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Background: Although maternal anaemia often stems from malaria infection during pregnancy, its effects on foetal haemoglobin levels are not straightforward. Lower-than-expected cord haemoglobin values in malarious versus non-malarious regions were noted by one review, which hypothesized they resulted from foetal immune activation to maternal malaria. This study addressed this idea by examining cord haemoglobin levels in relation to maternal malaria, anaemia, and markers of foetal immune activation. Methods: Cord haemoglobin levels were examined in 32 malaria-infected and 58 uninfected women in Blantyre, Malawi, in relation to maternal haemoglobin levels, malaria status, and markers of foetal haematological status, hypoxia, and inflammation, including TNF-α, TGF-β, and ferritin. All women were HIV-negative. Results: Although malaria was associated with a reduction in maternal haemoglobin (10.8 g/dL vs. 12.1 g/ dL, p < 0.001), no reduction in cord haemoglobin and no significant relationship between maternal and cord haemoglobin levels were found. Cord blood markers of haematological and hypoxic statuses did not differ between malaria-infected and uninfected women. Maternal malaria was associated with decreased TGF-β and increased cord ferritin, the latter of which was positively correlated with parasitaemia (r = 0.474, p = 0.009). Increased cord ferritin was associated with significantly decreased birth weight and gestational length, although maternal and cord haemoglobin levels and malaria status had no effect on birth outcome. Conclusion: In this population, cord haemoglobin levels were protected from the effect of maternal malaria. However, decreased TGF-β and elevated ferritin levels in cord blood suggest foetal immune activation to maternal malaria, which may help explain poor birth outcomes. © 2005 Abrams et al; licensee BioMed Central Ltd.
Ninety pregnant women pre-labor or in the latent phase of labor attending the Labour Ward, Queen Elizabeth Central Hospital, Blantyre, Malawi, were recruited from February to October 2002 as part of a prospective cohort study investigating the impact of maternal malaria on HIV vertical transmission [27]. All women with positive peripheral malaria blood smears in the larger study were enrolled, along with the next two sequential uninfected women. Women were excluded from this study if they had HIV, preeclampsia, or multiple gestations. Maternal socio-economic (marital status, educational level, maternal and paternal occupations, and house construction), health (antenatal clinic attendance and iron tablets usage) and anthropometric (height, weight, and mid-upper arm circumference (MUAC)) data were collected. Neonatal anthropometrics (head, abdominal, and arm circumferences, recumbent length, and weight) were evaluated within the first 24 hours after birth. Gestational age was determined by the New Ballard Score [28]. Maternal peripheral blood samples were taken at recruitment, and cord and placental samples were collected at delivery. Thick blood films were prepared from these samples for malaria microscopy. Placental blood was collected into EDTA by incising the cleaned maternal surface of the placenta and aspirating blood welling from the incision with a sterile pipette. Samples were separated within one hour, and plasma was stored at -70°C. Placental biopsies from a pericentric area (approximately 1 cm from the cord on the maternal side) were placed into 10% neutral buffered formalin to be used for placental malaria histopathology. Thick blood films were air-dried and Field's stained. Plasmodium falciparum malaria parasitaemia per μl was determined by counting parasites per 200 leukocytes, assuming 6000 leukocytes per μl [29]. Placental malaria histopathology was determined by SJR as described [13]; stage of infection and degree of malaria pigment in placental monocytes and fibrin were evaluated to determine disease severity [13]. For the purposes of data analysis, malaria infection was defined as either maternal (any parasites on maternal peripheral thick blood film) or placental (any parasites on placental thick blood film or histopathology). All subjects were consented and received HIV pre-test and post-test counseling before the onset of active labor. HIV status was determined by Serocard rapid test for HIV-1 and 2 (Trinity Biotech, Wicklow, Ireland) and Determine HIV1/2 (Abbott Diagnostics, Abbott Park, Illinois, USA); disagreement between tests was settled by HIV-SPOT ELISA (Genelabs Diagnostics, Singapore). Haemoglobin (Hb) concentration were measured in peripheral and cord blood samples using a Hemocue® haemoglobinometer (HemoCue AB, Ängelholm, Sweden); some results were not available as samples clotted. Maternal peripheral and cord plasma soluble transferrin receptor (sTfR) and ferritin were assayed by enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer's instructions (Ramco Laboratories, Stafford, TX; and IBL, Hamburg, Germany; respectively). Data were analysed in Microsoft Excel using a log-log standard curve. Limits of detection were as follows: 1 μg/ml (sTfR) and 0.59 ng/ml (ferritin). Maternal peripheral, placental and cord plasma tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) and maternal peripheral and cord plasma C reactive protein (CRP) levels were also assayed by ELISA (R&D Systems, Minneapolis, MN, USA). Color was developed by SigmaFast o-phenylenediamine dihydrochloride tablets (Sigma, St. Louis, MO, USA). Data were analysed as described above. Limits of detection were as follows: 4 pg/ml (TNF-α), 13 pg/ml (TGF-β), and 1 μg/ml (CRP). Because scalp blood gas analysis, the most accurate means of assessing foetal hypoxia, was not feasible in the study setting, other proxies for chronic foetal hypoxia were measured: erythropoietin (Epo) [19], and cortisol and corticotrophin-releasing hormone (CRH) [20]. Maternal peripheral, placental, and cord plasma erythropoietin (Epo) levels were assayed by ELISA according to manufacturer's instructions (IBL, Hamburg, Germany). Placental and cord cortisol (CRT) and placental CRH were determined by ELISA as well (cortisol: R&D Systems, Minneapolis, MN, USA; CRH: Phoenix Pharmaceuticals, Belmont, CA, USA, respectively). Data were analysed as described above. Limits of detection were as follows: 0.4 mU/mL (EPO), 0.16 ng/ml (cortisol), and 0.19 ng/ml (CRH). This study was approved by the College of Medicine Research Committee, University of Malawi, and the Institutional Review Boards of the University of Michigan and the University of North Carolina. Data were entered in MS-Access and analyses were performed using SPSS v.10 and Intercooled Stata 8.0. Skewed data were log or (log+1) transformed before certain analyses. Maternal characteristics in malaria-infected and uninfected women (Table (Table2)2) were compared by t-tests (continuous variables) and chi-squared tests (dichotomous variables). Mann-Whitney U tests were used to compare maternal and cord characteristics in malaria-infected and uninfected women (Tables (Tables33 and and4).4). Bonferroni's method was used as a more conservative measure of significance for multiple comparisons [30]; p values reported reflect pre-adjustment values. The 5% significance level was used to determine significance. Maternal characteristics in women with and without maternal peripheral malaria infection Mann-Whitney U test of effect of maternal peripheral malaria on maternal and neonatal haematological and inflammatory status aIQR: Inter-quartile range b Significant after Bonferroni's adjustment for multiple comparisons. Mann-Whitney U test of effect of placental malaria on markers of neonatal haematological, inflammatory, and hypoxic status aIQR: Inter-quartile range
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