Placenta 34 (2013) 162e167
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Placenta
journal homepage: www.elsevier.com/locate/placenta
Complement activation in primiparous women from a malaria endemic area
is associated with reduced birthweight
A. Khattab a, *, P.G. Kremsner b, c, S. Meri a, d
a
Infection Biology Program, Department of Bacteriology and Immunology, Haartman Institute, University of Helsinki, Haartmaninkatu 3, P.O. Box 21, 00014 Helsinki, Finland
Institute for Tropical Medicine, University of Tübingen, Tübingen, Germany
c
Medical Research Unit, Albert Schweitzer Hospital, Lambaréné, Gabon
d
Helsinki University Central Hospital, Helsinki, Finland
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Accepted 22 November 2012
The hallmark of placental malaria (PM) due to Plasmodium falciparum infection is the accumulation of
mature-stage parasites, monocytes and macrophages in the maternal vascular bed of the placenta. The
mechanisms leading to morbidity and mortality in PM are incompletely understood. However, an
inflammatory response in the placenta has been related to both severe anemia in the mother and low
birthweight (<2500 g) in the newborn. In this study we analyzed whether complement activation as
a mediator of inflammation could contribute to poor pregnancy outcome in PM. The concentrations of
the soluble terminal complement complex (TCC) were measured as an indicator of complement activation in placental, cord and peripheral blood samples from 146 women from a malaria endemic area.
Placental and cord plasma samples of primiparous women, a group vulnerable to PM, showed significantly higher levels of TCC than multiparous women. Additionally, in women with malaria history during
pregnancy or placental infection by P. falciparum at delivery, the TCC levels in the corresponding
placental and cord plasma samples were significantly higher than in the malaria negative group. In
multiple regression analysis parity was shown to be the main determinant of TCC levels. Placental plasma
samples corresponding to babies weighing less than 2700 g had significantly higher levels of TCC than
babies carrying more weight. In conclusion, both primiparity and P. falciparum infection were related to
a local increase of complement activation in the placentas. Association between reduced birthweight and
higher levels of TCC in placental blood suggests a role for complement activation in influencing the
pregnancy outcome in malaria exposed women.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Placenta
Malaria
Innate immunity
Complement
Birthweight
Intrauterine growth retardation
1. Introduction
Adults living in malaria endemic areas have normally been
exposed to repeated infections with Plasmodium falciparum and
are, therefore, clinically immune to the disease [1]. However, this is
not true in the case of pregnant women who can suffer from serious
disease despite apparent immunity. The disease, known as
placental malaria (PM), is more pronounced in primiparous women
than in multiparous women [2]. The severity of placental malaria is
attributed mainly to the lack of antibodies against a particular
protein (var2CSA) from the multicopy variant antigens, PfEMP1s,
encoded by the var gene family [3]. These variant antigens are
expressed on the surface of P. falciparum-infected erythrocytes (IEs)
* Corresponding author. Tel.: þ358 468834423.
E-mail address: ayman.khattab@helsinki.fi (A. Khattab).
0143-4004/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.placenta.2012.11.030
and mediate cytoadherence to endothelial cell receptors such as
CD36 and ICAM-1 [4]. Cytoadherence of IEs in the microvasculature
can lead to tissue damage and organ dysfunction thereby contributing to the pathology of malaria [5]. In pregnant women, the
placenta provides a new niche for the selective cytoadhesion of IEs.
The IEs bind via var2CSA to chondroitin sulfate A, a glycosaminoglycan, expressed on the surface of the syncytiotrophoblast layer
lining the placental chorionic villi [3,6]. Since var2CSA is an
unusually conserved protein among other PfEMP1s [7], immunity
to PM is acquired relatively soon and one or two pregnancies in
malaria endemic areas are generally enough to confer protection
[8]. Cytoadhesion properties of IEs displaying var2CSA explain the
accumulation of IEs in the maternal vascular area of P. falciparuminfected placentas in primiparous women [9]. The infected erythrocytes and consequent inflammation could lead to the recruitment
of monocytes and macrophages into the placenta during infection
[10]. The resulting possible outcomes of PM are maternal anemia,
A. Khattab et al. / Placenta 34 (2013) 162e167
abortion, stillbirth, prematurity, intrauterine growth retardation
and low birthweight (LBW). LBW is the greatest risk factor for death
during the first month of life [11]. With up to 125 million pregnancies at risk of malaria infection every year [12] there is an urgent
need for a better understanding of the pathophysiological mechanisms leading to poor outcomes associated with PM.
Increased complement activation has been identified in noninfectious pathological pregnancies [13], as well as in malaria in
general [14]. The complement system is a specific effector arm of the
humoral immunity. It plays a key role in destroying foreign invading
microbes and in the clean-up of endogenous waste products. The
complement system can be activated by antibodies binding C1q in
the classical pathway or by mannan binding lectin or ficolins to sugar
residues on the target surface in the lectin pathway. The alternative
pathway is spontaneously activated by binding of C3b to foreign
surfaces. Activation results in the assembly of C3-cleaving enzymes
(C3 convertases) on target surfaces. Amplification via the alternative
pathway leads to more C3b deposition on the target. The terminal
pathway is activated when C3b forms a complex with the C3convertases and generates C5-convertases that can cleave C5,
thereby yielding one anaphylatoxin C5a and one C5b molecule.
Association of C5b with C6, C7, C8, and multiple C9 molecules ultimately leads to the generation of C5b-9, also known as the membrane
attack complex (MAC). However, much of the C5b generated does not
result in the formation of MAC, but is diverted by control proteins (e.g.
clusterin and S protein) to form a soluble, lytically inert complex
called soluble C5b-9. C5b-9 is referred to commonly as Terminal
Complement Complex or TCC [15]. Despite the strong cytolytic
activity of complement many microbes including the malaria parasite, are able to escape complement attack. Complement can attack
microbes or human cells that are marked with antibodies or have an
activating altered cell surface. Paternal alloantigens in the fetoplacental unit can trigger antibody production by the maternal immune
system [16]. Antibodies could thus activate complement in the
placental compartment through immune complex formation.
However, the placental cells are usually protected by a panel of
complement regulators expressed by the fetal cytotrophoblast cells
and by the syncytiotrophoblast. In healthy pregnancies, these regulators such as CR1 (CD35), DAF (CD55), MCP (CD46) and protectin
(CD59) down regulate complement activation and protect the
placenta from complement-mediated damage [17]. In addition, the
soluble complement inhibitors, factor H (FH) and C4b binding protein
(C4bp) can keep complement activation under control in the fluid
phase and to some extent on surfaces, as well. In certain pathological
situations, pregnancy complications might occur. Particularly in
preeclampsia and in antiphospholipid syndrome, complement activation is believed to contribute to the pathogenetic processes [13,18].
In PM, the IEs bind to the syncytiotrophoblasts of the placenta [6]. The
bound IEs display parasite antigens on their surfaces. These could
thus be targets for maternal antibodies and activate the complement
system [19]. Merozoite egress from an infected erythrocyte is
accompanied by the release of Plasmodium proteins (e.g. GPIanchored proteins) and toxins (e.g. hemozoin e a Plasmodiumgenerated hemoglobin degradation product) into the placental
intervillous space. These could either directly or via immune complex
formation also lead to complement activation. Collectively, the above
considerations have led us to suspect that complement activation in
the placenta in connection with P. falciparum infection could lead to
a poor pregnancy outcome in PM.
The complement C3a and C5a anaphylatoxins and the soluble
TCC are widely used markers for complement activation. Their use
singly or in combination with each other depends largely on the
questions and conditions of the measurements. It is important to
note that C3a and C5a, in particular, bind actively to specific cell
receptors upon their generation. Therefore, the concentration of
163
C3a rapidly decreases over time and C5a cannot be measured from
clinical samples [20]. On the other hand, the soluble TCC generated
from complement activation remains in its measurable free form at
a steady concentration for a longer period of time [20]. Thus, in the
present study the level of complement activation was estimated by
measuring the concentration of the soluble TCC in placental,
peripheral and cord plasma samples collected from pregnant
women at delivery from a malaria endemic area. TCC levels were
then analyzed with respect to PM manifestations and outcomes.
2. Materials and methods
2.1. Study participants
The study cohort has been described elsewhere in an earlier study [21]. Briefly,
one hundred and fifty women, who presented at the maternity ward of the Albert
Schweitzer Hospital in Lambaréné, Gabon for delivery between May and October
2002, were recruited in the study. Malaria in the region is hyperendemic and
perennially transmitted, and the disease is predominantly due to P. falciparum [22].
Whenever possible, demographic data (e.g. ethnic group, date of birth, weight) were
collected from the study participants upon admission. At delivery, data regarding
characteristics of the newborn baby (vital status at birth, birthweight, sex and the
presence of twins) were collected. For those attending prenatal care, information
regarding malaria infections during the course of pregnancy was obtained from the
maternity record book, which documented peripheral parasitemia by thick blood
smears (TBS). Written informed consents were obtained from all of the women and
the study was approved by the ethics committee of the International Foundation of
the hospital. A more detailed summary of the study cohort characteristics, that was
not shown in the earlier study describing the cohort [21], is shown in Table 1.
2.2. Blood samples
Peripheral blood was collected from the study participants within 1 h after
delivery in an EDTA-containing monovette. Blood was also extracted directly from
the placenta. This was done by making several 1-cm deep incisions into the maternal
side of the placenta and the flowing blood was drawn into an EDTA syringe (monovette without needle). Cord blood was collected immediately after placental
expulsion following delivery. This was done by drawing venous blood into an EDTAcontaining monovette via puncture of the ethanol-sterilized umbilical vein at a site
distal to the placenta, to minimize the possibility of cross-contamination of maternal
and cord blood. Blood samples collected from the three different compartments
were used for preparing TBS to detect the presence of malaria parasites and to
determine parasite densities using a previously described method [23]. EDTAplasma samples were obtained from the withdrawn blood by centrifugation and
preserved at 70 C until further use.
2.3. Sandwich ELISA for the determination of the fluid-phase TCC in peripheral,
placental and cord plasma
For the TCC ELISA, 96-well microtiter plates (Maxisorp NUNC, Thermo Scientific)
were coated with the anti-C9 neoepitope Ab WU13-15 (5 mg/ml in 0.2 M Na2CO3, pH
10.6; Hycult Biotech, Uden, The Netherlands) and incubated overnight at 4 C. After
Table 1
Characteristics of the study group.
All women
(n ¼ 150)
Age mean years, (SD)a
P. falciparum positive:
Placental TBS at delivery,
n (%)
Reportedc, n (%)
Total, n (%)
Hemoglobin:
Mean g/dl, (SD)a
Anemic women, n (%)
Birthweight:
2500 g, n (%)
2700 g, n (%)
Primiparous Multiparous P value
women
women
(n ¼ 45)
(n ¼ 105)
25.65 (7.32) 18.91 (3.32)
28.57 (6.6)
<0.001*,b
21 (14)
9 (20)
12 (11.4)
0.16d
39 (26)
55 (37)
18 (40)
23 (51)
21 (20)
32 (30.4)
0.01*,d
0.016*,d
11.03 (1.99) 11.23 (2.37)
71 (47)
19 (42.2)
16 (10.6)
32
*Indicates significant value.
a
Standard deviation.
b
Wilcoxon rank-sum test.
c
extracted from maternity record book.
d
Pearson c2 test.
8 (17.7)
14 (31.1)
10.94 (1.80)
52 (49.5)
8 (7.6)
18 (17.1)
0.46d
0.37d
0.065d
0.056d
164
A. Khattab et al. / Placenta 34 (2013) 162e167
the incubation, antibody coating buffer was removed, and free binding sites on the
wells were blocked with 200 ml 1% BSA (SigmaeAldrich, Steinheim, Germany) in PBS
during incubation for 2 h at room temperature. Microtiter plates were then washed
three times with PBS containing 0.05% Tween 20 (PBS-T). The plasma samples
(100 ml diluted 1:20 in PBS-T) were added, the plates were incubated for 2 h at 4 C,
washed five times, and incubated for 1 h at room temperature with 1:1000 diluted
goat anti-C7 antibody (Organon Teknika, West Chester, PA). After five washes, HRPconjugated donkey anti-goat IgG antibody (1:3000; Jackson ImmunoResearch, West
Grove, PA) in PBS-T was incubated for 1 h at room temperature, followed by five
washes and the addition of HRP substrate (OPD; DAKO, Glostrup, Denmark). The
reaction was stopped with 100 ml 0.5 M H2SO4, and absorbances detected at 490 nm.
Arbitrary units (AU/ml) were calculated using a standard curve obtained by activating normal human serum with Zymosan A (SigmaeAldrich; 1 mg/ml, 60 min at
37 C). The TCC concentration in the undiluted standard was defined as 1000 AU/ml.
2.4. Statistical analysis
The data analyses were performed using the JMP (SAS) statistical software. For
comparing differences between proportions, the Pearson c2 test was employed, and
the Odds Ratio (OR) and the 95% confidence intervals (CI) for each comparison were
also calculated. Groupwise medians were compared for significant differences
between two groups by the Wilcoxon rank-sum test. SEM is used to describe the
variability of means. Multiple linear regression analysis models were used to estimate the association of TCC levels, entered into the models as a continuous response
variable, with parity and malaria infection, entered into the models as nominal
predictor variables. p values < 0.05 were considered statistically significant.
3. Results
3.1. Characteristics of the study population
The total number of women who gave their consent to participate in the study was 150. From them 142 peripheral, 146 cord and
142 placental plasma samples were available for the measurement
of TCC levels. In our study cohort the primiparous women were
significantly more often infected with P. falciparum than multiparous women (OR 2.38, 95% CI 1.16e4.88, P ¼ 0.016). Moreover,
babies born to women with a history of malaria during pregnancy
or with TBS positive placenta tended to be in the LBW (<2500 g)
category more often than those born to non-infected women (OR
3.3, 95% CI 1.02e11.01, P ¼ 0.023). A similar trend, albeit not
a significant one, was also obtained for the effect of parity on the
pregnancy outcome (LBW): in our study cohort primiparous
women tended to deliver babies in the LBW category more often
than multiparous women (OR 2.62, 95% CI 0.82e8.41, P ¼ 0.065).
infection rate [2]. Therefore, parity is an important parameter to
consider when evaluating the levels of complement activation in
PM. In the current study cohort, parity had a significant influence
on the levels of TCC in the placental and cord plasma samples. TCC
levels were significantly higher in the placental (Fig. 1A, Wilcoxon,
p ¼ 0.02) and cord (Fig. 1B, Wilcoxon, p ¼ 0.03) plasma samples of
primiparous women than in those of the multiparous women.
However, peripheral plasma samples did not show any significant
difference between the two groups.
3.4. Influence of malaria infection on TCC levels
Parasite sequestration in the placenta of P. falciparum infected
pregnant women could lead to complement activation and increase
in the TCC level. Therefore, the whole data set from each
compartment was stratified into malaria parasite-positive and
malaria parasite-negative groups and analyzed statistically. The
malaria parasite-positive group is created by adding the malaria
infection at delivery to the recorded past infection cases to exclude
any possible contribution of submicroscopic chronic infection from
the latter group to the measured TCC levels in the malaria parasitenegative group. To warrant validity of this grouping TCC levels of
the two groups were compared statistically and found to be similar
i.e. no significant difference between TCC levels in the two groups
in placental (Wilcoxon, p ¼ 0.9), cord (Wilcoxon, 0.8) and peripheral (Wilcoxon, p ¼ 0.5) plasma samples. As expected, placental
plasma samples of women with placental infection or malaria
history had significantly higher levels of TCC (Fig. 2A, Wilcoxon,
3.2. TCC levels in peripheral, placental and cord plasma
Since, PM is presented by poor pregnancy outcomes we wanted
to examine whether excessive complement activation in the
placenta could contribute to PM complications. Here, the TCC levels
were measured in plasma samples from three different compartments, the placental, cord and the peripheral blood from women
attending the maternity ward at Albert Schweitzer hospital for
delivery. The mean value of the measured TCC levels was highest in
the peripheral blood plasma samples (10.1 0.44 AU/ml) followed
by placental samples (6.9 0.44 AU/ml) and the cord samples
(1.5 0.23 AU/ml). These levels were significantly different
between all possible pairs of compartments data (Wilcoxon,
p < 0.0001). TCC levels in peripheral, placental and cord plasma
samples showed no correlation between each other (data not
shown).
3.3. Influence of parity on TCC levels
PM in stable malaria transmission areas affects mainly women
in their first pregnancies. In the following pregnancies, PM incidence declines [2]. This is reflected as a lower morbidity and
mortality in the mothers and their babies and as a smaller placental
Fig. 1. Parity and TCC levels in placental (A) and cord (B) plasma samples. TCC levels in
placental and cord plasma were significantly higher in the primiparous women’s than
in multiparous women’s group. The top, bottom and line through the middle of the box
correspond to the 75th, 25th, and 50th percentile, respectively. Statistical significances
of the differences between the groups were calculated by the Wilcoxon rank-sum test.
A. Khattab et al. / Placenta 34 (2013) 162e167
165
a birthweight of less than 2500 g at delivery. The TCC levels of
the LBW group (<2500 g, n ¼ 16) were compared with those of
the rest of the cohort. Data from the three compartments
(periphery, placenta and cord) showed no significant differences
between the two groups (data not shown). As the number of
samples in the LBW group was small we also studied babies
weighing <2700 g (<25% percentile, n ¼ 32). Placental plasma
samples from births of babies with a birthweight <2700 g
(n ¼ 32) had significantly higher levels of TCC than those of the
normal BW babies (Fig. 3A, Wilcoxon, p ¼ 0.007). Yet again, the
levels of TCC in the peripheral (Table 2, Wilcoxon, p ¼ 0.11) and cord
plasma samples (Fig. 3B, Wilcoxon, p ¼ 0.11) did not show any
significant differences between the low and normal baby weight
groups. Additionally, when the non-stratified data was used to
examine for a correlation between birthweight, as a continuous
variable, and TCC levels, weak negative correlations were observed
between birthweight and both placental and cord TCC levels,
r ¼ 0.2, p ¼ 0.013 and r ¼ 0.2, p ¼ 0.015, respectively. Altogether,
these findings suggest that complement activation could be related
to the delivery of smaller babies in malaria endemic areas.
4. Discussion
Complement activation in the placenta could be one of the
mechanisms that lead to poor pregnancy outcome in pregnancy
associated diseases. In the current study, the levels of complement
Fig. 2. Placental (A) and cord (B) plasma TCC levels in women with or without
P. falciparum-malaria infection. TCC levels in both placental and cord plasma samples
were significantly higher in the infected women than in the non-infected women.
P. falciparum-malaria infection was defined as thick blood smear positive placental
blood or malaria during pregnancy documented in the maternity record book. The top,
bottom and line through the middle of the box correspond to the 75th, 25th, and 50th
percentile, respectively. Statistical significances of the differences between the groups
were calculated by the Wilcoxon rank-sum test.
p ¼ 0.03) than women with no records of malaria at delivery or
during pregnancy. Additionally, cord plasma samples matched with
women with infected placenta or history of malaria during pregnancy had significantly higher TCC levels than those matched with
the counter group (Fig. 2B, Wilcoxon, p ¼ 0.01). However, once
again peripheral plasma TCC levels failed to show any significant
difference between the two groups. So far high TCC levels were
found to be significantly associated with both primiparous women
and malaria infection. However, these two variables were also
shown to be associated with each other (OR 2.38, 95% CI 1.16e4.88,
P ¼ 0.016). Therefore, multiple linear regression models have been
used to estimate which of these two variables could significantly
predict TCC levels. The multiple regression fitting models suggested
that only parity can significantly predict TCC levels after adjusting
for parity and malaria for both placental (F ¼ 4.35, p ¼ 0.039) and
cord (F ¼ 5.71, p ¼ 0.018) plasma.
3.5. TCC levels and birthweight
One profound manifestation of PM is the induction of premature
delivery or delivery of full term LBW babies. The pathophysiology
leading to these conditions is not yet fully understood. Most
newborns weigh 2700e4000 g with the average weight being
about 3400 g. Newborns who weigh below 2500 g are classified
as LBW babies. The study cohort included 16 babies with
Fig. 3. TCC levels in placental (A) and cord (B) plasma samples according to weights of
babies delivered in malaria endemic areas. TCC levels in placental plasmas from babies
with a birthweight < 2700 g were significantly higher than those in the placental
plasmas of babies with normal birthweight. No significant difference between the two
groups was observed when TCC levels from cord plasmas were analyzed. The top,
bottom and line through the middle of the box correspond to the 75th, 25th, and 50th
percentile, respectively. Statistical significances of the differences between the groups
were calculated by the Wilcoxon rank-sum test.
166
A. Khattab et al. / Placenta 34 (2013) 162e167
Table 2
Peripheral plasma TCC levels.
Variable
Group A
B
N
TCC
(AU/ml)
median
25e75 Percentile
(AU/ml)
P value
Parity
Primiparous
Multiparous
Positive
Negative
<2700 g
>2700 g
42
100
49
93
29
113
8.8
9.4
8.6
9.6
8.8
9.6
7.4e10.8
7.4e12.6
7.4e11.0
7.4e12.0
6.6e11.6
7.4e12.2
0.32
Malaria history
Birthweight
0.44
0.11
activation were measured in placental, peripheral and cord plasma
samples collected from pregnant women at delivery in a malaria
endemic area. Moderate levels of TCC in the placental plasma
samples, relative to the slightly higher levels of TCC in the
peripheral plasma samples, were detected. This could be explained
by the fact that complement activation is more controlled in the
placenta than in the periphery. It is worth mentioning that the
higher levels of TCC in the maternal peripheral plasma samples did
not show any significant differences between the investigated
groups as opposed to what was seen with the placental and cord
plasma samples. This suggests that TCC levels in the peripheral
plasma samples do not completely reflect the actual TCC levels in
the placenta produced in response to the sequestered parasites
despite the fact that the placental blood space is connected with the
peripheral blood circulation. This phenomenon could be explained
by the continuous activation of complement in the placenta due to
parasite sequestration in active infections, combined with the
slower blood flow and delayed clearance from the intervillous
spaces. Thus, the local variations in the levels of TCC would reflect
the pathological changes in the placenta. Thereby, the contribution
of the placental TCC levels that result from placental malaria
infection to the peripheral blood TCC levels could be minimal when
intervillous blood is mixed with peripheral blood. Significant
differences between levels of certain hormones [24] and cytokines
[25] in placental and peripheral blood were also previously reported. A similar scenario could also be proposed for past infection
cases in which any uncleared placental infection or placental
damage could remain as a source for continuing complement
activation.
On the other hand, the much lower TCC levels detected in the
cord samples could be explained by the immaturity of the
complement system in the fetus. For example, the concentrations of
C8 and C9 in full-neonates are in the range of 10e25% of the levels
in adults [26]. However, despite these very low levels of TCC in cord
plasma, stratification of the TCC levels according to either parity or
malaria status showed significant differences between groups.
Since there were no parasites detected in all cord blood samples
tested by TBS in our study cohort, the most probable explanation
for this finding is immune complexes transfer from the maternal to
the fetal circulation that would lead to complement activation in
the fetal blood. Antibody-dependent transplacental transfer of
a malaria blood-stage antigen in the form of immune complexes
has been reported previously in PM [27].
PM complications are more serious in primiparous than multiparous women in malaria endemic areas [8]. Our study has shown that
placental plasma samples of primiparous women had significantly
higher levels of TCC than those of multiparous women. This could be
simply explained by the higher susceptibility of primiparous women
in malaria endemic areas to malaria infection than of multiparous
women. In fact, primiparous women in our study cohort were more
likely to have malaria than multiparous women (OR 2.38, 95% CI
1.16e4.88, P ¼ 0.016). In addition, we have shown that pregnant
women with TBS positive placenta at delivery or TBS positive
peripheral blood during pregnancy presented a significantly higher
level of complement activation (TCC) in placental samples than
women without evidence for malaria. However, when the abovementioned arguments for explaining the higher levels of TCC in
primiparous women were statistically evaluated by multiple
regression models, parity had unexpectedly a stronger prediction
power for TCC than malaria infection. This indicates that the TCC
levels had stronger association with parity than malaria infection but
does not preclude the role of malaria infection in increasing the levels
of complement activation. It could also point out to some other
unknown conditions associated with primiparous women and have
contributed to complement activation.
The most interesting finding in our study was that placental
plasma samples from deliveries of babies with smaller birthweight
(<2700 g) had significantly higher levels of TCC than those corresponding to normal weight babies. This finding suggests that
complement activation in the placenta could play a role in the
pathogenesis of PM. It is worth pointing out that malaria might not
entirely account for this finding as there was no significant difference between TCC levels in malaria negative and malaria positive
groups in the <2700 g birthweight category (Wilcoxon, p ¼ 0.63,
data not shown). This is in addition to the results from the multiple
regression models pointing to a stronger role of parity than of
malaria infection in increasing TCC levels. However, increasing the
number of cases in a future study cohort and the detection of the
malaria infection status by PCR and histological section of the
placentas at deliver should address the role of other factors in this
association and overcome the limitations of our study.
A similar role of the involvement of complement activation in
inducing fetal growth restriction was previously suggested in
preeclampsia, another pregnancy-related disease that is more
profound in primiparous women. It has been shown that preeclamptic
patients with intrauterine growth retardation (IUGR) had significantly
higher plasma TCC levels than those without IUGR [28]. Since
preeclampsia was not an exclusion criterion in our study it is possible
that preeclampsia could have contributed to the increased levels of
TCC in placental plasmas of primiparous women. This argument could
also explain why primiparity rather than malaria infection was the
main determinant of the TCC levels when both were entered in the
logistic regression analysis models as predictor variables.
In PM, there is a massive sequestration and cytoadhesion of
parasitized red blood cells to the placental syncytiotrophoblast [2]
and this was shown earlier to be accompanied by C3b deposition in
the placenta as consequence of complement activation [29]. Parasite sequestration is usually accompanied by massive monocyte
and macrophage infiltrations into the placental intervillous spaces
[10]. An association between monocytes, macrophages and their
secreted products with LBW was previously reported in PM [30e
34]. In line with the current study, the latter observation could be
explained by the possibility that sequestered parasites in the
placenta could activate complement and the resulting anaphylatoxins C3a and C5a (complement activation split products) would
then activate and recruit more monocytes and macrophages to
a newly formed inflammation site. The consequences of these
events are the secretion of abnormal levels of proinflammatory
cytokines that can lead to impairment of vascular angiogenesis and
inhibition of trophoblast invasion of the decidua resulting in IUGR.
In fact, overproduction of TNF-a [25,33,34], IFN-g [34] and IL-8 [33]
has been previously related to LBW in PM. Also, C5a release caused
by the activation of complement by P. falciparum parasite material
was shown to induce an amplified inflammatory (ex. IL-6 and TNFa) and anti-angiogenic response (sFlt-1) by primary human
monocytes, in-vitro [35]. In the same study [35], C5a concentration
was shown to be significantly higher in placental plasma samples of
primiparous women with PM than in those without PM confirming
A. Khattab et al. / Placenta 34 (2013) 162e167
our finding on PM cases showing higher levels of complement
activation. Interestingly, a recent study performed in a malaria
mouse model has shown that C5a could be generated independent
of the known pathways of complement activation that converge at
the C3 step [36]. The latter finding suggests that generation of C5a
can proceed via two different mechanisms in malaria infected
individuals and this possibly can result in a much higher levels of
C5a causing more inflammatory responses to the disease.
Furthermore, in an experimental malaria infection, blocking of C5a
or the C5a receptor (C5aR) by antibodies protected mice form
cerebral malaria [37] suggesting that blocking complement activation at the terminal pathway level could protect from disease
severity. Additionally, a mutation in MASP2, a component of the
lectin pathway of complement activation, that could eventually
lead to a reduced level of C5a showed a trend towards protection
from LBW in PM [38].
In conclusion, the current study shows an association between
complement activation and poor fetal outcome in PM. This finding
suggests a role for complement activation as one of the underlying
mechanisms. Parasite-mediated complement activation could
release the anaphylatoxins C3a and C5a that would recruit and
activate monocytes and other inflammatory cells in the placenta.
The consequences of these events are the secretion of abnormal
levels of proinflammatory cytokines that can lead to impairment in
vascular angiogenesis and inhibition of trophoblast invasion of the
decidua resulting in intrauterine retardation [39].
Acknowledgment
We are grateful to the volunteers for their participation in the
study and the staff of the maternity ward of the Albert Schweitzer
Hospital for their cooperation. This work was supported by Helsinki
University Central Hospital funds (EVO), Helsinki University Three
Years Research grant program, Sigrid Jusélius Foundation and the
Academy of Finland.
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