Adverse pregnancy outcomes are associated with Plasmodium vivax malaria in a prospective cohort of women from the Brazilian Amazon

Background Malaria in Brazil represents one of the highest percentages of Latin America cases, where approximately 84% of infections are attributed to Plasmodium (P.) vivax. Despite the high incidence, many aspects of gestational malaria resulting from P. vivax infections remain poorly studied. As such, we aimed to evaluate the consequences of P. vivax infections during gestation on the health of mothers and their neonates in an endemic area of the Amazon. Methods and findings We have conducted an observational cohort study in Brazilian Amazon between January 2013 and April 2015. 600 pregnant women were enrolled and followed until delivery. After applying exclusion criteria, 329 mother-child pairs were included in the analysis. Clinical data regarding maternal infection, newborn’s anthropometric measures, placental histopathological characteristics, and angiogenic and inflammatory factors were evaluated. The presence of plasma IgG against the P. vivax (Pv) MSP119 protein was used as marker of exposure and possible associations with pregnancy outcomes were analyzed. Multivariate logistic regression analysis revealed that P. vivax infections during the first trimester of pregnancy are associated with adverse gestational outcomes such as premature birth (adjusted odds ratio [aOR] 8.12, 95% confidence interval [95%CI] 2.69–24.54, p < 0.0001) and reduced head circumference (aOR 3.58, 95%CI 1.29–9.97, p = 0.01). Histopathology analysis showed marked differences between placentas from P. vivax-infected and non-infected pregnant women, especially regarding placental monocytes infiltrate. Placental levels of vasomodulatory factors such as angiopoietin-2 (ANG-2) and complement proteins such as C5a were also altered at delivery. Plasma levels of anti-PvMSP119 IgG in infected pregnant women were shown to be a reliable exposure marker; yet, with no association with improved pregnancy outcomes. Conclusions This study indicates that P. vivax malaria during the first trimester of pregnancy represents a higher likelihood of subsequent poor pregnancy outcomes associated with marked placental histologic modification and angiogenic/inflammatory imbalance. Additionally, our findings support the idea that antibodies against PvMSP119 are not protective against poor pregnancy outcomes induced by P. vivax infections.


Methods and findings
We have conducted an observational cohort study in Brazilian Amazon between January 2013 and April 2015. 600 pregnant women were enrolled and followed until delivery. After applying exclusion criteria, 329 mother-child pairs were included in the analysis. Clinical data regarding maternal infection, newborn's anthropometric measures, placental histopathological characteristics, and angiogenic and inflammatory factors were evaluated. The presence of plasma IgG against the P. vivax (Pv) MSP1 19 protein was used as marker of exposure and possible associations with pregnancy outcomes were analyzed. Multivariate logistic regression analysis revealed that P. vivax infections during the first trimester of pregnancy are associated with adverse gestational outcomes such as premature birth (adjusted odds ratio [aOR] 8.12, 95% confidence interval [95%CI] 2.69-24.54, p < 0.0001) and reduced head circumference (aOR 3.58, 95%CI 1. .97, p = 0.01). Histopathology analysis showed marked differences between placentas from P. vivax-infected and non-infected a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Introduction Pregnant women are highly susceptible to malaria and its deleterious effects. Recently, the World Health Organization Malaria Report pointed the often-neglected malaria cases in pregnancy (MiP) that occur mainly in Africa, where around 11 million pregnant women were estimated to have developed the disease during 2018 [1]. Africa is characterized by a high and stable transmission of Plasmodium (P.) falciparum. Despite being frequently under the spotlight of malaria control campaigns, infections during pregnancy are still a burden associated with maternal anemia, abortion, premature delivery, and low birth weight (LBW) [2][3][4][5][6][7]. Few studies have focused on the burden of MiP in low transmission regions like Latin America, where P. vivax is the most prevalent species [1]. Its infections are often considered benign, a paradigm that is frequently supported by inconsistent reports that tend to focus on P. falciparum malaria episodes during pregnancy; this makes the evaluation of the real impact of P. vivax monoinfections somehow challenging [8][9][10][11].
In P. falciparum MiP, poor pregnancy outcomes are mainly linked to severe placental inflammation triggered by the sequestration of infected erythrocytes (which characterize placental malaria, PM) [12]. This process results in dramatic histological changes of the placental structure [11,13], which ultimately dysregulate local homeostasis and contribute to poor pregnancy outcomes [2]. This phenomenon is not yet clearly elucidated in P. vivax MiP mainly due to scarce and controversial evidence concerning: (1) placental adhesion [14,15]; (2) severe placental histopathology [9][10][11]; and (3) subsequent poor gestational outcomes [16][17][18]. The harmful effects of P. vivax infections are most likely a consequence of systemic inflammation and production of cytokines [19], such as TNF-α and IFN-γ, which are linked to disease severity, or IL-10 that is associated with better disease prognosis [20]. Accordingly, P. vivax-induced inflammation during pregnancy may be implicated in placental structural abnormalities and leukocytes infiltration, as well as in angiogenic imbalance, which altogether can directly or indirectly impact fetal development [11,13,21]. Nevertheless, to our knowledge, studies addressing these parameters together are still missing.
Attenuation of clinical symptoms frequently occurs after naturally acquiring immunity due to frequent exposures to Plasmodium spp. In P. falciparum MiP, loss of protection is mainly attributed to lack of immunity against the P. falciparum erythrocyte membrane protein 1 (PfEMP-1) variant VAR2CSA that binds to chondroitin sulfate A (CSA) in the placenta. In subsequent pregnancies, the development of PfEMP1 var2csa specific antibodies is associated with protection from MiP [22][23][24][25][26]; yet, in P. vivax MiP, the establishment of a clear association between pre-existing acquired immunity and improved pregnancy outcomes is yet to be achieved.
Herein, we report the deleterious relationship between P. vivax monoinfections, poor pregnancy outcomes, placental histopathology, cytokine and angiogenic imbalance, and humoral immune response in pregnant women from the Brazilian Amazon. Our results suggest that P. vivax infections during the first gestational trimester are associated with poor pregnancy outcomes. The presence of IgG against P. vivax merozoite surface protein 1 (P. vivax MSP1 19 ) is not associated with protection against the deleterious effects of P. vivax MiP.

Ethics statement
This study was approved by the Ethics Committee from the University of São Paulo (Plataforma Brasil, CAAE: 03930812.8.0000.5467 and 90474318.4.0000.5467), according to resolution n˚466/12 of the Brazilian National Health Committee and in compliance with the Declaration of Helsinki. All subjects or their legal guardians (if minors) gave written informed consent. This study followed a specified analysis plan (S1 Text) and the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) statement guidelines (S1 STROBE Checklist).

Setting and study design
We have conducted an observational cohort study in five municipalities (Cruzeiro do Sul, Mâncio Lima, Rodrigues Alves, Marechal Thaumaturgo, and Porto Walter) in the Juruá Valley, located in the Brazilian Amazon southwest region (Acre State, Brazil). This region is considered highly endemic for malaria, with an annual parasite incidence (API) above 100 cases per 1,000 inhabitants. Juruá Valley is also characterized by a significant prevalence of P. vivax infections, responsible for 70-80% of total malaria cases [27,28]. A complete region characterization with detailed procedures and methods used for data and sample collection has been described elsewhere [11,13,21]. Briefly, 600 pregnant women were recruited during their first antenatal care (ANC) visit. These women were followed until delivery, between January 2013 and April 2015. Data was collected on socioeconomic, clinical, and obstetric variables at the time of recruitment, together with peripheral blood samples. In parallel, thick and thin peripheral blood smears were performed to screen for malaria parasites by microscopy, which were later confirmed by PCR. Each pregnant woman was followed by a trained nurse, which involved at least two domiciliary visits, at the second and third trimester, to monitor their clinical state and collect a peripheral blood sample, in addition to the usual prenatal care visit to the health care services. An additional blood sample was collected in each malaria episode during pregnancy. At the time of delivery, clinical data were collected from mother and newborn and performed a placental biopsy and blood sampling. Only women with P. vivax monoinfections and their non-infected counterparts were considered for this study (Fig 1A). We evaluated mother-child pairs at delivery in the Women and Children's Hospital of Juruá Valley located in Cruzeiro do Sul.

Malaria screening and treatment
Malaria during pregnancy was first diagnosed from thin and thick blood smears by microscopy analysis, which was performed by the endemic surveillance team of Juruá Valley. Upon diagnosis, all women infected with P. vivax during pregnancy were treated with chloroquine (25mg/kg of body weight) in divided doses over a 3-day period. Successful treatment was confirmed subsequently. No hypnozontocidal treatment with primaquine was performed, following the Brazilian Ministry of Health (MoH) guidelines regarding medical prescription in pregnant women [29]. Of note, the Intermittent Preventive Treatment in pregnancy (IPTp) is not employed in Brazil. All samples collected throughout pregnancy (recruitment, malaria episodes, and domiciliary visits) and at delivery were further screened by molecular diagnosis using the real-time PCR technique (PET-PCR) as described elsewhere [13,30].

Exclusion criteria
Pregnant women were excluded from further analysis if they self-informed any alcohol consumption, smoking, and/or if presenting any other diagnosed infectious diseases (e.g. Toxoplasmosis, Rubella, Cytomegalovirus and Herpes simplex (TORCH), HIV, Hepatitis B and C virus, Syphilis), and/or other comorbidities (e.g. hypertension, pre-eclampsia/eclampsia, diabetes mellitus, and newborn with congenital malformation). Likewise, pregnant women infected with other Plasmodium species (P. falciparum or mixed infections) were excluded after confirmation by PET-PCR (Fig 1A).

Laboratory procedures
Peripheral and placental blood (from the maternal side) was collected aseptically in heparinized tubes and separated into plasma and whole blood cells after centrifugation. All samples

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were stored at -80˚C. Total DNA was obtained from whole blood cells using a commercially available extraction kit (QIAmp DNA Mini Kit, Qiagen), following the manufacturer's instructions. Fragments of placental tissue were collected and stored in 10% neutral buffered formalin at 4˚C, until processing. Placental tissue was sectioned and embedded in paraffin using standard procedures; afterward, it was stained with Hematoxylin-Eosin (H&E) or Giemsa for histological examination. All histological evaluation methods were optimized by our group and are described elsewhere [11,13]. Placental images were captured using a Zeiss Axio Imager M2 light microscope equipped with a Zeiss Axio Cam HRc camera. Placental parameters were evaluated and analyzed using Image J software (http://imagej.nih.gov/ij). All measurements were performed by two investigators blinded to group and outcome. Cases that proved to be contradictory between observers were re-evaluated until consensus was reached.

Placental weight and newborn's anthropometric measurements
Placental weight was measured (grams (g)) immediately after expulsion/extraction using a digital semi-analytical scale (Shimadzu), with a capacity of 3.2 kilograms (kg) and a precision of 0.01 g. Umbilical cord, membranes, and fetal blood contained within the large chorionic vessels and intervillous space, were included in weight measurement. Newborns' anthropometric measures were obtained after birth (maximum within a 24-hour period) by trained obstetrician nurses. Weight was measured (g) using digital pediatric scales (Toledo), with a maximum capacity of 15 kg and a precision of 5 g. Length, head and chest circumferences were measured using a non-stretching flexible measuring tape and expressed as centimeters (cm). Rohrer's ponderal index was calculated as the newborn's weight (g) divided by the cube of the length (cm).

Gestational age estimation and other definitions
Gestational age (GA) was assessed during the first trimester of pregnancy using crown-rump length measured by trans-abdominal ultrasound. In the absence of this examination within this period, the GA was estimated by the woman's last menstrual period (LMP) together with the earliest ultrasound performed. The LMP method is recommended by the Brazilian MoH for GA calculation. Low birth weight (LBW) was defined as birth weight below 2500 g. Preterm birth was defined as birth before the 37th week of gestation, abortion as birth occurring before the 22nd week of gestation, and stillbirth as fetal death occurring between the 22nd week of gestation and time of delivery (Fig 1B). Term LBW was defined as term birth (after 37th week of gestation) with weight below 2500 g. Small for gestational age (SGA), reduced head circumference, and reduced body length were defined as: birth weight, head circumference and length below the 10th percentile sex-specific for GA according to the INTERGROWTH-21st standards [31]. Maternal anemia was defined as hemoglobin levels lower than 11 g/dL. Placental malaria was diagnosed according to a previously established classification [12].

Measurement of total IgG antibodies against Plasmodium vivax merozoite surface protein 1 (PvMSP1 19 )
For the study of total IgG antibodies against PvMSP1 19 , samples collected at the first timepoint (recruitment) were used; yet, we excluded infected pregnant women who were recruited after having already one or more malaria episodes during that pregnancy. Enzyme-linked immunosorbent assay (ELISA) was performed using high-binding 96-well microplates (Costar, Cambridge, MA) coated with Glutathione S-transferase (GST)-PvMSP1 19 (31.25 ng/ well), produced as described elsewhere [32,33]. In parallel, GST was used as a control for nonspecific binding. The optimal concentrations were determined by plasma serial titration of positive and negative controls. Plates were incubated at 4˚C overnight and washed four times with phosphate buffered saline with 0.05% Tween 20 (PBS-T). PBS-T containing skimmed milk was used for blocking (4%) and diluting plasma and antibodies (1%). Each plasma sample (50 μL/well) was diluted 1:200. After a 1-hour incubation at room temperature, antibody-antigen complexes were detected with HRP-conjugated mouse anti-human IgG (IG266) (Novus, Cat# NBP2-34648H) at a 1:3,000 dilution. Enzyme-substrate reaction was revealed with tetramethylbenzidine (TMB)/H 2 O 2 at room temperature (dark conditions). After 10 minutes, the reaction was interrupted with 2N H 2 SO 4 . Absorbance values were measured at 450/595 nm using a CLARIOstar Plus plate reader (BMG Labtech). Corrected absorbance values were obtained by subtracting absorbance readings with GST ran on the same microplate. Absorbance data used in statistical analyses correspond to corrected values. Reactivity indices (RIs) were calculated as the ratio between each test sample's corrected absorbance values and a cutoff value for antigen, corresponding to the average corrected absorbance for samples from 10 malaria-naïve blood donors plus 3 standard deviations. Positive samples were considered as having RIs greater than 1.

Statistical analysis
Data were analyzed using Stata/SE 14.2 (Stata, College Station, TX, USA) and GraphPad Prism 6.0 software (GraphPad Software Inc., San Diego, CA, USA). Continuous variables were summarized using mean and standard deviation (SD) values as well as median values and interquartile ranges (IQRs). Categorical variables were summarized using frequencies and percentages. Differences between groups were evaluated using the nonparametric Kruskal-Wallis test followed by the Dunn's post hoc multiple comparison test and the Mann-Whitney test as appropriate. Differences between categorical data and proportions were analyzed using Pearson 0 s chi-squared test or Fisher's exact test depending on the type of variables. All p values were 2-sided and interpreted at a significance level of 0.05. To compare the association between malaria and other variables (clinical characteristics, newborns' anthropometric measurements, placental parameters, angiogenic and inflammatory factors, and anti-PvMSP1 19 IgG), we used linear regression, adjusting for maternal age, number of gestations, residence, education level, and occupation. To assess associations between malaria and adverse pregnancy outcomes, the presence of anti-PvMSP1 19 IgG and outcomes in the context of MiP, adjusted odds ratios (aOR) with 95% confidence intervals (CI) were estimated using multiple logistic regression, which included infection by malaria (no / yes), maternal age (� 18 years old / � 17 years old), number of gestations (two or more / one), residence (urban / rural), education level (� secondary / � primary), occupation (others / housewife) and presence of anti-PvMSP1 19 IgG (no / yes) as explanatory variables and adverse pregnancy outcomes (yes / no) as response variables. The first category for each explanatory variable was considered as the reference.

Study population and baseline characteristics
From 600 pregnant women enrolled in the major study, 329 (54.8%) were eligible for further analysis after applying the exclusion criteria and malaria parasite species diagnosis by PET-PCR (Fig 1A). Of these, 170 (51.7%) were classified as non-infected and 159 (48.3%) as P. vivax (Pv)-infected during gestation (Fig 1A). The overall socio-demographic profile of the stratified participants is depicted in Table 1. Group stratification was performed by the trimester in which the first infection occurred, considering that time of infection is known to be associated with MiP poor pregnancy outcomes [34]. Fig 1B illustrates a standard gestational window with the hypothetical poor outcomes in unsuccessful pregnancies. Table 1 shows some heterogeneity between the groups since pregnant women in the infected groups tend to be younger, with reduced educational level and rural residence. Occupation also varies significantly between the groups, but no difference was found regarding the number of previous pregnancies. The aforementioned characteristics were used as explanatory variables in the following multiple logistic and linear regressions analysis due to reported heterogeneity and their possible impact as confounding effects.

P. vivax infections during the first trimester are associated with MiP poor pregnancy outcomes
Malaria in pregnancy is known to impact proper fetal development [2]. We assessed pregnancy outcomes and newborns' anthropometric measurements to ascertain if P. vivax infections at a given gestational trimester are associated with impaired fetal development and poor outcomes. Multiple logistic regression analysis revealed that P. vivax infections during the first trimester of pregnancy increase the odds of premature birth (aOR 8.12, 95%CI 2.69-24.54, p < 0.0001), LBW (aOR 4.34, 95%CI 1.49-12.62, p = 0.007), as well as LBW in those born at term (aOR 4.75, 95%CI 1.06-21.35, p = 0.04). First trimester infections were also associated with reduced head circumference (aOR 3.58, 95%CI 1.29-9.97, p = 0.01) and newborn's length (aOR 4.48, 95%CI 1.66-12.06, p = 0.003) (Fig 2). No differences were observed for the overall birth weight and length (Fig 3A and 3D and S1 Table). Though, in line with our findings, we observed statistically significant differences in head and chest circumference of children born from women infected with P. vivax during the first trimester and those born from non-infected women  Fig 3B and 3C and S1 Table). Our results suggest that P. vivax infections during the first gestational trimester promote preterm birth and impair fetal development, possibly due to clinical manifestations associated with MiP.

Clinical and infection characteristics of pregnant women with P. vivax MiP
As for the aforementioned outcomes, pregnant women infected with P. vivax had some clinical characteristics that are worth mentioning (Table 2). Women infected during the first trimester had reduced gestational age at delivery when compared with non-infected counterparts   [34.5-38.6], p = 0.008) and increased clinical anemia in those infected during the third trimester (Pv-3 rd tri 28.3% vs. NI 14.1%, p = 0.02). Additionally, women in the Pv-infected group reported more malaria episodes before current pregnancy than their non-infected counterparts regardless the infection trimester (Table 2). Pregnant women infected during the first trimester had higher chances of having three or more infections than infected women from other groups. Parasitemia tend to be similar for the first infection across gestational trimesters; yet, it seems to decrease on average from the second infection onward (Table 3). 8.1% of women infected in the first trimester had past PM, while 4.6 and 4.3% of women infected during the second and third trimesters had active acute

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PM, respectively. This was established by the presence of hemozoin in milder or moderate amounts. However, it was absent in more than 90% of the examined placentas, regardless of the infection's gestational trimester. Our observations support the idea that systemic effects rather than PM are linked to the outcomes observed during P. vivax MiP in our cohort.

P. vivax infections across gestational trimesters lead to diverse placental histologic alterations and angiogenic and cytokine imbalance
Reduced PM prevalence is frequently observed in P. vivax infections because, contrary to P. falciparum infections, P. vivax sequestration in the placenta is controversial [14,15] and studies Forest plot of odds ratios of adverse events in newborns from women infected during pregnancy compared with newborns from non-infected women is shown. n/Nnumber of events by total number of individuals in each group; aOdds Ratio-adjusted Odds Ratio; CI-confidence interval; tri-gestational trimester. Odds ratios were adjusted (aOR) for maternal age, gravidity status, residence, education level and occupation. Preterm birth-birth < 37 th week of gestation; Low birth weight-birth weight < 2500 g; Term low birth weight-birth weight < 2500 g from 37 th week of gestation; Small for gestational age, Reduced head circumference and Reduced body length-birth weight, head circumference and length (respectively) < 10 th centile for sex-specific gestational age. P-values were estimated through multiple logistic regression methods.
https://doi.org/10.1371/journal.pntd.0009390.g002 PLOS NEGLECTED TROPICAL DISEASES that report this event frequently have reduced sample sizes. Nevertheless, placental histologic changes had been observed [9][10][11]. To determine whether Pv-infections across the gestational trimesters result in considerable histologic and cytological alterations, we have evaluated several histologic parameters in more than 100 placentas from Pv-infected women (Fig 4 and S2  Table). We observed that placentas from Pv-infected women had a significant increase in syncytial nuclear aggregates (SNA), fibrin deposition, monocytes and leukocytes infiltrate  (Fig 4 and S2  Table). Differences between placentas from non-infected women and those from women infected in the first trimester were observed regarding SNA and monocyte infiltrate (median  (Fig 4A and 4C and S2 Table). Interestingly, Weight is given in grams (g). Head and chest circumferences, and body length are informed in centimeters (cm). Data are represented as Tukey boxplots, with the bottom and top of each box representing the first and third quartiles; the line inside the box is the median, and the whiskers represent the lowest and the highest data within 1.5 IQR of the first and upper quartiles; and the circles, outliers. Differences between groups were determined by multiple liner regression (adjusted for maternal age, gravidity status, residence, education level, and occupation).

Clinical outcomes
Gestational age at delivery, weeks, median (IQR) e

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infections closer to term tend to yield higher levels of fibrin deposition and monocytes infiltrate at delivery (Fig 4B and 4C and S2 Table). These findings are representative of placental anomalies that can be extended to angiogenic and inflammatory mediators in placental plasma (S3 and S4 Tables). First and third trimester infections led to reduced levels of angiopoietin-2 (ANG-2) and increased levels of soluble TEK receptor tyrosine kinase (sTIE-2), respectively (median [IQR], ANG-2 -Pv-1 st tri 4.4 ng/mL [1.0-6.6] vs. NI 6.8 ng/mL [3.5-12.4], p = 0.03; sTIE-2 -Pv-3 rd tri 18.5 ng/mL [11.7-24.7] vs. NI 13.9 ng/mL [9.1-19.0], p = 0.008). Overall, factors such as sFlt-1, sVEGFR-2, and the hormone leptin were also altered in placentas from Pv-infected women (S3 Table). Regarding inflammatory mediators, only IL-12 and C5a showed more consistent and significant alterations, as they were reduced at delivery due to first and third trimester infections (median  Table). Altogether, our observations suggest that poor pregnancy outcomes associated with first trimester infections may be linked to placental inflammation, structural abnormalities and angiogenic imbalance during P. vivax MiP.

Antibodies against P. vivax MSP1 19 are not associated with protection from poor pregnancy outcomes
The impact of gravidity on P. vivax MiP outcomes is still inconclusive [16,35,36]. Possibly the absence of pregnancy-specific antigens in P. vivax and its restricted distribution to areas of low

P. vivax (N = 159) P. vivax -1 st tri (N = 52) P. vivax -2 nd tri (N = 54) P. vivax -3 rd tri (N = 53)
Gestational age at 1st infection, weeks, median (IQR) 21 ( malaria transmission may impair immunity acquisition across pregnancies [8,37]. As such, other antigens might be targeted by the immune system to confer some degree of protection from P. vivax MiP. Therefore, we measured antibodies against the C-terminal fragment of the P. vivax merozoite surface protein 1 (PvMSP1 19 ). We used it as a surrogate marker of previous exposures (Fig 5A and S5 Table) and sought possible associations between antibody titers and improved pregnancy outcomes. Infected pregnant women who were recruited with more than one malaria episode during their current pregnancy were removed from the analysis, to target antibodies acquired upon any malaria episode occurred before pregnancy. We observed that Pv-infected women previously exposed to the parasite (Pv-PvMSP1 Ab+ ) had higher titers of IgG when compared with non-infected pregnant women with prior exposure (NI-PvMSP1 Ab  (Fig 5A and S5 Table). However, no associations were observed between IgG  NI-PvMSP1 Ab--noninfected non-exposed (N = 106-112); NI-PvMSP1 Ab+ -non-infected exposed (N = 54-58); Pv-PvMSP1 Ab--P. vivax-infected non-exposed (N = 19); Pv-PvMSP1 Ab+ -P. vivax-infected exposed (N = 80-95). PvMSP1-Plasmodium vivax merozoite surface protein 1; Ab-Antibody; LBW-low birth weight; SGA -small for gestational age; HC-head circumference. RI-Reactivity indices were calculated as the ratio between each test sample's corrected absorbance values and a cut-off value for antigen, corresponding to the average corrected absorbance of samples from 10 malaria-naïve blood donors plus 3 standard deviations. Weight in grams (g). Head and chest circumferences, and body length in centimeters (cm). Data are represented as Tukey boxplots, with the bottom and top of each box representing the first and third quartiles; the line inside the box is the median, and the whiskers represent the lowest and the highest data within 1.5 IQR of the first and upper quartiles; and the circles, outliers. Odds ratios were adjusted (aOR) for maternal age, gravidity status, titers and outcomes, suggesting that the presence of these antibodies do not protect pregnant women from the adverse outcomes of P. vivax MiP (Fig 5B). Accordingly, no statistically significant differences were observed for birth weight, head and chest circumference, and body length, between P. vivax-infected women who had PvMSP1 19 -specific antibodies and those who did not have them (Fig 5C-5F and S5 Table).
Overall, our results suggest that P. vivax infections during the first gestational trimester are associated with poor pregnancy outcomes most likely due to placental histologic and physiologic abnormalities, which are unlikely to be prevented by the presence of IgG against PvMSP1 19, acquired during previous P. vivax-malaria episodes.

Discussion
MiP is considered a public health problem worldwide, as it is a significant risk factor for abortion, stillbirth, LBW, and maternal anemia [2][3][4][5][6]. Here, we describe an important association between P. vivax MiP and adverse gestational events, placental histopathological changes, and a reduction of the neonate's anthropometric profile. However, a protective association between IgG against PvMSP1 19 and improved pregnancy outcomes was not observed.
P. vivax is widely distributed worldwide, particularly in Asia, Pacific regions, and Latin America. Though, longitudinal studies on gestational P. vivax-malaria are still limited, especially regarding placental histopathological analysis [8][9][10][11]. Although P. vivax MiP is poorly associated with severe malaria, this species' infections are far from benign and may have severe consequences for maternal and fetal health [16,17,35,36,[38][39][40]. The gestational outcomes associated with MiP can vary not only with the species but also with variables such as transmission intensity and stability, number of previous pregnancies, and trimester of infection [7,37]. In our study, no association was found between gravidity and improved pregnancy outcomes (S6 Table), suggesting that gestational-dependent mechanisms that govern immunity against P. falciparum during pregnancy may not be relevant in infections caused by P. vivax. In low transmission settings, where P. vivax is more prevalent, gravidity-dependent immunity acquisition is uncertain and inconsistently reported [8,16,35,39]. This could be related to the inexistence of known pregnancy-related P. vivax proteins targeted by the immune system [14,15].
The burden of first trimester infections, was scarcely addressed by researchers until recent years [7] despite knowing that perturbation in early pregnancy imposes severe fetal development consequences [41,42]. In our study, P. vivax first trimester infections were found associated with LBW and reduced newborn's length. The proportion of pregnancies with LBW observed by us was consistent with other studies conducted in Latin America [38,43] and other P. vivax transmission areas [7]. These findings are relevant as LBW is strongly associated with neonatal morbidity and mortality, growth inhibition and cognitive impairment in children, and chronic diseases in adulthood [44]. Similar to what we reported recently on P. falciparum MiP, P. vivax infections also impact fetal cephalic perimeter development [13]. However, a previous report using the same study population reported that one antenatal P. vivax malaria episode in another trimester than the first trimester was enough to impair fetal development, which was aggravated by recurrent parasite exposure during pregnancy [36].
A distinctive characteristic of P. vivax is the occurrence of multiple malaria episodes due to reinfections or relapses, with the latter being potentiated by the absence of primaquine treatment in pregnant women due to its teratogenic nature [45]. Taken that and the higher prevalence of peripheral parasitemia observed during the second gestational trimester, also residence, education level and occupation using multiple logistic regression. Differences among the groups were determined by multiple linear regression (adjusted for maternal age, gravidity status, residence, education level and occupation).
The mechanisms by which P. vivax infections lead to MiP outcomes are still unclear; yet, systemic events such as maternal anemia and generalized inflammation have been discussed as relevant to P. vivax MiP pathology [16,35,40]. Our data evidence a significant hematocrit reduction in pregnant women with P. vivax; though, no severe maternal anemia was observed. Only a significant frequency of anemia at delivery was detected among women with third trimester infections. Although maternal anemia is a well-known risk factor for intrauterine growth retardation (IUGR) and LBW in P. vivax-infected women [2,[16][17][18]35,36,40], this is unlikely to be influencing pregnancy outcomes in our cohort.
Contrary to P. falciparum infections, where pregnancy complications and adverse outcomes are often associated with PM [4,[48][49][50], in P. vivax infections, PM is unlikely to be the reason why infected women experience complications during pregnancy. Previous studies indicate that PM caused by P. vivax is modest and not associated with placental sequestration of infected erythrocytes [9][10][11]40], despite evidence of binding to CSA and placental tissue sections [14,15]. Our observations corroborate previous studies, as only 8.6% of the analyzed placentas were classified as having PM. Nonetheless, most placentas from P. vivax-infected pregnant women present histopathological changes. Placental lesions and histopathological changes are often difficult to observe in P. vivax-infected pregnant women, probably due to the low sample size reported in the studies [9][10][11]40]. By performing histologic analysis of a considerable number of placentas, we have shown that P. vivax infections during the first trimester promote the increase of SNA and monocyte infiltrate at delivery. Monocyte infiltrate and inflammation have been frequently associated with reduced birth weight and preterm birth in pregnant women infected with P. falciparum [51][52][53][54]. Therefore, we hypothesize that this may also occur in P. vivax infections and that placental inflammation independent of PM may be linked to P. vivax MiP adverse outcomes, opposing previous observations suggesting that P. vivax monoinfections do not promote placental inflammation and that adverse outcomes have an alternative etiologic nature [55].
Dysregulation of angiogenic and inflammatory factors are often associated with placental insufficiency and impaired fetal development in P. falciparum MiP [2]. The imbalance of angiopoietins has been associated with PM and LBW in pregnant women infected with P. falciparum [56] and observed in P. vivax-infected patients with thrombocytopenia [57]. In fact, in P. vivax infections, we observed diminished placental levels of ANG-2, sFlt-1, and leptin and increased sTIE-2 and sVEGFR-2 levels at delivery. It is unclear whether placental vasomodulation is being promoted or inhibited in our cohort, as diminished sFlt-1 and increased sVEGFR-2 support angiogenesis and vasodilation [58], while decreased ANG-2 and increased sTIE-2 suggest an anti-angiogenic environment [59]. On the other hand, in our study, the placental levels of classic inflammatory cytokines at delivery were not altered, reflecting a picture from past events. It is plausible that cytokines at delivery are already downregulated, which is in line with previous observations that showed inflammatory cytokines associated with episodes of P. vivax malaria during pregnancy that are lost at delivery [60]. Yet, a decrease in the plasma levels of IL-12 and C5a at delivery, particularly upon infections that occur during the first and third trimesters, was observed by us. IL-12 has been previously associated with P. vivax infections during pregnancy [60], and C5a is considered a hypothetical key driver of PM pathogenesis, being associated with placental insufficiency and SGA babies, and correlated negatively and positively with ANG-1 and ANG-2, respectively [61,62]. Moreover, our study shows that both placental C5a and ANG-2 are diminished at delivery when women are infected for the first time during the first trimester. These observations oppose a previous hypothesis assuming that P. vivax infections would promote the same augmented levels of C5a as those observed during P. falciparum MiP [61]. Altogether, our data suggest that inflammatory responses acting against P. vivax infections in the first trimester may contribute to chronic placental damage and insufficiency. This might occur through angiogenic and vascular remodeling mechanisms, which, when altered, promote abnormal placental/fetal development and poor pregnancy outcomes, despite early pharmacological intervention with antimalarials.
Currently, the main strategy to control/treat Plasmodium spp. infections relies on antimalarials and vaccines, channeling the research resources to develop these approaches. Regarding P. falciparum MiP, vaccines such as PAMVAC have been tested to induce immunity acquisition to VAR2CSA. However, there are no known orthologs in P. vivax that the immune system can directly target to protect women during pregnancy. Alternatively, the combat of disease symptoms in pregnant women can be done through the natural generation of antibodies against blood-stage parasite antigens. Antibodies against P. vivax MSP1 19 were shown to be associated with protection against symptomatic malaria in non-pregnant individuals [63,64] and those against P. vivax VIR, and Duffy-binding protein (PvDBP) were shown protective of birth weight reduction in pregnant women infected with P. vivax [65,66], suggesting that targeting non-pregnancy related antigens may also improve gestational outcomes. Nonetheless, in our study, we did not observe a statistically significant association between antibodies against P. vivax MSP1 19 and improved pregnancy outcomes, which is in line with previous observations showing that P. vivax MSP1 19 antibodies are associated with infection but do not correlate with protection from maternal anemia or reduced birth weight [66].
P. vivax MiP is still poorly understood, representing major challenges for pregnant women and their concepts. Here, we have shown that P. vivax MiP impairs newborn's development and leads to placental tissue damage. We conclude that P. vivax infections during the first trimester are associated with poor pregnancy outcomes and that the presence of antibodies against P. vivax MSP1 19 does not protect pregnant women from adverse gestational outcomes. Abnormal pregnancies in this context might occur due to placental inflammation and dysregulation of local homeostasis, regardless the absence of parasite sequestration and prompt treatment administration given to pregnant women in the endemic areas of the Brazilian Amazon.