Haemozoin Deposits Influence Fetal Weight of Pregnant Mice Infected by Plasmodium berghei

Low birth weight is commonly attributed to malaria in pregnancy, but the cellular and molecular mechanisms that underlie this are incompletely understood. Many of hormones and cytokines are dysregulated in this case and it alters histological structure of placenta which known as placenta malaria. In the placenta malaria, there is an accumulation of infected erythrocytes, macrophages and malarial pigment (haemozoin). This study was conducted to compare the levels of plasma and placenta interferon-gamma (IFN-γ) and haemozoin deposit in pregnant mice that infected by Plasmodium berghei (treatment group) to the normal pregnant mice (control group) and its association with fetal weight. This in vivo experimental laboratory study used pregnant Balb/c mice which divided to control and treatment group. Placentas were staining with Haematoxylin-Eosin (HE) for haemozoin deposits examination. Plasma and placenta levels of IFN-γ examined with ELISA assay. Levels of IFN-γ were higher in plasma than placenta and slightly higher in treatment group than control group, but the differences were not significant (p>0,05). Fetal weight of treatment group was lower than those of control group (p=0,002) however there was no correlation between fetal weight and plasma as well as placenta levels of IFN-γ (p>0,05). Haemozoin deposit was found only in treatment group and influenced weight of fetuses (Spearman=-0,633, p=0,006). Weights of fetuses are more interfered by haemozoin deposit and seemly not by plasma and placenta levels of IFN-γ during malaria infection.


Placenta and the Fetus
Malaria is a significant health and development concern Pregnant mice were intra-peritoneally (IP) infected at day- 6 facing millions of people and its control and prevention 9 pregnancy with 10 iRBC, and parasitemia was recorded are part of the United Nations Millenium Development every other day. Non-infected pregnant females were used Goals (MDGs) (1). According to Annual Parasite Incidence as controls in pregnancy infection experiments as some areas in Indonesia are still in high level of malaria appropriate. Part of the pregnant females (both infected and the other areas are in moderate and low level (2). It is and controls) were caesarian section at day-18 of a major public health program in Indonesia with 6 million pregnancy to placenta pathology observation and the clinical cases and 700 deaths each year (3). Malaria in fetus were scaled using analytical scale (Mettler AE 50). pregnant women could cause severe anemia to the Tissue Preparation and Histopathological Analysis mother (4) that will lead to morbidity in fetus such as low birth weight (5), preterm delivery and died after birth (6).
Placentas from infected and non-infected females were treated in a similar way. Placentas were separated in two Malaria in pregnancy has a particular feature that is halves, one half was fixed in formaldehyde for further accumulation of infected red blood cells (iRBC) within tissue processing (sectioning) and the other half collected placenta (7) which is referred as placental malaria (PM).
in freeze for measurement of IFN-γ level. Paraffin-Placental malaria (PM) revealed a number of embedded non-consecutive placenta sections were abnormalities to placental tissue such as excess of s t a i n e d w i t h H a e m a t o x i l l i n -E o s i n ( H E ) a n d fibrinoid deposits and malarial pigment deposits immunohistochemically using purified anti-mouse (haemozoin) (8). Accumulation of iRBC in placenta related Interferon-gamma (BioLegend) and examined under a light to placenta inflammation by activated placental microscope (Olympus CX 21 LED FSI). macrophage that could induce placental damage through releasing inflammatory cytokines such as Tumour Measurements of Haemozoin Deposit and IFN-γ Necrosis Factor-Alfa (TNF-α), Interferon-gamma (IFN-γ) Placental sections were stained with H-E and examined (9), Interleukin-beta (IL-β) and Interleukin-2 (IL-2) (10).
under light microscope under supervised of Anatomical Interferon-gamma (IFN-γ) as inflammatory cytokine was Pathologist. Placental plasma and tissue were measured suggested as part of carefully regulated cytokine network.
using IFN-γ Quantikine ELISA (Enzyme Immuno Assay) Inc The elevated expression of IFN-γ in multigravid women From BioLegend, catalog 505801. with PM-negative suggests that this cytokine is important in controlling the parasitemia in placenta (11). To further Statistical Analysis investigate the associations among the haemozoin Analyses were performed using SPSS 16 software. deposit, IFN-γ and fetal weight, we conducted an Lavene's test was used to see the distributed data and experimental study to analyze the level of IFN-γ in plasma Saphiro wilk was used for evaluated the data homogeneity. and placental tissue, the histopathological change in Statistical differences between groups of mice used in this placenta (haemozoin deposit) and weight of the fetus in study were evaluated by t test for normally distributed pregnant mice that infected with Plasmodium berghei.
data. Correlation test was done using Pearson and Spearman. METHOD

Research Design and Samples RESULTS
An in vivo experimental laboratory study was conducted Levels of IFN-γ in Plasma and Tissue Placentas by comparing data obtained from two groups of Balb/c There were eight samples of control group and nine strain pregnant mice those were a study group which samples of treatment group. All data have been test for infected by Plasmodium berghei on ninth day post mating normality and homogeneity (Saphiro Wilk>0,05). Levels of and a control group (non-infected pregnant mice) . The IFN-γ were higher in plasma than in tissue placentas and pregnant mice were mice of 20-30 grams weight and 13slightly higher in treatment group than control group, 15 weeks old. The mice were evaluated daily, especially however the differences were not significant both in their body weight and pregnancy symptoms also the plasma (p=0,807) and tissue (p=0,424) ( Figure 1). parasitemia of the study group. On the day 18 post mating the mice were terminated.

Ethical Considerations
Ethical clearance was provided by the committees of research of the Medical Faculty Brawijaya University (No. 104/EC/KEPK-S2/03/2013). All animals in was treated well during this research and buried after research.

Animals and Parasites
The BALB/c mice were obtained from Gadjahmada University and maintained in conventional housing at the Parasitology Laboratorium Faculty of Medicine Brawijaya University. Infection experiments were performed in adult females, between 13-16 weeks of age. Plasmodium berghei ANKA was provided by Parasitology Laboratory Brawijaya University. All animals were fed with regular  We also found increasing of trophoblast expressing IFN-γ Correlation Test between Level of IFN-γ, Haemozoin in placenta of treatment group. Figure 2 showed that Deposits and Fetal weight the expressions of IFN-γ from the treatment group are There was no correlation between levels of IFN-γ and more than the control group.
weight of fetus. Both were not significant (p>0,05). Table  below shows the data.

Haemozoin Deposit
There was a negative strong correlation between Haemozoin deposits were found in all samples of haemozoin deposits and weight of fetus (Spearman=treatment group and none of the control group shows 0,705, p=0,002) and this correlation was very significant. haemozoin deposit. Haemozoin was variable in treatment group, from less than 10% up to more 40% per high-power field.

DISCUSSION
Plasma levels of IFN-γ were higher than tissue level of IFN-γ in this study, and the level of IFN-γ in treatment group was slightly higher than the control group. IFN-γ is one of the pro-inflammatory cytokine that should be increased if there is an inflammatory process including malarial infection (11,12). IFN-γ expression in response to soluble malarial antigen stimulation tended to be exclusive. PMpositive primi/secundigravid produced high level of IFN-γ and PM-positive multigravid produced low level. The elevated expression of IFN-γ by PM-negative multigravid by PM-positive multigravid especially after stimulation with malarial antigen, lends further support to this hypothesis. Also, primi/secundigravid cells mounted a Fetal Weight slightly elevated IFN-γ response in the presence of PM Mean fetal weight in this study was 0,9392 g for the infection, yet they were unable to clear their parasitemia. control group and 0,6480 g for the treatment group. Using Taken together, these data suggest that women whose are independent t test there was a significant differences committed to constitutively produce high levels of IFN-γ (p=0,002).
(i.e., PM-negative multigravidae) can effectively control parasitemia upon exposure, and those who are low producers tend to be susceptible to PM (11). Different result has shown by Barasa et al that used female baboons to measure IFN-γ. In this study, the mean concentrations of IFN-γ cytokine were significantly lower (P<0,05) in sera samples from P. knowlesi infected placentas (21,2 pg/mL) than concentrations in sera from uninfected placentas (68,3 pg/mL). Even their result is not supporting other, Barasa et al agreed that IFN-γ has a crucial protective role against PM since reduced levels of this cytokine were detected in the malaria infected placentas. Cytokine responses in the placentas were significantly altered following the onset of PM leading to a shift in immunity (13). addition to acute-phase, the production of which is decrease the fetal birth weight significantly in this study. induced rapidly by parasite toxins, IFN-γ levels can This finding supporting the previous research from increase very early during malaria. Human NK cells also Menendez et al (2000) that the association between become activated early during malaria and are activated haemozoin and birth weight was not affected by the rapidly by parasites in vitro, which requires direct contact location of the pigment, except in the case of the of infected red blood cells with NK cells and results in IFN-γ syncytiotrophoblast. Haemozoin in this site may reflect a production. (14). Increasing of IFN-γ in early phase of severe infection, possibly accompanied by inflammation, malaria also shown by study from Poovassery & Moore in the layer that is in closest contact with fetal circulation (15). IFN-γ was significantly higher in infected pregnant and this haemozoin possibly has the greatest potential to mice on day 9 and decline on day 10 when the infected interfere with intrauterine growth (5). Another research in pregnant mice begin to abort. IFN-γ levels did not differ human also shows that women with high amount of between the infected pregnant mice and uninfected.
haemozoin had babies with lower birth weights and lower level of haemoglobin, which is haemoglobin tends to Weight of fetuses are significantly different between decrease with the amount of haemozoin in the placenta control and treatment group, this is supporting by many (17). This study finding concurred that placental malaria previous studies (6,11,16). But it is seemly not interfered alters placenta histology such as haemozoin deposit and just by IFN-γ levels. Even the levels of IFN-γ in plasma or disturbs many cytokines in pregnant mice including IFN-γ. tissue placentas from treatment group were slightly Placental malaria also interfere fetal weight by the higher than the control group, they have no significantly presence of haemozoin deposits, but there is no correlation with fetal weight. This data supporting correlation between plasma and placenta IFN-γ with fetal previous study from Rogerson et al (9) that conclude TNFweight in this study. α but not IFN-γ may be implicated in impaired fetal growth (study in Malawian women).

CONFLICT OF INTERESTS Inflammatory infiltration of the intervillous spaces was
The authors declare that there is no conflict of interests. associated with a reduction in birth weight, particularly when mononuclear cells were highly increased. The presence of massive mononuclear intervillositis in the ACKNOWLEDGMENTS placenta also was associated with a 4-fold increase in risk The authors would like to thank the University of of low birth weight. These findings strongly indicate that Brawijaya, Malang, Indonesia for financial support of this that placental insufficiency attributable to physical project. We express our appreciation to our partners in blockage by infected red blood cells may not be the only, study of placental malaria: Zanibur Rahmah S.Si, M.Si; or indeed even the most important mechanism mediating Yuliyanik, Amd.Keb.SKM; dr. Sujarot Dwi Sasmito, Nur intra uterine growth retardation. This massive monocyte Fahma Pradiptasari, Adila Ulfiati, Bougenvil Ungu, the staff infiltration of the intervillous space is likely to be a source of the Parasitology Laboratory Faculty of Medicine of cytokines including IFN-γ which is considered University of Brawijaya for the animal housing and Surya detrimental to pregnancy in association to poor Kurnia Hayati, Ssi from Biomedical Laboratory Faculty of pregnancy outcomes (5).
Medicine University of Brawijaya for her good assistance in The presence of haemozoin deposits in placental tissue ELISA assay and immunohistochemistry. 281