5)
(6.9)
TNF-␣
48.5 ± 105.2
63.4 ± 124.6
36.7 ± 86.6
0.012
45.7 ± 107.6
43.9 ± 99.3
45.8 ± 111.1 0.61
0.17
( 13.5)
( 29.8)
( 11.0)
( 12.4)
( 13.4)
( 10.9)
IL− 4
9.5 ± 6.4
8.6 ± 6.2
10.5 ± 6.6
0.19
12.8 ± 29.3
15.6 ± 43.1
11.0 ± 15.5
0.23
0.43
( 8.3)
( 6.4)
( 9.1)
( 7.5)
( 8.3)
( 6.6)
IL− 10
229.5 ± 556.3 449.1 ± 764.2
42.8 ± 85.1 <0.0001 51.9 ± 93.4
72.5 ± 107.0
37.1 ± 77.5
0.003
0.0003
(41.9)
(113.2)
(21.4)
(21.1)
(27.8)
(18.0)
* IFN ⳱ interferon; TNF ⳱ tumor necrosis factor; IL ⳱ interleukin. Bold numbers indicate statistical significance.
† Comparison between all women with PTD and FTD.
‡ Mean ± SD.
§ Median (25th percentile, 75th percentile).
¶ In this study, 26.5% of women with placental malaria were peripheral blood smear negative, consistent with a previous report.47
# Mean ± SD (median).
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SUGUITAN AND OTHERS
variate between-group comparisons of binomial variables, including dichotomized age, gravidity, chemoprophylaxis usage
during pregnancy, and anemia. The Wilcoxon signed-rank
test was used to compare peripheral and placental parasitemias for each subject. The correlations among IL-10, TNF-␣,
PCV, and placental parasitemia were obtained by determining the Spearman’s rank coefficient. The crude and adjusted
odds ratios with 95% confidence intervals, along with P values from the partial likelihood ratio statistics, were determined using a logistic regression model.
RESULTS
Study population. The distribution of women with respect
to pregnancy outcome and placental malarial status is shown
in Table 1. No significant difference in mean maternal age,
gravidity, and reported anti-malarial drug usage during pregnancy was found between infected and non-infected women
with PTDs and FTDs. Women with placental malaria in the
PTD and FTD groups were both more prone to anemia, as
demonstrated by significantly lower mean PCV (PTD: P >
0.001; FTD: P ⳱ 0.05) compared with non-infected women
(Table 1). As expected from previous studies, parasitemias
were significantly higher in the placenta than in the peripheral
blood of both groups of women (P < 0.0001 for both PTDs
and FTDs). In comparison, women with PTDs had significantly higher placental parasitemias than women with FTDs
(median ⳱ 2.1% versus 0.7%, respectively; P < 0.0001). More
than 70% of women with PTDs had placental parasitemias
greater than 1%, compared with 41.2% of women in the
FTD group (P ⳱ 0.003) (Figure 1). Overall, 75.8% of placental malaria-positive women with PTDs had anemia
(i.e., had a PCV less than 30%) (Table 1). Since there was a
strong correlation between placental and peripheral parasitemias (Spearman’s ␳ ⳱ 0.87, P > 0.0001), high peripheral
parasitemias were also found in women with PTDs (median
⳱ 0.12% versus 0.03%, PTD and FTD, respectively; P ⳱
0.022).
FIGURE 1. Distribution of placental parasitemia in women with
pre-term deliveries (PTDs) and full-term deliveries (FTDs). Women
with PTDs (black bars) and FTDs (shaded bars) were stratified into
five categories based on their level of placental parasitemia. Only
30% of women in the PTD group had parasitemias less than 1%
compared with 59% of women in the FTD group.
Placental malaria in women with PTDs did not have a significant impact on either infant birth weight (P ⳱ 0.91) or
mean gestational age (P ⳱ 0.47) (Table 1). However, placental malaria was associated with a reduction in infant birth
weight among women with FTDs (P ⳱ 0.01) (Table 1).
Cytokine levels in placental plasma. The distribution of
IFN-␥, TNF-␣, IL-4, and IL-10 in placental plasma of women
with PTDs and FTDs is shown in Figure 2. No significant
differences in cytokine levels were observed between malarianegative women with PTDs and FTDs for the four cytokines
(P > 0.05 in all comparisons). Thus, PTD, by itself, was not
associated with an alteration in the placental cytokine balance.
Conversely, both TNF-␣ and IL-10 were significantly elevated in malaria-positive women with PTDs compared with
women with FTDs (Figure 2). The median level of TNF-␣ in
malaria-positive women with PTDs was more than twice that
found in infected women with FTDs (P ⳱ 0.012). In contrast,
the median IL-10 level of malaria-infected women with PTDs
was four-fold higher than that in infected women with FTDs
(P < 0.0001) (Table 1 and Figure 2). These data support the
hypothesis that TNF-␣ levels are elevated in malaria-infected
women with PTDs, but reject the hypothesis that IL-10 synthesis is suppressed. In fact, infected women with PTDs had
very high levels of IL-10 (Figure 2). Although IL-10 levels
were also elevated 1.5-fold in infected women with FTDs
(27.8 pg/mL) compared with non-infected women (18.0 pg/
mL; P ⳱ 0.003), the increase was significantly less than that
observed in PTDs (Table 1 and Figure 2). However, a significant correlation between TNF-␣ and IL-10 levels exists within
the same individual of all infected women (Spearman’s ␳ ⳱
0.62, P < 0.0001).
Since both TNF-␣ and IL-10 were significantly elevated in
malaria-infected women with PTDs, the ratio of TNF-␣ to
IL-10 for each woman in the study was determined and TNF␣:IL-10 ratios were compared. No significant differences were
found in median values for malaria-negative women with
FTDs and PTDs (median ⳱ 0.59 versus 0.53, respectively;
P ⳱ 0.94) or malaria-positive women with FTDs (median ⳱
0.49). However, significantly lower TNF-␣:IL-10 ratios were
found in malaria-positive women with PTDs compared with
those with FTDs (median ⳱ 0.28 versus 0.49, respectively;
P ⳱ 0.004). Thus, TNF-␣:IL-10 ratios were similar in women
with successful FTDs and in malaria-negative women with
PTDs, but they were significantly lower in women with PTDs
with placental malaria.
Risk factors associated with PTDs in malaria-positive
women. Our results show that malaria-infected women with
PTDs had higher parasitemias, (Table 1 and Figure 1), lower
PCV values (Table 1), higher levels of placental TNF-␣ and
IL-10 (Table 1 and Figure 2), and lower TNF-␣:IL-10 ratios.
Therefore, risk factors for PTD were assessed by calculating
the odds ratios associated with these variables (Table 2). Univariate analysis showed that both peripheral and placental
parasitemias greater than 1%, maternal anemia, elevated levels of IL-10, and low TNF-␣:IL-10 cytokine ratios were all
significant risk factors for PTDs (Table 2). After adjusting for
other covariates, only maternal anemia remained significantly
associated with PTDs (P ⳱ 0.005). Significant correlations
between IL-10 and placental parasitemia (Spearman’s ␳ ⳱
0.38, P < 0.0001) and between TNF-␣ and placental parasitemia (Spearman’s ␳ ⳱ 0.10, P ⳱ 0.02) were also found.
PLACENTAL MALARIA, CYTOKINES, AND PREMATURITY
577
FIGURE 2. Cytokine levels in placental plasma. Placental plasma levels of interferon-␥ (IFN-␥), tumor necrosis factor-␣ (TNF-␣), interleukin-4
(IL-4), and IL-10 are displayed in box plots with the median values represented by the horizontal line within each box for malaria-negative and
malaria-positive women with pre-term deliveries (PTDs) and full-term deliveries (FTDs). The shaded regions represent the interquartile ranges
while the upper and lower vertical bars represent the 90th and 10th percentiles, respectively. The Wilcoxon sum rank test was used to detect
significant differences between the groups compared. P < 0.05 was considered significant.
When women were categorized based on their placental
parasitemias and stratified according to anemia, anemia did
not appear to enhance the prevalence of PTD among malarianegative women (Figure 3). Among malaria-infected women
with low placental parasitemias (< 1.0%), the proportion of
women who delivered prematurely was significantly higher in
the anemic compared with the non-anemic group (24.1% versus 7.1%, respectively; P ⳱ 0.045). However, the most dramatic increase in the proportion of women with PTDs was
observed among women with high placental parasitemias
(> 1.0%) who were also anemic (i.e., 60.7% of women in this
group have PTDs), which represents a 3.6-fold increase compared with the proportion of women with PTDs in nonanemic group with similar levels of placental parasitemias
(18.5%; P < 0.001) (Figure 3).
Since anemia was a significant risk factor for PTDs, risk
factors for maternal anemia were also evaluated. Univariate
analyses showed that risk factors for maternal anemia included placental parasitemias > 0.1% and elevated levels of
IL-10 (Table 3). After adjusting for other covariates, only
placental parasitemias > 0.1% was significantly associated
with anemia. Because cytokines have been implicated in the
development of anemia20,21 and high parasite densities,22,23
correlations between 1) TNF-␣ and PCV, 2) IL-10 and PCV,
3) TNF-␣ and placental parasitemia, and 4) IL-10 and placental parasitemia were determined. A significant negative
correlation was found between IL-10 and PCV (Spearman’s
␳ ⳱ -0.20, P ⳱ 0.002), while there was no correlation between
TNF-␣ and PCV (Spearman’s ␳ ⳱ -0.004, P ⳱ 0.94). Taken
together, these results suggest that among malaria-infected
women, high placental parasitemias and maternal anemia
dramatically increase the risk of PTD and that these risk factors are associated with elevated IL-10 levels.
DISCUSSION
The identification of factors contributing to malariaassociated PTD are extremely important because low birth
weight babies born prematurely have an increased risk of
dying during the first year of life.6 Pregnant women who become infected with bacteria often produce pro-inflammatory
cytokines including TNF-␣.24 Production of TNF-␣, in the
absence of compensatory production of IL-10, has been associated with spontaneous abortions and PTDs.14,25 Since P.
falciparum parasites sequester in the intervillous space and
stimulate the infiltration of monocytes/macrophages26 that secrete TNF-␣, it was logical to speculate that increased levels
of placental TNF-␣ and low levels of IL-10 would be contrib-
578
SUGUITAN AND OTHERS
TABLE 2
Odds ratios (ORs) and 95% confidence intervals (CIs) for risk factors associated with pre-term delivery*
Risk factor
Category
Prevalence of PTD (%)
Crude OR (95% CI)
Maternal age (years)
>20
ⱕ20
21.7
21.2
Multigravidae
Primigravidae
20.4
22.7
>30% PCV
ⱕ30% PCV
16.7
30.6
0
<0.1%
18.5
8.3
0.1–1.0%
18.4
>1.0%
40.0
Below median
Above median
19.2
31.1
Below median
Above median
20.1
21.5
Above median
Below median
17.9
31.0
Referent
1.0
(0.5–1.8)
Referent
1.1
(0.7–1.9)
Referent
2.2
(1.3–3.7)
Referent
0.4
(0.1–1.8)
1.0
(0.4–2.2)
2.9
(1.6–5.3)
Referent
1.9
(1.1–3.3)
Referent
1.1
(0.7–1.8)
Referent
2.1
(1.2–3.6)
Gravidity
Anemia
Placental parasitemia
IL-10
TNF-␣
TNF-␣/IL-10
Crude P
Adjusted OR (95% CI)
Adjusted P†
0.93
0.9
(0.4–2.1)
0.82
0.61
1.3
(0.7–2.5)
0.47
0.004
2.5
(1.3–4.6)
0.005
NA
NA
0.002
NA
0.02
1.5
(0.8–3.0)
0.20
0.75
0.9
(0.5–1.7)
0.79
0.01
Not included
* PTD ⳱ pre-term delivery; PCV ⳱ packed cell volume; NA ⳱ not estimable due to small sample size in one of the parasitemia categories; IL ⳱ interleukin; TNF ⳱ tumor necrosis factor;
IFN ⳱ interferon. Bold numbers indicate statistical significance.
† By partial log-likelihood ratio test in a multivariate logistic regression model including all the variables in the table.
uting factors to PTDs in malaria-infected women. Results
from the present study suggest that this scenario is not correct. That is, women with placental malaria who have PTDs
produce significantly higher amounts of IL-10 than women
with FTDs (Figure 2), and their placental TNF-␣:IL-10 ratio
was significantly biased towards a Th2-profile. Thus, inadequate IL-10 production dos not appear to be a contributing
factor to PTD in the context of placental malaria. Since a
significant correlation between TNF-␣ and IL-10 (within the
same individual) was found in women with placental malaria,
it is likely that a feedback mechanism has been induced to
help regulate the inflammatory process at the fetal-maternal
interface.
The present study identified several risk factors for malaria-related PTD after adjusting for age and gravidity (Table
2). The major risk was having a placental parasitemia of
greater than 1% at the time of delivery, confirming reports by
other investigators that high placental parasitemias significantly influences PTD.8 In addition, anemia, which is one
of the most common complications of malaria during pregnancy, especially in primigravidae,27,28 was also identified as a
risk factor for PTD (Table 2). This combination of factors is
an important predictor for PTD (Figure 3). Since parasite
densities and the development of anemia are influenced by
cytokines,20–23 it was not surprising that changes in cytokine
levels were also found to be risk factors for PTD. These included a low placental TNF-␣:IL-10 ratio due to excessive
production of IL-10, and high placental IL-10 levels alone
(Table 2).
The immunologic relevance of the ratio of TNF-␣ and IL10 concentrations found in peripheral plasma samples remains controversial in the literature. Several studies have reported that high TNF-␣ and low IL-10 levels are associated
with severe malaria in children,29–31 whereas other studies
have found an association between elevated levels of IL-10
and high parasitemias.22,23,32,33 Studies in murine models of
malaria have also suggested that IL-10 response during infection may be associated with disease exarcebation.34 It is be-
FIGURE 3. Placental parasitemia, anemia, and the prevalence of
pre-term deliveries (PTDs). The proportion of women who delivered
prematurely was not significantly different in non-infected women
who had (10 of 55) or did not have anemia (32 of 166) at delivery
(P ⳱ 0.92). Among women with placental parasitemias < 1.0%, the
proportion of women with PTDs was significantly higher in the anemic group (7 of 29) compared with those without anemia (3 of 42)
(P ⳱ 0.045). The proportion of women with PTDs was highest in
anemic women with placental parasitemias > 1.0% (17 of 28), a 3.6fold increase compared with the proportion of women who had PTDs
among non-anemic women with similar levels of placental parasitemias (5 of 30) (P < 0.001). Pearson’s chi-square test was used to
determine any significant differences in the proportion of cases in the
groups compared. P < 0.05 was considered significant.
579
PLACENTAL MALARIA, CYTOKINES, AND PREMATURITY
TABLE 3
Odds ratios (ORs) and 95% confidence intervals (CIs) for risk factors associated with anemia*
Risk factor
Category
Prevalence of anemia (%)
Crude OR (95% CI)
Maternal age (years)
>20
ⱕ20
30.7
35.5
Multigravidae
Primigravidae
31.3
32.4
0
<0.1%
24.4
34.8
0.1–1.0%
43.8
>1.0%
48.3
Below median
Above median
26.0
40.2
Below median
Above median
28.2
35.1
Above median
Below median
34.1
31.6
Referent
1.2
(0.7–2.1)
Referent
1.0
(0.7–1.7)
Referent
1.6
(0.7–4.1)
2.4
(1.3–4.6)
2.9
(1.6–5.3)
Referent
1.9
(1.1–3.2)
Referent
1.4
(0.9–2.2)
Referent
0.9
(0.5–1.5)
Gravidity
Placental parasitemia
IL-10
TNF-␣
TNF-␣/IL-10
Crude P
Adjusted OR (95% CI)
Adjusted P†
0.44
1.0
(0.5–2.1)
0.99
0.84
1.2
(0.7–2.3)
0.48
0.002
1.4
(0.5–4.0)
2.4
(1.2–5.0)
2.3
(1.1–4.6)
0.042
0.02
1.6
(0.9–2.8)
0.14
0.17
1.0
(0.6–1.7)
0.94
0.67
Not included
* IL ⳱ interleukin; TNF ⳱ tumor necrosis factor; IFN ⳱ interferon. Bold numbers indicate statistical significance.
† By partial log-likelihood ratio test in a multivariate logistic regression model including all the variables in the table.
lieved that high IL-10 levels impair the inflammatory response required for parasite killing,35 resulting in higher parasitemias. However, information on the balance between
TNF-␣ and IL-10 and its role in malaria protection/
pathogenesis remains to be elucidated.
In the present study, P. falciparum-infected women with
PTDs had elevated levels of both TNF-␣ and IL-10 (Figure 2),
but the TNF-␣:IL-10 ratio was significantly decreased, suggesting a Th2-biased response. During pregnancy, the overall
immune response of the mother is Th-2 biased to prevent
fetal allograft rejection.36,37 While both maternal leukocytes
and fetal trophoblasts are capable of producing IL-10 during
P. falciparum infection,16,17 maternal monocytes and macrophages are the most likely source of placental IL-10 in malaria-infected women.17 Massive monocytic infiltration is a
hallmark of placental malaria,38–40 and increased migration of
monocytes and macrophages to the placental intervillous
space has been identified as a risk factor for PTD.8 In women
with malaria-associated PTDs, it is possible that elevated levels of IL-10 suppress anti-parasite inflammatory responses
resulting in high placental parasitemias and ultimately anemia. In this study, a significant correlation was found between
IL-10 and placental parasitemia, as well as a negative correlation between IL-10 levels and PCV. In addition, a significant correlation was also found between TNF-␣ and placental
parasitemia but not with PCV. The latter result was somewhat
surprising given that the role of TNF-␣ in the development of
anemia in the context of malaria is well established.41 However, recent evidence suggest that both pro- and antiinflammatory cytokines are involved in the pathogenesis of
anemia of chronic diseases.20,42 Elevated levels of IL-10 can
increase the acquisition and retention of iron by monocytes
and macrophages and increase ferritin synthesis, thereby reducing the amount of iron in the plasma and thus contributing
to anemia.20,42 Since many cases of placental malaria are
chronic in nature43 and anemia places a stress on iron me-
tabolism, it is probable that the elevated levels of IL-10 contribute significantly to the development of maternal anemia, a
known risk factor for PTD.44 Human IL-10 has also been
shown to suppress the production of granulocyte-macrophage
colony−stimulating factor by T cells resulting in the inhibition
of erythroid progenitor cells,45 presenting another mechanism
on how IL-10 may contribute to anemia. Thus, both elevated
levels of pro-inflammatory TNF-␣ and regulatory IL-10 may
contribute to anemia in pregnant women.
Information from the present study should be applicable to
women living in urban cities throughout Cameroon and other
African countries where malarial transmission is perennial.
The pregnant women in the study represented a diverse group
of women, with different ages (15−50 years old), numbers of
pregnancies (1−11), and a range of economic and social backgrounds (Table 1). Their malariometric profile, however, is
similar to that reported for pregnant women in other African
countries.1,2 The influence of other diseases on the results is
difficult to assess, including infection with human immunodeficiency virus, which infects an estimated 4.0−13.6% of
women attending antenatal clinics in urban areas of Cameroon in 2001.46 However, because all consecutively recruited
women with PTDs were studied and women with FTDs were
randomly selected, it is unlikely other diseases had a major
impact on the results. There is no reason to suspect that placental malaria was not the key factor influencing the cytokine
changes observed in this study (Figure 2).
In summary, results from this study are consistent with the
following scenario. In pregnant women, malarial parasites sequester in the placenta and stimulate the accumulation of
activated macrophages in the intervillous space where they
secrete large amounts of TNF-␣.17 In response to this inflammatory challenge, maternal and fetal cells secrete IL-10 to
limit pathology and to protect the fetal allograft. Although
IL-10 is important in modulating the possible deleterious effects of inflammatory cytokines, its over-expression may have
580
SUGUITAN AND OTHERS
detrimental consequences resulting in PTD by enhancing maternal anemia and suppressing anti-parasite inflammatory responses leading to persistent placental parasitemias.
13.
Received June 12, 2003. Accepted for publication September 18,
2003.
Acknowledgments: We express our gratitude to all the Cameroonian
women who participated in this study. We are indebted for the immense help of the entire staff of the Biotechnology Center of the
University of Yaounde, including the medical students who participated in the sample collection.
14.
15.
Financial support: This work was supported by the National Institute
of Allergy and Infectious Diseases, National Institutes of Health
(grant numbers UO1A153 and UO1AI-43888).
16.
Authors’ addresses: Amorsolo L. Suguitan, Jr., Simon Metenou, Lucy
Thuita, and Diane Wallace Taylor, Room 406, Reiss Science Building, Department of Biology, Georgetown University, 37th and O
Streets, NW, Washington, DC 20057. Timothy J. Cadigan, Department of Biology, University of Scranton, Scranton, PA 18510, Telephone: 570-941-4348. Thu A. Nguyen, Mayo Medical Center, Rochester, MN 55905, Telephone: 617-388-1938. Ainong Zhou, AZ Data
Clinic, Inc., Rockville, MD, 20850, Telephone: 240-476-2148. Robert
J. I. Leke, Rosette Megnekou, Josephine Fogako and Rose G. F.
Leke, The Faculty of Medicine and Biomedical Sciences, Biotechnology Center, University of Yaounde I, Yaounde, Cameroon, Telephone: 237-223-7479.
17.
Reprint requests: Diane Wallace Taylor, Room 406, Reiss Science
Building, Department of Biology, Georgetown University, 37th and
O Streets, NW, Washington, DC 20057, Telephone: 202-687-5972,
Fax: 202-687-5662, E-mail: [email protected].
18.
19.
20.
21.
22.
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