Does malaria affect placental development? Evidence from in vitro models

Abstract

Background: Malaria in early pregnancy is difficult to study but has recently been associated with fetal growth restriction (FGR). The pathogenic mechanisms underlying malarial FGR are poorly characterized, but may include impaired placental development. We used in vitro methods that model migration and invasion of placental trophoblast into the uterine wall to investigate whether soluble factors released into maternal blood in malaria infection might impair placental development. Because trophoblast invasion is enhanced by a number of hormones and chemokines, and is inhibited by pro-inflammatory cytokines, many of which are dysregulated in malaria in pregnancy, we further compared concentrations of these factors in blood between malaria-infected and uninfected pregnancies.

Methodology/principal findings: We measured trophoblast invasion, migration and viability in response to treatment with serum or plasma from two independent cohorts of Papua New Guinean women infected with Plasmodium falciparum or Plasmodium vivax in early pregnancy. Compared to uninfected women, serum and plasma from women with P. falciparum reduced trophoblast invasion (P = .06) and migration (P = .004). P. vivax infection did not alter trophoblast migration (P = .64). The P. falciparum-specific negative effect on placental development was independent of trophoblast viability, but associated with high-density infections. Serum from P. falciparum infected women tended to have lower levels of trophoblast invasion promoting hormones and factors and higher levels of invasion-inhibitory inflammatory factors.

Conclusion/significance: We demonstrate that in vitro models of placental development can be adapted to indirectly study the impact of malaria in early pregnancy. These infections could result in impaired trophoblast invasion with reduced transformation of maternal spiral arteries due to maternal hormonal and inflammatory disturbances, which may contribute to FGR by limiting the delivery of maternal blood to the placenta. Future prevention strategies for malaria in pregnancy should include protection in the first half of pregnancy.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. The effect of pooled serum from uninfected and P. falciparum infected women at enrolment on HTR8/SVneo invasion index over 48 h.

Invasion index indicates the proportion of cells invaded through the Matrigel™, normalized to baseline invasion of trophoblast cells treated with complete media (untreated group). Serum from infected women inhibited median (IQR) invasion by 65% (Mann Whitney-U test P = .1) compared to serum from uninfected women, and by 37% compared with complete media treatment (P = .06). The malaria-specific reduction in invasion was comparable to that of 10 ng/ml TGFβ (positive control for invasion inhibition), which inhibited invasion by 31% (Mann Whitney U-test, P = .06). Normal uninfected pregnancy serum enhanced trophoblast invasion by 80% (P = .06) compared with complete media alone. Both viability and invasion assays were repeated three times in triplicate. Data shown are expressed as median and (IQR) of the mean of each of three independent experiments, repeated in triplicate.

Figure 2. Migration of Swan cells was determined over time using Wimasis software.

Swan 71 trophoblast cells were plated, and scratched 12 h later. At the time of treatment (0 h) with either 10% FBS, serum free media (SFM, not shown), or LPS 500 ng/ml in SFM, or with 10% plasma in SFM (not shown); three independent images were taken at 10x magnification. The same frames were imaged subsequently at 6 and 24 h following treatment. Images were up-loaded to www.ibidi.com, and the percent cell coverage (green) compared to the scratched area (black) was determined in each frame using WIMASIS analysis software. The percent increase in migration over time (Figure 3 A and B) was determined by subtracting cell coverage at 0 h from subsequent time points.

Figure 3. Plasma collected from Plasmodium falciparum infected women in early pregnancy inhibits Swan cell migration at 6 h (A) and 24 h (B).

Median (IQR) percent increase in migration (relative to 0 h) of Swan 71 cells was measured over 24 h in response to control treatments (10% fetal bovine serum [FBS] in serum free medium, untreated [SFM], and 500 ng/ml LPS in SFM, open bars) or with 10% plasma from individuals (black bars) that were malaria-uninfected (n = 13), P. vivax-infected (n = 9) or P. falciparum-infected (n = 13), over 4 independent experiments. (A) At 6 h, plasma collected from women with P. falciparum infection inhibited migration compared with plasma from uninfected women (*P = .01). Treatment with LPS significantly inhibited migration (*P = .03) compared with 10% FBS treatment. There was no difference between uninfected and P. vivax plasma treatments (P = .35). (B) At 24 h, compared with cells treated with uninfected plasma, plasma collected from women with P. falciparum infection significantly reduced migration (**P = .004), while migration with plasma from P. vivax infection was unchanged (P = .64). LPS significantly inhibited migration compared to cells treated with 10% FBS (*P = .03).

Figure 4. Plasmodium falciparum infection status does not affect trophoblast viability.

(A) Mean and SD fold change in HTR8/SVneo viability relative to that of cells treated with complete media (open bars) was measured by percent reduction in AB dye after 4 h, following incubation with serum treatments (black bars) for 48 h. Compared with complete medium, complete medium supplemented with 10% volume pooled serum from women with or without infection had no effect on trophoblast viability (ANOVA P = .4). Treatment with SRM (open bar, positive control for a negative effect on trophoblast viability) reduced cell viability compared to trophoblasts treated with complete media (Mann Whitney test P = .06). Figure 4A indicates the mean and SD of each of three independent experiments, repeated in triplicate. (B) Median (IQR) fold change in viability of Swan 71 following treatment with plasma from individual women in early pregnancy. Swan 71 cells were treated once in triplicate for 24 h with either complete media (open bar), or serum free media supplemented with 10% plasma from women with (n = 13) and without (n = 11) P. falciparum infection in early pregnancy (black bars). Following treatment, Swan 71 cells were incubated with AB and cell metabolism (as a proxy for viability) was normalized to that of cells treated with complete media. There was no difference in viability between plasma treatments with infection (P = .4), but cells cultured in complete media had relatively higher measure of viability than those treated with 10% plasma (*P = .01 in both cases).

Figure 5. Parasite density negatively influences Swan 71 cell migration.

Parasite densities from all P. falciparum and P.vivax infected participants (n = 22) were log transformed, and correlated with respective Swan 71 migration data at 24 h post-treatment. There was a significant negative association between density of infection in early pregnancy with migration (P = .02). No other clinical parameters were found to correlate with Swan 71 cell migration in response to plasma treatment.