IEs adhere to placental CSA in vivo

The ability of Plasmodium falciparum-infected erythrocytes (IE) to sequester in the placenta is a key determinant of predisposition to malaria in pregnancy, and immune responses to antigens expressed on the surface of these IE appear to mediate protection from malaria (Fried and Duffy, Reference Fried and Duffy1996; Fried et al. Reference Fried, Nosten, Brockman, Brabin and Duffy1998b). Recent reviews in this special issue (Duffy; Hviid and Salanti; Scherf) and elsewhere (Beeson and Duffy, Reference Beeson and Duffy2005; Rogerson et al. Reference Rogerson, Hviid, Duffy, Leke and Taylor2007) summarize current understanding regarding how IE sequester in the placenta and how pregnancy specific malaria immunity develops, in detail. The accumulation of IE in the maternal circulation or intervillous spaces of the placenta occurs through adhesion of IE to placental receptors such as chondroitin sulphate A on the syncytiotrophoblast surface or within the intervillous space. There is compelling evidence that the VAR2CSA form of PfEMP1 binds to chondroitin sulphate A, and that placental parasites almost always transcribe the gene encoding this protein (Duffy et al. Reference Duffy, Caragounis, Noviyanti, Kyriacou, Choong, Boysen, Healer, Rowe, Molyneux, Brown and Rogerson2006; Salanti et al. Reference Salanti, Staalsoe, Lavstsen, Jensen, Sowa, Arnot, Hviid and Theander2003; Tuikue Ndam et al. Reference Tuikue Ndam, Salanti, Bertin, Dahlback, Fievet, Turner, Gaye, Theander and Deloron2005, and see also Hviid and Salanti, in this special issue). Few IE are apposed to the syncytiotrophoblast on histology; they may adhere to secreted chondroitin sulphate A in the intervillous space (Muthusamy et al. Reference Muthusamy, Achur, Bhavanandan, Fouda, Taylor and Gowda2004). There may be artefacts from the histological preparation process or it may be that IE are frequently trapped in fibrin deposits initiated by activated macrophages expressing Tissue Factor (Imamura et al. Reference Imamura, Sugiyama, Cuevas, Makunde and Nakamura2002). Immunoelectron microscopy and immunochemical studies could resolve some of these questions regarding site of sequestration, while probing histological sections with antibodies to specific VAR2CSA epitopes will allow comparison of parasites in different sites in the same placenta, and between placentas.

The value of placental histology examination

The association between placental histology and pregnancy outcome has recently been reviewed in detail (Brabin et al. Reference Brabin, Romagosa, Abdelgalil, Menendez, Verhoeff, McGready, Fletcher, Owens, D'Alessandro, Nosten, Fischer and Ordi2004). Placental malaria is associated with accumulation in the intervillous space of mononuclear cells, predominantly monocytes and macrophages that often contain the malaria pigment haemozoin (Walter, Garin and Blot, Reference Walter, Garin and Blot1982), and deposition of haemozoin in fibrin. These features of placental pathology can only be accurately detected using histology, which is also more sensitive than peripheral or placental blood microscopy at detecting infection. Both current infection (presence of parasites) and past infection (haemozoin without parasites) have been associated with reduced birth weight (Rogerson, Mkundika and Kanjala, Reference Rogerson, Mkundika and Kanjala2003b). Dense monocyte infiltrates are particularly common in primigravid women, and are associated with LBW due to FGR, whereas high parasitaemia is associated with PTD (Menendez et al. Reference Menendez, Ordi, Ismail, Ventura, Aponte, Kahigwa, Font and Alonso2000; Ordi et al. Reference Ordi, Ismail, Ventura, Kahigwa, Hirt, Cardesa, Alonso and Menendez1998). Whenever possible, intervention studies should use histology as an outcome measure, while studies of pathogenesis should examine the relationship between timing of infection and subsequent pathology (reviewed below).

The importance of malaria in early pregnancy

In one of the few studies to recruit women in early pregnancy, malaria parasite prevalence peaked at 13–16 weeks gestation (Brabin, Reference Brabin1991b). A recent study suggests that women most frequently present to antenatal clinics with symptomatic malaria (febrile symptoms accompanied by parasitaemia) in the second trimester, but that such episodes are not uncommon in first trimester (Bardaji et al. Reference Bardaji, David, Amos, Romagosa, Maixenchs, Sigauque, Banda, Bruni, Sanz, Aponte, Alonso and Menendez2006). This is important both for prevention – should we start antenatal care and intermittent preventive therapy (IPTp) in the first trimester? – and for understanding pathogenesis. Placental blood flow only begins at 10–12 weeks gestation, so IE cannot sequester in the placenta before this time (Jauniaux, Gulbis and Burton, Reference Jauniaux, Gulbis and Burton2003). We need further studies examining malaria infection and disease in early pregnancy, to determine the significance and consequences of these early infections, the characteristics of the parasites infecting women in early pregnancy and (if there is a predilection to malaria before placental circulation develops) what the immunological or hormonal basis may be for this susceptibility.

Age and gravidity are separate components of susceptibility to malaria in pregnancy

In a community-based study in Senegal, age-related disease incidence and parasite density data showed that acquisition of immunity to malaria extends into adulthood (Trape et al. Reference Trape, Rogier, Konate, Diagne, Bouganali, Canque, Legros, Badji, Ndiaye, Ndiaye, Brahimi, Faye, Druilhe and da Silva1994); thus young women are more susceptible to malaria than their older peers, and this susceptibility may be exacerbated in pregnancy. In several recent studies, age has been a more important independent predictor of malaria parasitaemia in pregnant women than gravidity, and, in some studies, women of equal gravidity but younger age were shown to be more likely to carry malaria infection (Rogerson et al. Reference Rogerson, van den Broek, Chaluluka, Qonqwane, Mhango and Molyneux2000; Saute et al. Reference Saute, Menendez, Mayor, Aponte, Gomez-Olive, Dgedge and Alonso2002; Tako et al. Reference Tako, Zhou, Lohoue, Leke, Taylor and Leke2005; Wort et al. Reference Wort, Warsame and Brabin2006). The mechanisms underlying the age-dependent predisposition to malaria are worthy of exploration. Practically, these observations provide strong support for programmes that target adolescent girls for antenatal interventions including malaria prevention (Brabin and Brabin, Reference Brabin and Brabin2005; Wort et al. Reference Wort, Warsame and Brabin2006).

Symptomatic and severe malaria in pregnancy

In areas of low malaria transmission, almost all malaria episodes in pregnant women become symptomatic if untreated and pregnant women are especially susceptible to severe malaria, with high case fatality rates (Nosten et al. Reference Nosten, Rogerson, Beeson, McGready, Mutabingwa and Brabin2004; Wickramasuriya, Reference Wickramasuriya1935). In sub-Saharan Africa, much malaria infection is asymptomatic, but febrile symptoms are more common in women with malaria infection than in uninfected women (Diagne et al. Reference Diagne, Rogier, Cisse and Trape1997; Rogerson et al. Reference Rogerson, Pollina, Getachew, Tadesse, Lema and Molyneux2003c). Symptoms of febrile illness have a low positive predictive value for malaria parasitaemia, so parasitological diagnosis is important (Bardaji et al. Reference Bardaji, David, Amos, Romagosa, Maixenchs, Sigauque, Banda, Bruni, Sanz, Aponte, Alonso and Menendez2006). Symptomatic malaria requires prompt treatment, as it may otherwise lead to miscarriage or premature labour, while fever without parasitaemia should lead to the search for alternative explanations.

Maternal mortality is a devastating problem in sub-Saharan Africa, such that in many countries a woman's lifetime risk of dying during pregnancy or the puerperium is over 5%. Effective management of obstetric complications is absolutely critical (Campbell and Graham, Reference Campbell and Graham2006), but 50% or more of deaths may be clinically of infectious aetiology (Lema et al. Reference Lema, Changole, Kanyighe and Malunga2005), and in Mozambique malaria was the third most common cause of maternal death at autopsy. Malaria and other treatable infectious diseases were frequently misdiagnosed in life. Autopsy studies, combined with facility based descriptions of the spectrum of symptomatic disease and with community based surveillance for symptomatic disease are required to delineate the importance of symptomatic, severe and fatal malaria in pregnancy.

Malaria and HIV

HIV infection increases maternal susceptibility to malaria, and this effect is most pronounced in multigravid women (ter Kuile et al. Reference ter Kuile, Parise, Verhoeff, Udhayakumar, Newman, van Eijk, Rogerson and Steketee2004). This susceptibility may be explained by a failure to develop adequate variant-specific immunity (Mount et al. Reference Mount, Mwapasa, Elliott, Beeson, Tadesse, Lema, Molyneux, Meshnick and Rogerson2004) or by altered cellular immune responses (reviewed by Ned et al. Reference Ned, Moore, Chaisavaneeyakorn and Udhayakumar2005). HIV impairs response to antimalarial therapy (Kamya et al. Reference Kamya, Gasasira, Yeka, Bakyaita, Nsobya, Francis, Rosenthal, Dorsey and Havlir2006; Shah et al. Reference Shah, Smith, Obonyo, Kain, Bloland, Slutsker and Hamel2006; Van Geertruyden et al. Reference Van Geertruyden, Mulenga, Mwananyanda, Chalwe, Moerman, Chilengi, Kasongo, Van Overmeir, Dujardin, Colebunders, Kestens and D'Alessandro2006), and HIV infected women may need more frequent administration of IPTp (Parise et al. Reference Parise, Ayisi, Nahlen, Schultz, Roberts, Misore, Muga, Oloo and Steketee1998), although clear benefit of increased dosing has yet to be demonstrated (Meshnick, Mwapsa and Rogerson, Reference Meshnick, Mwapasa and Rogerson2006). Cotrimoxazole prophylaxis improves health and prolongs life in highly immunosuppressed HIV infected adults (Mermin et al. Reference Mermin, Ekwaru, Liechty, Were, Downing, Ransom, Weidle, Lule, Coutinho and Solberg2006), and may improve neonatal outcomes in women with low CD4 counts (Walter et al. Reference Walter, Mwiya, Scott, Kasonde, Sinkala, Kankasa, Kauchali, Aldrovandi, Thea and Kuhn2006). Moreover, cotrimoxazole has antimalarial activity, decreasing malaria episodes among HIV infected and uninfected recipients (Mermin et al. Reference Mermin, Lule, Ekwaru, Malamba, Downing, Ransom, Kaharuza, Culver, Kizito, Bunnell, Kigozi, Nakanjako, Wafula and Quick2004; Thera et al. Reference Thera, Sehdev, Coulibaly, Traore, Garba, Cissoko, Kone, Guindo, Dicko, Beavogui, Djimde, Lyke, Diallo, Doumbo and Plowe2005), and appears safe in pregnancy (Forna et al. Reference Forna, McConnell, Kitabire, Homsy, Brooks, Mermin and Weidle2006). Therefore, malaria-exposed HIV-infected pregnant women should receive cotrimoxazole and sleep under insecticide-treated bed nets (Brentlinger, Behrens and Micek, Reference Brentlinger, Behrens and Micek2006). If receiving antiretroviral drugs, they should be monitored closely for interactions between antimalarials and these agents (Brentlinger et al. Reference Brentlinger, Behrens and Micek2006). Whether they should receive additional IPTp (with non-sulphur based drug combinations), and whether antiretroviral therapy will diminish the particular susceptibility of HIV infected women to malaria in pregnancy are important and unresolved questions.

Nutrition and malaria

Women with short stature (<145 cm), low body weight (<45 kg), and/or mid-upper arm circumference<22 cm are at increased risk of adverse pregnancy outcomes. These parameters may reflect acute or chronic under-nutrition. Maternal under-nutrition and malaria frequently occur together in poor rural women, and each condition is most common in the rainy season, when work demands are greatest, malaria transmission is highest and food is scarcest (Rayco-Solon, Fulford and Prentice, Reference Rayco-Solon, Fulford and Prentice2005). Nutritional supplementation at these times of food scarcity increased birth weight (Ceesay et al. Reference Ceesay, Prentice, Cole, Foord, Weaver, Poskitt and Whitehead1997). To date, these studies have not systematically examined malaria and maternal nutrition in parallel, and an important challenge is to determine whether combining nutritional and malarial interventions has additive or synergistic effects on pregnancy outcomes.

MiP outside of Africa

A large proportion of pregnant women at risk of acquiring malaria reside in Asia and South America, but we know little about the burden of disease or its pathophysiology in these regions. Important questions include disease manifestations in women with little prior malaria exposure; the role of P. vivax in pregnancy (which has been shown to decrease birth weight in one Asian study (Nosten et al. Reference Nosten, McGready, Simpson, Thwai, Balkan, Cho, Hkirijaroen, Looareesuwan and White1999)); and the optimal strategies for prevention. The burden of malaria in pregnancy outside Africa is reviewed in detail by Desai et al. (Reference Desai, ter Kuile, Nosten, McGready, Asamoa, Brabin and Newman2007), and malaria in pregnancy is compared between high and low transmission regions by Nosten et al. (Reference Nosten, Rogerson, Beeson, McGready, Mutabingwa and Brabin2004).

Defining the role of intervillous monocytes as independent markers of LBW

Monocyte infiltrates can develop in the intervillous space in response to malaria (Walter et al. Reference Walter, Garin and Blot1982), and have been associated with FGR and anaemia (Menendez et al. Reference Menendez, Ordi, Ismail, Ventura, Aponte, Kahigwa, Font and Alonso2000; Rogerson et al. Reference Rogerson, Pollina, Getachew, Tadesse, Lema and Molyneux2003c). IE sequestered in the placenta induce the secretion of β-chemokines by maternal mononuclear cells (Abrams et al. Reference Abrams, Brown, Chensue, Turner, Tadesse, Lema, Molyneux, Rochford, Meshnick and Rogerson2003; Chaisavaneeyakorn et al. Reference Chaisavaneeyakorn, Moore, Mirel, Othoro, Otieno, Chaiyaroj, Shi, Nahlen, Lal and Udhayakumar2003; Suguitan et al. Reference Suguitan, Leke, Fouda, Zhou, Thuita, Metenou, Fogako, Megnekou and Taylor2003) and by foetal syncytiotrophoblast (Abrams et al. Reference Abrams, Brown, Chensue, Turner, Tadesse, Lema, Molyneux, Rochford, Meshnick and Rogerson2003; Lucchi et al. Reference Lucchi, Peterson and Moore2006a), attracting monocytes to the placenta. Macrophage migration inhibitory factor is found in increased levels in women with placental malaria (Chaisavaneeyakorn et al. Reference Chaisavaneeyakorn, Lucchi, Abramowsky, Othoro, Chaiyaroj, Shi, Nahlen, Peterson, Moore and Udhayakumar2005), and it may help to retain the recruited monocytes and to activate them to secrete β-chemokines, setting up a positive feedback loop. How these monocytes are activated, what molecules they secrete and how this affects birth weight is largely not understood.

Activation of placental monocytes and cytokine secretion

Placental monocytes are relatively more activated than circulating monocytes (Diouf et al. Reference Diouf, Fievet, Doucouré, Ngom, Gaye, Dumont, Ndao, Le Hesran, Chaouat and Deloron2004). Several factors present in the intervillous space of malaria-infected placentae could activate monocytes. In particular, IEs (Bate, Taverne and Playfair, Reference Bate, Taverne and Playfair1988), malaria pigment (haemozoin) (Pichyangkul, Saengkrai and Webster, Reference Pichyangkul, Saengkrai and Webster1994), glycosylphosphatidylinositol (Krishnegowda et al. Reference Krishnegowda, Hajjar, Zhu, Douglass, Uematsu, Akira, Woods and Gowda2005) and fibrinogen (Smiley, King and Hancock, Reference Smiley, King and Hancock2001) have all been shown to induce TNF production by monocytes. Placental levels of TNF and interleukin 6 (which come mainly from monocytes) and of interferon γ (a major activator of monocytes) have been associated with LBW in at least one study (Fried et al. Reference Fried, Muga, Misore and Duffy1998a; Moormann et al. Reference Moormann, Sullivan, Rochford, Chensue, Bock, Nyirenda and Meshnick1999; Rogerson et al. Reference Rogerson, Brown, Pollina, Abrams, Tadesse, Lema and Molyneux2003a). The mechanism by which these cytokines might cause FGR is not known, and studies of pathological pregnancies leading to FGR (reviewed below) may hold relevant insights for understanding pathogenesis of FGR due to malaria.

Could malaria-activated macrophages impair placentation?

Pre-eclampsia is another pathological pregnancy condition that leads to LBW (Sibai, Dekker and Kupferminc, Reference Sibai, Dekker and Kupferminc2005). Similarities (and discrepancies) between pre-eclampsia and MiP have been recently reviewed (Brabin and Johnson, Reference Brabin and Johnson2005) and common pathogenic mechanisms may be involved. Pre-eclampsia is characterized by an impaired cytotrophoblast invasion, sub-optimal uterine arterial remodelling and defective replacement of maternal endothelium by endovascular cytotrophoblasts (Redman and Sargent, Reference Redman and Sargent2005), such that the normal development of a low-resistance, high-capacity blood flow through spiral arteries feeding the placenta is impaired (Goldman-Wohl and Yagel, Reference Goldman-Wohl and Yagel2002).

Malaria has been identified as a risk factor for pre-eclampsia and hypertension in pregnancy in some studies (Sartelet et al. Reference Sartelet, Rogier, Milko-Sartelet, Angel and Michel1996; Muehlenbachs et al. Reference Muehlenbachs, Mutabingwa, Edmonds, Fried and Duffy2006) but not others (Dorman et al. Reference Dorman, Shulman, Kingdom, Bulmer, Mwendwa, Peshu and Marsh2002). A recent study (reviewed by Duffy, in this special issue) showed an association between chronic placental malaria and hypertension in primigravidae (Muehlenbachs et al. Reference Muehlenbachs, Mutabingwa, Edmonds, Fried and Duffy2006). There are no published data on the effect of malaria during placentation on cytotrophoblast invasion and spiral artery remodelling, but the peak prevalence of P. falciparum parasitaemia early in the second trimester overlaps with the period when spiral arteries undergo remodelling (complete by 20–22 weeks of gestation). Thus, there is a window in time during which malaria infection could indeed impair the establishment of an optimal placental blood flow.

Trophoblast invasion of spiral arteries is vulnerable to factors causing activation of maternal cells in the uterine bed. In particular, TNF and interleukin 1β (both increased in placental malaria) dramatically increase secretion of the monocyte chemokine MCP-1 by uterine decidua (Lockwood et al. Reference Lockwood, Matta, Krikun, Koopman, Masch, Toti, Arcuri, Huang, Funai and Schatz2006), a chemokine found increased in placental malaria (Abrams et al. Reference Abrams, Brown, Chensue, Turner, Tadesse, Lema, Molyneux, Rochford, Meshnick and Rogerson2003; Suguitan et al. Reference Suguitan, Leke, Fouda, Zhou, Thuita, Metenou, Fogako, Megnekou and Taylor2003). It has been recently shown in vitro that activated macrophages could inhibit trophoblast invasion (Renaud et al. Reference Renaud, Postovit, Macdonald-Goodfellow, McDonald, Caldwell and Graham2005) probably through TNF production (Bauer et al. Reference Bauer, Pollheimer, Hartmann, Husslein, Aplin and Knofler2004; Renaud et al. Reference Renaud, Postovit, Macdonald-Goodfellow, McDonald, Caldwell and Graham2005).

Maternal malaria infection during placental development could thus increase numbers of activated macrophages in the maternal decidua, and reduce trophoblast invasiveness, impairing the remodelling of spiral arteries leading to a sub-optimal placentation and a possible placental hypoxia. Studies investigating the migration/invasion potential of first trimester cytotrophoblasts in the context of gestational malaria are critically needed.

Could various monokines trigger an impairment of nutrient transport?

In recent years, there have been marked improvements in our understanding of placental nutrient transport and its role in pathogenesis of FGR of other causes (Regnault et al. Reference Regnault, Friedman, Wilkening, Anthony and Hay2005; Sibley et al. Reference Sibley, Turner, Cetin, Ayuk, Boyd, D'Souza, Glazier, Greenwood, Jansson and Powell2005; Zamudio, Baumann and Illsley, Reference Zamudio, Baumann and Illsley2006) and it is now clear that amino acids and glucose act as regulators of placental and foetal development (Regnault et al. Reference Regnault, Friedman, Wilkening, Anthony and Hay2005). Foetal concentrations of amino acids are decreased in babies with FGR compared to normal babies (Cetin et al. Reference Cetin, Corbetta, Sereni, Marconi, Bozzetti, Pardi and Battaglia1990, Reference Cetin, Ronzoni, Marconi, Perugino, Corbetta, Battaglia and Pardi1996; Economides et al. Reference Economides, Nicolaides, Gahl, Bernardini and Evans1989), and FGR is associated with impairment of the active transport mechanisms operating at the level of the syncytiotrophoblast. The importance of changes in transcription, expression, localisation and function of nutrient transporters and their encoding genes is more and more recognised in FGR. Increasing numbers of techniques to examine them have become available in the last 5 years (Glazier and Sibley, Reference Glazier and Sibley2006; Regnault et al. Reference Regnault, Friedman, Wilkening, Anthony and Hay2005; Zamudio et al. Reference Zamudio, Baumann and Illsley2006) but these have yet to be applied to studies of the pathogenesis of MiP. For example, interleukin 1β, which is increased in MiP (Moormann et al. Reference Moormann, Sullivan, Rochford, Chensue, Bock, Nyirenda and Meshnick1999), inhibited amino acid uptake of the trophoblast-like cell line (BeWo) in a dose-dependent manner (Thongsong et al. Reference Thongsong, Subramanian, Ganapathy and Prasad2005), while TNF has been shown to down-regulate amino acid transport across the placenta in vivo (Carbo, Lopez-Soriano and Argiles, Reference Carbo, Lopez-Soriano and Argiles1995). Local inflammation, triggered by malaria infection and supported by maternal monocyte infiltrates, could thus cause a decrease in amino acid transfer across the placenta, impairing foetal growth.

Beside amino acids, glucose transport across the placenta could also be reduced during MiP. GLUT1 is the main glucose transporter in the placenta and sits in the microvillous and basal membranes of the syncytiotrophoblast. Since the microvillous membrane contains more transporter than the basal membrane, alteration in the density of transporters in the basal membrane is the main factor regulating glucose transplacental transport. For example, at high altitude the amount of basal membrane GLUT1 decreases relative to microvillous membrane levels (Zamudio et al. Reference Zamudio, Baumann and Illsley2006). Glucose transport is impaired in FGR (Baumann, Deborde and Illsley, Reference Baumann, Deborde and Illsley2002) and recently, the insulin-insulin growth factor axis has been identified as a major pathogenic mechanism of LBW (Bajoria et al. Reference Bajoria, Sooranna, Ward and Hancock2002; Kajimura, Aida and Duan, Reference Kajimura, Aida and Duan2005). The basal membrane expression of GLUT1 is positively regulated by insulin-like growth factor I, low levels of which have been reported in human malaria (Mizushima et al. Reference Mizushima, Kato, Ohmae, Tanaka, Bobogare and Ishii1994). This could lead to a decrease of glucose flux across the placenta, possibly leading to FGR.

Determining the activity of the different nutrient transport pathways in MiP would greatly advance our understanding of the pathogenesis of LBW and could provide avenues for intervention strategies.

Hypoxia during pregnancy: a double-edged sword

Hypoxia is a physiological driving force for cytotrophoblast migration during implantation (Graham et al. Reference Graham, Postovit, Park, Canning and Fitzpatrick2000), but in term placental tissue, hypoxia leads to apoptosis (Levy et al. Reference Levy, Smith, Chandler, Sadovsky and Nelson2000), increased production of pro-inflammatory cytokines (Benyo, Miles and Conrad, Reference Benyo, Miles and Conrad1997) and impaired amino acid (Nelson et al. Reference Nelson, Furesz, Ganapathy, Parvin and Smith2002; Zamudio et al. Reference Zamudio, Baumann and Illsley2006) and glucose (Zamudio et al. Reference Zamudio, Baumann and Illsley2006) transport.

Placental hypoxia could be caused by sub-optimal placental blood flow due to inadequate placentation, as discussed above, or by the massive monocyte and IE infiltrates in the intervillous spaces. Moreover, by adhering to the syncytiotrophoblast, IEs and monocytes could physically decrease the surface of exchange between maternal and foetal blood leading to a further decrease in nutrient and oxygen transport across the placenta. From peak oxygen saturation of 60% in mid pregnancy, foetal blood oxygen saturation falls to around 40% by term, due to increasing foetal demand (Soleymanlou et al. Reference Soleymanlou, Jurisica, Nevo, Ietta, Zhang, Zamudio, Post and Caniggia2005). Any fall in oxygen saturation in uteroplacental blood, or significant decrease in blood supply, will result in decreased oxygen availability to the foetus.

Placental hypoxia appears to be a well-defined cause of LBW whether it is normobaric hypoxia in animal models of FGR (Regnault et al. Reference Regnault, de Vrijer, Galan, Wilkening, Battaglia and Meschia2006) or hypobaric hypoxia in high-altitude pregnancies (Zamudio, Reference Zamudio2003). Studies addressing the potential role of placental hypoxia in MiP-associated FGR would provide valuable insights in our understanding of MiP pathogenesis.

MiP-associated FGR: a complex network of potential pathogenic mechanisms

In the previous sections, we have tried to present MiP-associated FGR as a condition induced by different pathogenic processes. It is increasingly evident that the pathogenesis of FGR (associated with MiP but also with other pathological pregnancies) is multifactorial and that all those processes interact in a complex network. For example, hypoxia can induce a decrease in amino acid transport activity (Nelson et al. Reference Nelson, Furesz, Ganapathy, Parvin and Smith2002) and so do pro-inflammatory cytokines (Thongsong et al. Reference Thongsong, Subramanian, Ganapathy and Prasad2005). But, in addition, hypoxia can induce the release of pro-inflammatory cytokines (Benyo et al. Reference Benyo, Miles and Conrad1997). Deciphering the relative importance of each of these potential pathogenic mechanisms in MiP-associated FGR is a challenging but necessary task that may require prospective studies.

Animal models and cell lines: strengths and limitations

Due to ethical reasons, ex vivo studies on the pathogenesis of MiP-associated FGR have primarily used placental tissue collected at delivery. If valuable insights have been gained from these studies, such samples give no information on the kinetics of the pathogenic processes leading to LBW and, clearly, longitudinal studies are needed (as discussed above).

Our knowledge of the pathogenesis of FGR in MiP is also limited by the relative lack of relevant animal models easily amenable to experimentation. The P. coatneyi – Macaca mulatta model has proven useful (Davison et al. Reference Davison, Cogswell, Baskin, Falkenstein, Henson and Krogstad2000), but it cannot be used in most laboratories due to its cost and requirement for specific facilities. Rodent models of MiP have also been developed (Oduola et al. Reference Oduola, Phillips, Spicer and Galbraith1986; Vinayak et al. Reference Vinayak, Pathak, Asnani, Jain and Malik1986; Desowitz et al. Reference Desowitz, Shida, Pang and Buchbinder1989; Hioki, Hioki and Ohtoma, Reference Hioki, Hioki and Ohtomo1990) including a mouse model in which an accumulation of IEs in the placenta was observed together with a spontaneous abortion early in pregnancy (Poovassery and Moore, Reference Poovassery and Moore2006). However, anatomical and functional differences between primate (Benirschke and Miller, Reference Benirschke and Miller1982), mouse (Georgiades, Ferguson-Smith and Burton, Reference Georgiades, Ferguson-Smith and Burton2002) and human placentae together with the differences in the species of Plasmodium engaged imply many limitations: set-up of the model, type of questions that can be addressed, analysis and relevance of the data obtained.

The same limitations apply to cell lines, such as the choriocarcinoma cell line BeWo, which are used as models of certain cell populations in the placenta. A recent study showed that the binding of IEs to BeWo induced changes in these cells (Lucchi et al. Reference Lucchi, Koopman, Peterson and Moore2006b) suggesting that the syncytiotrophoblast might have an active role in MiP pathogenesis. Syncytiotrophoblast derived in vitro may be preferable to BeWo cells (Lucchi et al. Reference Lucchi, Koopman, Peterson and Moore2006b).

These previous remarks do not undermine the value of animal models or cell lines but they do support the necessity to conduct studies on human samples as much as possible, which may require the development and use of tools new to this field.

The example of laser capture microdissection

Placentae from LBW babies have various anatomical and histological differences from placentae from normal birth weight babies and MiP is also associated with a remodelling of the structure of placental tissue (Brabin et al. Reference Brabin, Romagosa, Abdelgalil, Menendez, Verhoeff, McGready, Fletcher, Owens, D'Alessandro, Nosten, Fischer and Ordi2004). A drawback of placental studies using whole tissue is that changes in the cellular composition of the tissue may lead to misleading differences between cases and controls. Laser capture microdissection allows the guided selection of specific areas of a tissue section for excision and analysis. The downstream applications include real-time RT-PCR (Chan et al. Reference Chan, Murray, Franklyn, McCabe and Kilby2004), genotyping (Rook et al. Reference Rook, Delach, Deyneko, Worlock and Wolfe2004), microarrays (Elkahloun, Gaudet and Robinson, Reference Elkahloun, Gaudet and Robinson2002) and proteomics (Craven and Banks, Reference Craven and Banks2001; Batorfi et al. Reference Batorfi, Ye, Mok, Cseh, Berkowitz and Fulop2003). We have recently adapted this technique to the study of syncytiotrophoblast gene expression using pressure-assisted laser capture microdissection. Captured material is collected directly into RNA extraction buffer and quantitative real-time RT-PCR is performed (Boeuf et al. Reference Boeuf, Vigan-Womas, Jublot, Loizon, Barale, Akanmori, Mercereau-Puijalon and Behr2005). This approach allowed us to identify differences in syncytiotrophoblast gene expression between Plasmodium-infected and control placentae that went unnoticed when we addressed the whole placental tissue. This example shows that technical tools such as laser capture can provide new insights in the pathogenesis of MiP by focusing on some of the many different cell types that compose the complex placental tissue architecture.