Table 1.
Stains and scoring system used to quantify malaria-associated placental parameter.
Table 2.
Characteristics of the women who participated in the study according to infection status†.
Figure 1.
The syncytial parameters evaluated by Plasmodium species during infection.
Syncytial knotting (A and B) and syncytial rupture (C and D) were evaluated on H&E-stained slides at 100× magnification. Placental barrier thickness (E and F) was evaluated on Masson's trichrome-stained slides at 1000× magnification after overlaying horizontal lines with 5 µm of interspace (see Methods and Table 1). For all parameters, placentas from the “no plasmodium” group (n = 41; white boxes) had the lowest values, followed by placentas from the “P. vivax” (n = 59; red boxes) and “P. falciparum” (n = 19; grey boxes) groups. Graphs (B, D and F) represent the transformed data. * ANOVA test, P-value≤0,006. The boxes represent the mean and standard deviation values. The whiskers represent the 5th and 95th percentiles. Photographs were taken using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc. The grid overlays and counts were conducted using Image J. Arrow heads on A, C and D point to syncytial knots, syncytial rupture and an example of a thickness measurement, respectively.
Figure 2.
The placental parameters evaluated by Plasmodium species during infection.
For all placentas, areas of necrosis (B) and intervillous space (C) were measured by overlaying a square grid (A) and counting the number of intersecting points that touched necrotic areas (yellow dots; the white circle indicates an example) or intervillous space areas (blue dots; the black circle indicates an example). The ratios of intervillous space area per necrosis (D) and intervillous space area per placental barrier thickness (E) were calculated. The placentas in the “no plasmodium” group (n = 41; white boxes) appear to have similar necrotic areas and more intervillous space than the placentas in the “P. vivax” group (n = 59; red boxes). The placentas in the “P. falciparum” group (n = 19; grey boxes) exhibited more necrotic areas and less intervillous space. Graphs (B, C, D and E) represent the transformed data. The boxes represent the mean and standard deviation values. The whiskers represent the 5th and 95th percentiles. The photograph was taken using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc. Grid overlays and counts were performed using Image J.
Table 3.
Results and univariate analysis of the placental parameters, evaluated by histology, according to the species of Plasmodium infection during pregnancy†.
Figure 3.
The immune-cell parameters evaluated by Plasmodium species during infection.
The percentage of immune cells present in the intervillous space of the placentas evaluated (A) was calculated after counting a total of 500 intervillous space cells. Total leucocytes percentage (B), mononuclear cells percentage (C) and polymorphonuclear cells percentage (D) were plotted against Plasmodium exposure during pregnancy, assessed by microscopy. The placentas from the “no plasmodium” group (n = 41; white boxes) appear to have less immune cells present in the intervillous space than the placentas from the “P. vivax” group (n = 59; red boxes) and the placentas from the “P. falciparum” group (n = 19; grey boxes). * ANOVA test, P-value = 0,039. Graphs (B, C, and D) represent the transformed data. The boxes represent the mean and standard deviation values. The whiskers represent the 5th and 95th percentiles. The photograph was taken using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc. Grid overlays and counts were performed using Image J.
Table 4.
Cuzick's trend test analysis of placental changes across ordered groups by number of P. vivax infections during pregnancy†.
Table 5.
Multivariate analysis of the association between placental histological parameters and malaria during pregnancy.
Figure 4.
The placental score differentiates the women who were exposed to P. vivax during pregnancy.
A score (termed the ‘vivax-score’) was developed and applied to all of the placental samples in this study (see main text for details). (A) The placental samples from the “no Plasmodium” group (n = 41, white box) revealed a significantly lower score than the placentas from the “P. vivax” group (n = 59, red box) (* Mann-Whitney, P = 0.027). (B) The vivax-score increased significantly (** Cuzick's trend test: z = 2.76, P = 0.006) with increased exposure to P. vivax during pregnancy. “No infection”, n = 41; “1 infection”, n = 39; “2+ infections”, n = 20).