Although the effects of malaria infection on the mother and the foetus depend upon multiple factors, the host immune response and the histopathology of the placenta are specifically associated with complications observed before birth and around the neonatal period . This study evaluated the important immunological aspects and molecular mechanisms associated with tissue damage in the placenta, which contribute to the understanding of the pathophysiology of placental infection. Complex cellular and molecular mechanisms, such as apoptosis, hypoxia and inflammation have not been evaluated in cases of placental infection by Plasmodium spp in this endemic region. Such mechanisms are important to a healthy pregnancy since they contribute to implantation and promote placental development .
This is a first exploratory study on the subject in women from a region where both P. falciparum and P. vivax are endemic and is the first study to report on apoptosis and tissue injury during submicroscopic placental infection. All studied groups came from the same malaria endemic region as to control for any socio-epidemiology associated bias. According to a recent report , most placental infections at delivery in this region were sub-clinical and thick smear microscopy examination failed to diagnose them. Based on these findings, the current study included subjects in which diagnosis of infection was confirmed by qPCR.
Changes in apoptosis have been scarcely studied in placental malaria. In other pathologies such as preeclampsia, the high rate of apoptosis has been directly linked with intrauterine growth restriction . Importantly, the apoptotic index in the PM + placentas herein studied was higher than in PM-. Early reports on apoptosis in malaria infected placentas showed no difference in the proportion of apoptotic cells when compared to uninfected ones . A possible explanation for the dissimilar results from those reported here might be the application of different techniques- TUNEL vs. haematoxylin-eosin staining.
Fas and FasL expression have been proposed as markers of apoptosis, and this is considered an important mechanism by which cytokines act locally and may influence critical signaling processes. In in vitro studies with cell models and placental tissue explants stimulated with pro-inflammatory cytokines, the expression of Fas and FasL was high and similar to the results reported here . Taken together, these observations implicate apoptosis as a host response or a downstream effect of malaria infection in the placenta.
In the current study, increased apoptosis in infected placentas was also associated with high expression of the pro-inflammatory cytokines IFN-γ and TNF. Similar observations with increased expression of Th1 cytokines such as IL-2, TNF, IFN-γ and decreased Th2 cytokines such as IL-6 and TGF-β have been reported by others , and haemozoin deposition and haemozoin-loaded macrophages have implicated in the induction of inflammation in the throphoblast [30, 32]. Ongoing studies are exploring the association between haemozoin deposits and the cytokine profile in infected placentas in Colombia.
Other authors, based on high levels detected in serum of women with gestational malaria, have proposed IL-10 as biomarker of placental infection  and a definitive marker of inflammation during placental malaria . This hypothesis is supported in the current study by the higher expression of IL-10 observed in the infected vs. uninfected placentas. The increased expression of this cytokine in cases of submicroscopic infection is a novel and promising finding.
Hypoxia has been associated with complications from placental malaria infection . However, few studies have explored the relationship between malaria and placental hypoxia . Common histological changes associated with infection such as basal membrane thickening, mononuclear infiltrates and presence of parasites in the intervillous space, affect oxygen transport across the placenta. Some hypoxia mechanisms include oxygen consumption by infiltrating cells, decreased blood perfusion and reduction of the effective foetal-maternal surface area . In consequence, a physiological adaptation to hypoxia results in syncytial knot formation, an alteration often reported during placental infection by Plasmodium. Similar to the current report, Boeuf and colleagues in 2008, observed increased expression of HIF-1α and VEGF during placental infection, however not direct association between placental malaria and hypoxia could be confirmed .
Cyclooxygenase (COX) and lipoxygenase (LOX) transform fatty acids into prostaglandins and leukotrienes, which play important roles in pregnancy and foetal development. In addition, COX-2 has been proposed as a marker of preeclampsia and recovery after infectious conditions . In a study published by Sarr et al., increased expression of COX-2 and IL-10 in chronic placental malaria was detected, and COX-2 was associated with maternal anaemia, placental macrophage infiltration and haemozoin deposition . These results are consistent with the findings herein reported regarding increased COX-2 and IL-10 in cases of placental infection. Furthermore, COX-1 is reported for the first time in association with placental malaria.
Interestingly, no differences were observed between the changes in the markers studied in P. vivax infected placentas compared with those infected with P. falciparum. Some complications of P. vivax infection might be explained by the production of pro-inflammatory cytokines, which alter the balance at the foetal-maternal interface. Based on this, it is hypothesized that P. vivax is as pathogenic as P. falciparum, regardless of the presence of sequestering parasites.
The knowledge gathered so far on the pathophysiology of placental malaria mainly describes cases of P. falciparum infection. The current results demonstrate that P. vivax might also be equivalent in the pathophysiology of placental damage. These results need to be complemented with a study of a large series of P. vivax infected placentas. In addition, the role of naturally acquired specific humoral immunity should complement these studies in order to clarify its implication on clinical status. Finally, studies of the consequences of P. vivax placental infection in Asia and America in the context of a pregnancy vaccine and intermittent preventive therapy should be a priority.
In conclusion, submicroscopic placental infection by malaria parasites failed to induce major clinical effects on mother or foetus but associated with a pro-inflammatory cytokine profile, regardless of the infecting species, and this was more evident in the placental tissue. Finally, increased apoptosis was common in Plasmodium spp. infected placentas.