Unraveling the Interplay of Parasitaemia, Cytokines, and Pregnancy Outcomes in Plasmodium bergheiInfection: Insights from a Murine Model

Unraveling the Interplay of Parasitaemia, Cytokines, and Pregnancy Outcomes in Plasmodium bergheiInfection: Insights from a Murine Model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Unraveling the Interplay of Parasitaemia, Cytokines, and Pregnancy Outcomes in Plasmodium berghei Infection: Insights from a Murine Model Prem Lata Manhas, Rakesh Sehgal, Bishan Dass Radotra, Abhishek Mewara, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3327515/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Malaria infection during pregnancy presents notable risks to both maternal and fetal health. Present study examines the impact of Plasmodium berghei NK-65 strain infection during different stages of pregnancy in mice. The study categorized mice into non-pregnant and pregnant groups (early, mid, and late pregnancy) and induced infection on specific gestation days. Parasitaemia, cytokine profiles, histopathological changes, and pregnancy outcomes were evaluated. Major findings include severe anemia in mid-pregnancy infection, fetal deaths in early and mid-pregnancy infections, stillbirths in mid-pregnancy infections, and premature deliveries in late-pregnancy infections. Cytokine profiles differed between pregnant and non-pregnant mice, indicating varied immune responses. Plasmodium berghei infection in pregnant mice provides valuable insights into understanding placental malaria and its implications for maternal and fetal health. Plasmodium berghei Pregnancy Parasitaemia Cytokines Histopathology Pregnancy outcomes Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Malaria remains one of the most significant global health burdens, particularly affecting vulnerable populations such as pregnant women and their unborn children. Pregnant women are at an increased risk of severe malaria infection due to alterations in their immune response, hormonal changes, and the presence of placental tissue, which provides a unique environment for parasite sequestration and growth (Rogerson et al., 2007 ). Malaria during pregnancy can result in adverse outcomes, including maternal anemia, intrauterine growth restriction, preterm births, stillbirths, and an increased risk of maternal mortality (Rogerson et al., 2007 ). Plasmodium falciparum and Plasmodium vivax are the most common species responsible for causing malaria during pregnancy in endemic regions (Dellicour et al., 2010 ). In malaria-endemic areas, around 25 million expectant mothers face the threat of infection annually, leading to approximately 200,000 infant deaths annually (Schantz-Dunn & Nour, n.d.). Placental malaria, which involves the accumulation of infected red blood cells in the placenta, plays a central role in these adverse outcomes (Hviid et al., 2010 ). The pathogenesis of placental malaria involves the adhesion of infected red blood cells to chondroitin sulfate A on the syncytiotrophoblast surface, leading to immune activation and inflammation in the placenta (Lekana Douki et al., 2002 ; Rogerson et al., 2007 ) . To better understand the impact of malaria during pregnancy and to identify potential interventions, animal models have been extensively used (Doritchamou et al., 2017 ). Mice are often used as models for studying malaria due to their ease of manipulation, genetic tools, and availability of well-characterized parasite strains(Craig et al., 2012 ; Vaughan & Kappe, 2017 ). Among these models, Plasmodium berghei infection in mice has been utilized to study various aspects of malaria pathogenesis, immunity, and potential vaccine candidates(Stephens et al., 2012 ). Plasmodium berghei , while not a direct human pathogen, shares several similarities with human malaria parasites and has been shown to induce placental malaria-like symptoms in mice (Franke-Fayard et al., 2005 ; Rodrigues-Duarte et al., 2012 ). This research paper aims to investigate the impact of Plasmodium berghei NK-65 strain infection during different stages of pregnancy in mice, simulating the gestational timeline observed in humans. The study specifically focuses on early, mid, and late stages of pregnancy to explore how the timing of infection influences maternal and fetal health outcomes. By evaluating parasitaemia, cytokine profiles, histopathological changes, and pregnancy outcomes, this study seeks to shed light on the complex interactions between the immune response, placental pathology, and pregnancy outcomes in the context of malaria infection. 2. Methodology 2.1. Ethical statement Ethical clearance for the study was obtained from the Animal Ethics Committee vide clearance no. 80/IAEC/507. Throughout the study, adherence to ‘The Committee for the Purpose of Control and Supervision of Experiments on Animals’ (CPCSEA) guidelines was ensured, encompassing humane treatment of mice models, housing, handling, and experimental procedures aimed at minimizing distress. Administration of anaesthesia and analgesia was performed as required, and efforts were undertaken to minimize animal use while maximizing scientific significance. The highest ethical standards and scientific integrity were upheld, with a primary focus on animal welfare in contributions to malaria and pregnancy research. 2.2. Animal Model and Study Design For this study, female mice were divided into two main groups: non-pregnant (NP) and pregnant (P) mice. Pregnant mice were further categorized based on their gestational stage as early pregnancy (EP), mid pregnancy (MP), and late pregnancy (LP). Each group comprised six experimental mice infected with the Plasmodium berghei NK-65 strain and six control mice that remained non-infected. The gestation days chosen for infection were the 6th, 10th, and 14th day for early, mid, and late pregnancy groups, respectively(Manhas et al., 2023). Parasitaemia and cytokine samples were collected on the 5 th day post-infection (5DPI) to represent moderate infection and on the 7th day post-infection (7DPI) to represent severe infection (fig.1). 2.3. Parasitaemia Count by Microscopy To determine the parasitaemia levels, blood smears were prepared from all experimental and control mice. Thin blood smears fixed with methanol were then stained with Giemsa stain. The slides were examined under a light microscope, and parasitaemia was calculated as a percentage of infected red blood cells (RBCs) over total RBCs (Rogerson et al., 2007). Parasitaemia levels were confirmed twice to ensure accuracy and consistency. 2.4. Histopathology Methodology After euthanizing the mice, liver, kidney, and placenta tissues were collected for histopathological analysis. The samples were preserved using 10% buffered formalin and subsequently embedded in paraffin wax. Subsequent steps involved the creation of thin 5 μm sections, which were then subjected to hematoxylin and eosin (H&E) staining for analysis using a light microscope. Histopathological changes, including inflammatory cell infiltration, parasite accumulation, and tissue damage, were evaluated. Infected tissues were further divided into mild, moderate, and high infection categories based on the severity of pathological changes observed. 2.5. Cytokine Analysis by Cytokine Bead array Method Cytokine analysis was performed using the Cytokine Bead Array (CBA) method. Blood samples were collected from all experimental and control mice at the specified time points, and serum was isolated by centrifugation. The levels of various cytokines, including interleukin-5 (IL-5), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), interleukin-4 (IL-4), interleukin-2 (IL-2), and interferon-gamma (IFN-γ), were measured. For the CBA, capture beads coated with specific antibodies for each cytokine were mixed with the serum samples. This mixture was then incubated to allow cytokine binding to the capture beads. After washing away unbound components, a detection antibody specific to each cytokine was added, followed by a streptavidin-phycoerythrin conjugate. The cytokine-bead complex was then analyzed using a flow cytometer BD FACS CANTO-II (BD Biosciences, USA), which detected the phycoerythrin fluorescence, quantifying the amount of each cytokine present in the sample. The cytokine levels for each sample was assessed using the LEGENDplexTM Data analysis software(Riley et al., 2006). 2.6. Statistical Analysis The statistical analysis was conducted using GraphPad Prism software. The data were expressed as mean ± standard deviation (SD) for data with a normal distribution. Group differences were assessed using the t-test and one-way analysis of variance (ANOVA), followed by post hoc tests for multiple comparisons. A significance level of p < 0.05 was used to determine statistical significance. 3. Results 3.1. Parasitaemia Count by Microscopy Microscopic examination of blood smears revealed varying levels of parasitaemia in different groups of mice, with significant differences observed between pregnant and non-pregnant mice. In non-pregnant mice, parasitaemia levels were observed to be 10-35% on the 5th day post-infection (5DPI) (fig 2A-b,c) and 30-70% on the 7th day post-infection (7DPI) (fig 2A). Increase in parasitaemia and a rise in total leukocyte count (TLC) was observed on the 7th day post-infection (fig 2A-d,e) Pregnant mice which were infected during early pregnancy (EP) exhibited a high parasitaemia of 50-70% on the 5th day post-infection (5DPI), with markedly low red blood cell (RBC) density and mostly fragmented RBCs . However, by the 6th day post-infection (6DPI), more than 70% parasitaemia ,massively infected RBCs , and a very low RBC density was observed in these mice, leading to the mortality of all mice infected in early pregnancy stage (fig 2B-a,b). In mid-pregnancy (MP) infected mice, the parasitaemia on the 5th day post-infection was 30-50% (fig 2B-c). Subsequent to the 6th day post-infection and after delivery (AD), all of these mice displayed marked anemia, very low RBC density, and the presence of late trophozoites and schizonts in RBCs (fig 2B-d,e). Additionally, the presence of fragmented RBCs and an increase in white blood cell (WBC) count, including elevated neutrophils, monocytes, and lymphocytes, were observed in these mice. Late pregnancy (LP) infected mice exhibited parasitaemia levels ranging from 50-60% on the 5th day post-infection, with mostly fragmented RBCs and a raised TLC. After delivery, some polychromatic RBCs were observed in LP-AD mice, along with decreased RBC density and raised reticulocytes (fig 2B-f,g). 3.2. Histopathology Histopathological examination of liver, kidney, and placenta tissues revealed varying degrees of infection severity in different groups. In early pregnancy infected mice on the 5th day post-infection (EP-5DPI), infected livers showed prominent Kupffer cells containing hemazoin within the sinusoids. Severe infection in early pregnancy infected mice on the 6th day post-infection led to liver infarction in 3 mice of this group (50% mice) The severity of infection was moderate in non-pregnant mice on the 7th day post-infection (NP-7DPI), whereas early and mid-pregnancy infected mice exhibited high degree of infection in the form of malaria pigment within 80% RBCs and extracellular pigment was seen because of lysed RBCs after day 6 post infection in these tissues (fig 3C). In the infected groups, kidney tissues showed congested RBCs within the glomerulus and peritubular capillaries and RBCs showed the presence of trophozoites . Late pregnancy infected mice was highly associated with moderate infection. 7th DPI exhibited increased infection severity in early and mid-pregnancy mice compared to non-pregnant mice (Fig 3A). Placenta tissues were categorized into moderate and high infection levels. Mid-pregnancy infected mice showed the maximum number of severe infection cases (25%), followed by EP-5DPI (17%) and LP-5DPI (14%). Placental histopathology showed trophoblastic malaria pigment deposition and congestion (Fig 3B) 3.3. Pregnancy Outcomes Pregnancy outcomes were significantly affected by the timing of infection. In early and mid-pregnancy infected mice, intrauterine fetal deaths were observed (66% and 50% respectively), while stillbirths were recorded in mid-pregnancy infected mice (83%), and late pregnancy infections resulted in all premature deliveries. 3.4. Cytokine Analysis Non Pregnant Mice: Following malaria infection, levels of IL-10, IL-5, TNF-α, IL-4, and IFN-γ increased in non-pregnant mice. On the 5th day post-infection, the maximum increase was observed in IL-10, an anti-inflammatory type 2 cytokine, followed by TNF-α and IFN-γ. IL-10 levels were significantly higher than other cytokines, indicating its crucial role in the immune response to the infection. A slight elevation in IL-4 and IL-5 levels was also observed on the 5th day post-infection, albeit lower compared to IL-10 (fig 4). However, by the 7th day post-infection, the levels of all cytokines decreased compared to the 5th day post-infection. Although there was an elevation in cytokine levels compared to the non-infected control, the increase was minimal compared to the levels observed on the 5th day post-infection, suggesting a decline in the immune response as the infection progressed to severe stages. Pregnant Mice: In early pregnancy (EP), the IFN-γ level was highest in the non-infected group. IL-5 levels decreased after infection, while IL-10 and TNF-α levels increased on the 5th day post-infection, with IFN-γ showing the maximum elevation (fig4). Similar trends were observed in mid-pregnancy (MP), but the IFN-γ level was lower compared to early pregnancy (fig4). In late pregnancy (LP), maximum elevation was seen in IFN-γ, TNF-α, and IL-10, and all their levels were comparable. Correlation of Parasitaemia with cytokines level: In early pregnancy infection, IL-4 and IL-5 showed a strong negative correlation with parasitaemia (table 1), indicating that as parasitaemia increased, the levels of these anti-inflammatory cytokines decreased. This suggests that as the infection progressed to severe stages, the anti-inflammatory response declined, leading to increased parasite load. In mid-pregnancy infection, IL-10, IL-4, and IL-5 showed a weak to moderate negative correlation with parasitaemia, while TNF-α and IFN-γ showed a positive correlation (table1). This shows that a positive correlation was observed with Th1 cytokines, while a negative correlation was noted with Th2 cytokines. It indicates that the maternal body prioritized clearing the infection, especially during mid-pregnancy when the fetus is in the developing stage. In late pregnancy infection, TNF-α showed a strong negative correlation, while IL-4 and IL-5 showed a weak positive correlation with parasitaemia (table 1). The decline in Th1 cytokines with rising parasitaemia suggests that the maternal body reduces Th1 response to prevent inflammatory conditions during late stages of pregnancy and protect the fetus from fatal outcomes. 4. Discussion 4.1 . Malaria remains a significant global health concern, especially for pregnant women, as it poses considerable risks to both maternal health and fetal development. In this study, we sought to investigate the impact of Plasmodium berghei infection during different stages of pregnancy in mice, with a focus on understanding the associated immune responses and pregnancy outcomes. By categorizing pregnant mice into early, mid, and late pregnancy stages and infecting them with Plasmodium berghei , we aimed to determine how the timing of infection influences the severity of the disease and the maternal immune response. Our major findings revealed diverse pregnancy outcomes, varying levels of parasitaemia, and distinct cytokine profiles in response to the infection, shedding light on the complex interactions between malaria and pregnancy. One of the key findings of this study was the varying severity of infection and pregnancy outcomes based on the timing of infection during pregnancy. Early and mid-pregnancy infections resulted in severe anemia and intrauterine fetal deaths, while late pregnancy infections led to premature deliveries. In contrast, non-pregnant mice displayed marked anemia 7 th day post infection, and mortality in all mice was observed 6 day post infection in pregnant mice groups infected during early and mid pregnancy due to the heavy accumulation of infected red blood cells (iRBCs) in various tissues and highly anemic conditions. Microscopy showed more than 80% fragmented RBCs and lysed RBCs in the severe infection groups of early and mid pregnancy (6 th and 7 th day post infection ). These observations are in line with previous studies that have highlighted the adverse effects of malaria on pregnancy, including anemia and poor fetal outcomes (Rogerson et al., 2007; Umbers et al., 2011). Furthermore, our study demonstrated distinct cytokine profiles in response to Plasmodium berghei infection, with important implications for the maternal immune response during malaria. The levels of IL-5 and IL-10, known for their protective roles in malaria, were found to be higher in moderate infection groups (5DPI) but declined in severe infection groups (7DPI). This decline in anti-inflammatory cytokines might have contributed to the increased parasite load and adverse pregnancy outcomes observed in early and mid-pregnancy infections. Our findings align with previous studies that have highlighted the significance of IL-5 and IL-10 in providing protection against malaria (Villegas-Mendez et al., 2012; Wunderlich et al., 2014) . In contrast, pro-inflammatory cytokines TNF-α and IFN-γ showed elevated levels in infected groups, indicating the activation of a pro-inflammatory response. The strong positive correlation of TNF-α and IFN-γ with parasitaemia suggests that these cytokines play a crucial role in controlling the infection but may also contribute to the severity of the disease. These observations are consistent with previous studies that have implicated TNF-α and IFN-γ in the immune response to malaria and the pathogenesis of severe disease (Neres et al., 2008; Villegas-Mendez et al., 2012) . Our study also provided valuable insights into the tissue-specific infections through histopathology analysis. The prominent infiltration of monocytes and macrophages in the placenta, along with the accumulation of infected RBCs and hemozoin in trophoblast cells, mirrors the characteristics of human placental malaria caused by P. falciparum (Poovassery et al., 2009). These similarities indicate that Plasmodium berghei infection in mice may be a valuable model for studying placental malaria. As we compare our findings with previous literature, we acknowledge the strengths and limitations of our study. One of the major strengths lies in the comprehensive examination of different stages of pregnancy and the associated immune responses and pregnancy outcomes. By using an animal model, we were able to directly investigate the effects of Plasmodium berghei infection on pregnancy. The use of histopathology provided detailed insights into tissue-specific infections, and cytokine analysis shed light on the immune response dynamics. However, one limitation of the study is the use of a lethal strain of Plasmodium berghei, leading to mortality in pregnant mice. Future studies could consider using non-lethal strains to avoid this limitation and allow for long-term observations. An unexpected finding in our study was the decline in IL-5 levels after infection during early pregnancy, despite its protective role against malaria. This unexpected decline might have contributed to the severity of infection and adverse pregnancy outcomes. Further investigation into the factors influencing this decline could offer valuable insights into potential therapeutic interventions for pregnant women with malaria. 4.3. Conclusion : The findings of this study shed light on the impact of Plasmodium berghei infection during pregnancy in mice, uncovering crucial insights into infection severity, immune response, and pregnancy outcomes. The results demonstrate that the timing of infection during pregnancy plays a significant role in determining the severity of outcomes. Early and mid-pregnancy infections resulted in severe anemia, intrauterine fetal deaths, and stillbirths, whereas late pregnancy infections were associated with premature deliveries. Histopathological analysis indicated the accumulation of infected RBCs in various tissues, contributing to mortality in both non-pregnant and pregnant mice. The cytokine analysis revealed the protective roles of IL-5 and IL-10 in malaria, as their levels remained higher in moderate infection groups but declined in severe infection groups. Conversely, TNF-α and IFN-γ showed elevated levels in infected groups, indicating pro-inflammatory responses. The study also emphasizes the significance of Plasmodium berghei as a better model for studying placental malaria compared to other species. It highlights the intricate relationship between cytokine levels, parasitaemia, and pregnancy outcomes, offering valuable implications for understanding malaria during pregnancy. Declarations Funding information: This work was supported by ICMR and PGIMER, Chandigarh. Prem Lata Manhas was a Ph.D. student at the Post Graduate Institute of Medical Education and Research and had an ICMR-JRF fellowship. Declaration of Competing Interest: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Author contribution: Study concept: PM, RS; Data collection: PLM; Data analysis and curation: PLM, BDR, AM; Writing of manuscript: PLM, PM; Revision of manuscript: PLM, PM; Study supervision: PM, RS. References Craig, A. G., Grau, G. 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Frontiers in Microbiology , 5 . https://doi.org/10.3389/fmicb.2014.00559 Tables Table 1 Correlation of Cytokines levels with parasitaemia Cytokine level Group Pearson’s correlation coefficient P value IFN-γ NP-5DPI 0.486 0.268 NP-7DPI 0.772 0.07 EP-5DPI 0.305 0.462 MP-5DPI 0.579 0.22 LP-5DPI 0.615 0.142 MPI-AD -0.696 0.082 LPI-AD 0.372 0.467 IL-2 NP -0.261 0.497 EP 0.013 0.978 MP 0.128 0.837 LP MPI-AD 0.423 0.345 LPI-AD IL-10 NP-5DPI 0.325 0.433 NP-7DPI 0.81 0.05 EP-5DPI 0.535 0.172 MP-5DPI -0.663 0.222 LP-5DPI -0.169 0.718 MPI-AD -0.632 0.128 LPI-AD 0.204 0.698 TNF-α NP-5DPI 0.571 0.108 NP-7DPI 0436 0.355 EP-5DPI 0.377 0.358 MP-5DPI -0.224 0.718 LP-5DPI -0.721 0.067 MPI-AD -0.62 0.135 LPI-AD -0.269 0.606 IL-4 NP-5DPI -0.296 0.439 NP-7DPI 0.406 0.424 EP-5DPI -0.657 0.077 MP-5DPI -0.118 0.850 LP-5DPI 0.047 0.921 MPI-AD -0.076 0.872 LPI-AD 0.518 0.293 IL-5 NP-5DPI 0.394 0.294 NP-7DPI -0.439 0.2 EP-5DPI -0.985 0 MP-5DPI -0.349 0.565 LP-5DPI 0.476 0.280 MPI-AD 0.365 0.421 LPI-AD 0.177 0.737 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3327515","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":231505930,"identity":"9895709a-052f-4b1a-a317-d52b947ae157","order_by":0,"name":"Prem Lata Manhas","email":"","orcid":"","institution":"Postgraduate Institute of Medical Education and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Prem","middleName":"Lata","lastName":"Manhas","suffix":""},{"id":231505931,"identity":"f17bc3dd-ea37-409c-b43b-2bace5497bfd","order_by":1,"name":"Rakesh Sehgal","email":"","orcid":"","institution":"Post Graduate Institute of Medical Education and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rakesh","middleName":"","lastName":"Sehgal","suffix":""},{"id":231505932,"identity":"f5d37bf5-9a58-4fed-95a1-1ecaac41c8ac","order_by":2,"name":"Bishan Dass Radotra","email":"","orcid":"","institution":"Post Graduate Institute of Medical Education and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bishan","middleName":"Dass","lastName":"Radotra","suffix":""},{"id":231505933,"identity":"00d29cf3-15a6-4772-b49b-fa6e1be7ece2","order_by":3,"name":"Abhishek Mewara","email":"","orcid":"","institution":"Post Graduate Institute of Medical Education and Research","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Abhishek","middleName":"","lastName":"Mewara","suffix":""},{"id":231505934,"identity":"ba84c771-fb9a-45c1-b954-c99bab8fb729","order_by":4,"name":"Pankaj Malhotra","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABEUlEQVRIiWNgGAWjYFAC5jY4i4GBxyYBzEwowKeFEaglAa4lLYGBDaTFgGgtDIchWhjwaNFtP9j24OOPe/Lm7GcPG/yQOZ/HL9+d+OGBAYM8v9gBrFrMziS2G85IKDbc2ZOXnNjDc7tYso13swTQYYYzZydg13IgsU2aJyGBccOBHOMDPDy3Ezcc490A0pJgcBuHlvMPwVrsN5x/Y3zwD885kJbNP/BquQGxJXHDjRzjZB6eAyAt2/DbcuNhm+SMtITknTPeGBvL8CQnzmzL3WaRYCCB2y/nk49JfLBJsN3On2Ms+bbHLrGf+ezmmz8qbOT5pbFrgQNwRDD2wPkS+JXDtTD8IKxwFIyCUTAKRh4AANMZYjp+6+xTAAAAAElFTkSuQmCC","orcid":"","institution":"Postgraduate Institute of Medical Education and Research","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Pankaj","middleName":"","lastName":"Malhotra","suffix":""}],"badges":[],"createdAt":"2023-09-05 11:44:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3327515/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3327515/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":43012477,"identity":"a8ab3c29-792e-4ed7-bff7-1b2c8f9f1c76","added_by":"auto","created_at":"2023-09-12 14:44:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":46876,"visible":true,"origin":"","legend":"\u003cp\u003eStudy Design: Gestation days are shown on horizontal scales, infection inoculation day is shown by injection and the sample collection days are shown by blood drop\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3327515/v1/efe70dae54d7b73462c7fe64.png"},{"id":43013251,"identity":"395a48b1-f6f9-4869-8db8-9c35b5061fa2","added_by":"auto","created_at":"2023-09-12 14:52:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1145874,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2A\u003c/strong\u003e: Microscopy of blood of non-pregnant groups of mice, uninfected RBCs (a), non-pregnant-5 day post infection (b,c), non-pregnant-7 day post infection (d,e)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2B\u003c/strong\u003e: Pregnant Mice : Microscopy of blood of pregnant groups of mice\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3327515/v1/e849e969b3f6ca21195576dc.png"},{"id":43012479,"identity":"b9902822-14e2-4ca1-a5ae-4b289077cbd7","added_by":"auto","created_at":"2023-09-12 14:44:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":835023,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3A\u003c/strong\u003e: Histology section of kidney showing glomerulus and proximal and distal tubules with mild infection load (a) focally seen in interstitium, moderate (b) with in the tubules and interstitium causing interstitial expansion and severe infection load (c) involving all three compartment interstitium, glomerulus and tubules in the form of malarial pigment laden RBCs\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3B\u003c/strong\u003e: Microphotograph of placenta showing markedly congested intervillous spaces and intra-villous blood vessels (a; H\u0026amp;E, 200X) and packed with Infected RBCs many of which are loaded with malarial pigment which can be better seen at higher magnification (b; H\u0026amp;E, 1000X)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3C \u003c/strong\u003e: Histological section of liver showing liver parenchyma with dilated sinusoids. The sinusoids are dilated and showing mild (a, b), moderate (c,d) and high (e,f) degree of infection in the form of malaria pigment with the RBCs and extracellular pigment because of lysed RBCs\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3327515/v1/5d9a8cc28dcc2726102af403.png"},{"id":43012478,"identity":"78f6bdb2-1b10-4741-bf82-55dbca4d83bb","added_by":"auto","created_at":"2023-09-12 14:44:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":36510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e4A\u003c/strong\u003e: Cytokine levels in non-pregnant mice, \u003cstrong\u003e4B\u003c/strong\u003e: Cytokine levels in Pregnant mice\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3327515/v1/1f81712f5db7a42e87440796.png"},{"id":43782734,"identity":"eb0b3e09-a3fc-4809-9ebc-0d8e494c4758","added_by":"auto","created_at":"2023-09-27 17:07:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2025369,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3327515/v1/66fcfe8a-f16d-45e0-93c0-2c44c4f85a04.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eUnraveling the Interplay of Parasitaemia, Cytokines, and Pregnancy Outcomes in \u003cem\u003ePlasmodium berghei\u003c/em\u003eInfection: Insights from a Murine Model\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eMalaria remains one of the most significant global health burdens, particularly affecting vulnerable populations such as pregnant women and their unborn children. Pregnant women are at an increased risk of severe malaria infection due to alterations in their immune response, hormonal changes, and the presence of placental tissue, which provides a unique environment for parasite sequestration and growth (Rogerson et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Malaria during pregnancy can result in adverse outcomes, including maternal anemia, intrauterine growth restriction, preterm births, stillbirths, and an increased risk of maternal mortality (Rogerson et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). \u003cem\u003ePlasmodium falciparum\u003c/em\u003e and \u003cem\u003ePlasmodium vivax\u003c/em\u003e are the most common species responsible for causing malaria during pregnancy in endemic regions (Dellicour et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn malaria-endemic areas, around 25\u0026nbsp;million expectant mothers face the threat of infection annually, leading to approximately 200,000 infant deaths annually (Schantz-Dunn \u0026amp; Nour, n.d.). Placental malaria, which involves the accumulation of infected red blood cells in the placenta, plays a central role in these adverse outcomes (Hviid et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The pathogenesis of placental malaria involves the adhesion of infected red blood cells to chondroitin sulfate A on the syncytiotrophoblast surface, leading to immune activation and inflammation in the placenta (Lekana Douki et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Rogerson et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) .\u003c/p\u003e \u003cp\u003eTo better understand the impact of malaria during pregnancy and to identify potential interventions, animal models have been extensively used (Doritchamou et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Mice are often used as models for studying malaria due to their ease of manipulation, genetic tools, and availability of well-characterized parasite strains(Craig et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Vaughan \u0026amp; Kappe, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Among these models, \u003cem\u003ePlasmodium berghei\u003c/em\u003e infection in mice has been utilized to study various aspects of malaria pathogenesis, immunity, and potential vaccine candidates(Stephens et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). \u003cem\u003ePlasmodium berghei\u003c/em\u003e, while not a direct human pathogen, shares several similarities with human malaria parasites and has been shown to induce placental malaria-like symptoms in mice (Franke-Fayard et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Rodrigues-Duarte et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2012\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis research paper aims to investigate the impact of \u003cem\u003ePlasmodium berghei NK-65\u003c/em\u003e strain infection during different stages of pregnancy in mice, simulating the gestational timeline observed in humans. The study specifically focuses on early, mid, and late stages of pregnancy to explore how the timing of infection influences maternal and fetal health outcomes. By evaluating parasitaemia, cytokine profiles, histopathological changes, and pregnancy outcomes, this study seeks to shed light on the complex interactions between the immune response, placental pathology, and pregnancy outcomes in the context of malaria infection.\u003c/p\u003e"},{"header":"2. Methodology","content":"\u003cp\u003e\u003cstrong\u003e2.1. Ethical statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical clearance for the study was obtained from the Animal Ethics Committee vide clearance no. 80/IAEC/507. Throughout the study, adherence to \u0026lsquo;The Committee for the Purpose of Control and Supervision of Experiments on Animals\u0026rsquo; (CPCSEA) guidelines was ensured, encompassing humane treatment of mice models, housing, handling, and experimental procedures aimed at minimizing distress. Administration of anaesthesia and analgesia was performed as required, and efforts were undertaken to minimize animal use while maximizing scientific significance. The highest ethical standards and scientific integrity were upheld, with a primary focus on animal welfare in contributions to malaria and pregnancy research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2. Animal Model and Study Design\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor this study, female mice were divided into two main groups: non-pregnant (NP) and pregnant (P) mice. Pregnant mice were further categorized based on their gestational stage as early pregnancy \u0026nbsp;(EP), mid pregnancy (MP), and late pregnancy (LP). Each group comprised six experimental mice infected with the \u003cem\u003ePlasmodium berghei NK-65\u003c/em\u003e strain and six control mice that remained non-infected. The gestation days chosen for infection were the 6th, 10th, and 14th day for early, mid, and late pregnancy groups, respectively(Manhas et al., 2023). Parasitaemia and cytokine samples were collected on the \u0026nbsp;5\u003csup\u003eth\u003c/sup\u003e day post-infection \u0026nbsp;(5DPI) to represent moderate infection and on the 7th day post-infection (7DPI) to represent severe infection (fig.1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3. Parasitaemia Count by Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the parasitaemia levels, blood smears were prepared from all experimental and control mice. Thin blood smears fixed with methanol were then stained with Giemsa stain. The slides were examined under a light microscope, and parasitaemia was calculated as a percentage of infected red blood cells (RBCs) over total RBCs (Rogerson et al., 2007). Parasitaemia levels were confirmed twice to ensure accuracy and consistency.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Histopathology Methodology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter euthanizing the mice, liver, kidney, and placenta tissues were collected for histopathological analysis. The samples were preserved using 10% buffered formalin and subsequently embedded in paraffin wax. Subsequent steps involved the creation of thin 5 \u0026mu;m sections, which were then subjected to hematoxylin and eosin (H\u0026amp;E) staining for analysis using a light microscope. Histopathological changes, including inflammatory cell infiltration, parasite accumulation, and tissue damage, were evaluated. Infected tissues were further divided into mild, moderate, and high infection categories based on the severity of pathological changes observed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5. Cytokine Analysis by Cytokine Bead array Method\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCytokine analysis was performed using the Cytokine Bead Array (CBA) method. Blood samples were collected from all experimental and control mice at the specified time points, and serum was isolated by centrifugation. The levels of various cytokines, including interleukin-5 (IL-5), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-\u0026alpha;), interleukin-4 (IL-4), interleukin-2 (IL-2), and interferon-gamma (IFN-\u0026gamma;), were measured.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor the CBA, capture beads coated with specific antibodies for each cytokine were mixed with the serum samples. This mixture was then incubated to allow cytokine binding to the capture beads. After washing away unbound components, a detection antibody specific to each cytokine was added, followed by a streptavidin-phycoerythrin conjugate. The cytokine-bead complex was then analyzed using a flow cytometer BD FACS CANTO-II (BD Biosciences, USA), which detected the phycoerythrin fluorescence, quantifying the amount of each cytokine present in the sample. The \u0026nbsp;cytokine levels for each sample was assessed using the LEGENDplexTM Data analysis software(Riley et al., 2006).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6. Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe statistical analysis was conducted using GraphPad Prism software. The data were expressed as mean \u0026plusmn; standard deviation (SD) for data with a normal distribution. Group differences were assessed using the t-test and one-way analysis of variance (ANOVA), followed by post hoc tests for multiple comparisons. A significance level of p \u0026lt; 0.05 was used to determine statistical significance.\u003c/p\u003e"},{"header":"3.\tResults","content":"\u003cp\u003e\u003cstrong\u003e3.1. Parasitaemia Count by Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMicroscopic examination of blood smears revealed varying levels of parasitaemia in different groups of mice, with significant differences observed between pregnant and non-pregnant mice.\u003c/p\u003e\n\u003cp\u003eIn non-pregnant mice, parasitaemia levels were observed to be 10-35% on the 5th day post-infection (5DPI) (fig 2A-b,c) and 30-70% on the 7th day post-infection (7DPI) (fig 2A). Increase in parasitaemia and a rise in total leukocyte count (TLC) was observed on the 7th day post-infection \u0026nbsp;(fig 2A-d,e)\u003c/p\u003e\n\u003cp\u003ePregnant mice which were infected during early pregnancy (EP) exhibited a high parasitaemia of 50-70% on the 5th day post-infection (5DPI), with markedly low red blood cell (RBC) density and mostly fragmented RBCs . However, by the 6th day post-infection (6DPI), more than 70% parasitaemia ,massively infected RBCs , and a very low RBC density was observed in these mice, leading to the mortality of all mice infected in early pregnancy stage (fig 2B-a,b).\u003c/p\u003e\n\u003cp\u003eIn mid-pregnancy (MP) infected mice, the parasitaemia on the 5th day post-infection was 30-50% (fig 2B-c). Subsequent to the 6th day post-infection and after delivery (AD), all of these mice displayed marked anemia, very low RBC density, and the presence of late trophozoites and schizonts in RBCs (fig 2B-d,e). Additionally, the presence of fragmented RBCs and an increase in white blood cell (WBC) count, including elevated neutrophils, monocytes, and lymphocytes, were observed in these mice.\u003c/p\u003e\n\u003cp\u003eLate pregnancy (LP) infected mice exhibited parasitaemia levels ranging from 50-60% on the 5th day post-infection, with mostly fragmented RBCs and a raised TLC. After delivery, some polychromatic RBCs were observed in LP-AD mice, along with decreased RBC density and raised reticulocytes (fig 2B-f,g).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2. Histopathology\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistopathological examination of liver, kidney, and placenta tissues revealed varying degrees of infection severity in different groups.\u003c/p\u003e\n\u003cp\u003eIn early pregnancy infected mice on the 5th day post-infection (EP-5DPI), infected livers showed prominent Kupffer cells containing hemazoin within the sinusoids. Severe infection in early pregnancy infected mice on the 6th day post-infection led to liver infarction in 3 mice of this group (50% mice) The severity of infection was moderate in non-pregnant mice on the 7th day post-infection (NP-7DPI), whereas early and mid-pregnancy infected mice exhibited high \u0026nbsp;degree of infection in the form of malaria pigment within 80% RBCs and extracellular pigment was seen because of lysed RBCs \u0026nbsp; after day 6 post infection in these tissues (fig 3C). In the infected groups, kidney tissues showed congested RBCs within the glomerulus and peritubular capillaries and RBCs showed the presence of trophozoites . Late pregnancy infected mice was highly associated with moderate infection. 7th DPI exhibited increased infection severity in early and mid-pregnancy mice compared to non-pregnant mice (Fig 3A). Placenta tissues were categorized into moderate and high infection levels. Mid-pregnancy infected mice showed the maximum number of severe infection cases (25%), followed by EP-5DPI (17%) and LP-5DPI (14%). Placental histopathology showed trophoblastic malaria pigment deposition and congestion (Fig 3B)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3. Pregnancy Outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePregnancy outcomes were significantly affected by the timing of infection. In early and mid-pregnancy infected mice, intrauterine fetal deaths were observed (66% and 50% respectively), while stillbirths were recorded in mid-pregnancy infected mice (83%), and late pregnancy infections resulted in all premature deliveries.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4. Cytokine Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNon Pregnant Mice:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing malaria infection, levels of IL-10, IL-5, TNF-\u0026alpha;, IL-4, and IFN-\u0026gamma; increased in non-pregnant mice. On the 5th day post-infection, the maximum increase was observed in IL-10, an anti-inflammatory type 2 cytokine, followed by TNF-\u0026alpha; and IFN-\u0026gamma;. IL-10 levels were significantly higher than other cytokines, indicating its crucial role in the immune response to the infection. A slight elevation in IL-4 and IL-5 levels was also observed on the 5th day post-infection, albeit lower compared to IL-10 (fig 4).\u003c/p\u003e\n\u003cp\u003eHowever, by the 7th day post-infection, the levels of all cytokines decreased compared to the 5th day post-infection. Although there was an elevation in cytokine levels compared to the non-infected control, the increase was minimal compared to the levels observed on the 5th day post-infection, suggesting a decline in the immune response as the infection progressed to severe stages.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePregnant Mice:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn early pregnancy (EP), the IFN-\u0026gamma; level was highest in the non-infected group. IL-5 levels decreased after infection, while IL-10 and TNF-\u0026alpha; levels increased on the 5th day post-infection, with IFN-\u0026gamma; showing the maximum elevation (fig4). Similar trends were observed in mid-pregnancy (MP), but the IFN-\u0026gamma; level was lower compared to early pregnancy (fig4). In late pregnancy (LP), maximum elevation was seen in IFN-\u0026gamma;, TNF-\u0026alpha;, and IL-10, and all their levels were comparable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorrelation of Parasitaemia with cytokines level:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn early pregnancy infection, IL-4 and IL-5 showed a strong negative correlation with parasitaemia (table 1), indicating that as parasitaemia increased, the levels of these anti-inflammatory cytokines decreased. This suggests that as the infection progressed to severe stages, the anti-inflammatory response declined, leading to increased parasite load.\u003c/p\u003e\n\u003cp\u003eIn mid-pregnancy infection, IL-10, IL-4, and IL-5 showed a weak to moderate negative correlation with parasitaemia, while TNF-\u0026alpha; and IFN-\u0026gamma; showed a positive correlation (table1). This shows that a positive correlation was observed with Th1 cytokines, while a negative correlation was noted with Th2 cytokines. It indicates that the maternal body prioritized clearing the infection, especially during mid-pregnancy when the fetus is in the developing stage.\u003c/p\u003e\n\u003cp\u003eIn late pregnancy infection, TNF-\u0026alpha; showed a strong negative correlation, while IL-4 and IL-5 showed a weak positive correlation with parasitaemia (table 1). The decline in Th1 cytokines with rising parasitaemia suggests that the maternal body reduces Th1 response to prevent inflammatory conditions during late stages of pregnancy and protect the fetus from fatal outcomes.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003e\u003cstrong\u003e4.1\u003c/strong\u003e. Malaria remains a significant global health concern, especially for pregnant women, as it poses considerable risks to both maternal health and fetal development. In this study, we sought to investigate the impact of \u003cem\u003ePlasmodium berghei\u003c/em\u003e infection during different stages of pregnancy in mice, with a focus on understanding the associated immune responses and pregnancy outcomes. By categorizing pregnant mice into early, mid, and late pregnancy stages and infecting them with \u003cem\u003ePlasmodium berghei\u003c/em\u003e, we aimed to determine how the timing of infection influences the severity of the disease and the maternal immune response. Our major findings revealed diverse pregnancy outcomes, varying levels of parasitaemia, and distinct cytokine profiles in response to the infection, shedding light on the complex interactions between malaria and pregnancy.\u003c/p\u003e\n\u003cp\u003eOne of the key findings of this study was the varying severity of infection and pregnancy outcomes based on the timing of infection during pregnancy. Early and mid-pregnancy infections resulted in severe anemia and intrauterine fetal deaths, while late pregnancy infections led to premature deliveries. In contrast, non-pregnant mice displayed marked anemia 7\u003csup\u003eth\u003c/sup\u003e day post infection, and mortality in all mice was observed 6 day post infection \u0026nbsp;in pregnant mice groups infected during early and mid pregnancy due to the heavy accumulation of infected red blood cells (iRBCs) in various tissues and highly anemic conditions. Microscopy showed more than 80% fragmented RBCs and lysed RBCs in the severe infection groups of early and mid pregnancy (6\u003csup\u003eth\u003c/sup\u003e and \u0026nbsp;7\u003csup\u003eth\u003c/sup\u003e day post infection ). These observations are in line with previous studies that have highlighted the adverse effects of malaria on pregnancy, including anemia and poor fetal outcomes\u0026nbsp;(Rogerson et al., 2007; Umbers et al., 2011).\u003c/p\u003e\n\u003cp\u003eFurthermore, our study demonstrated distinct cytokine profiles in response to \u003cem\u003ePlasmodium berghei\u003c/em\u003e infection, with important implications for the maternal immune response during malaria. The levels of IL-5 and IL-10, known for their protective roles in malaria, were found to be higher in moderate infection groups (5DPI) but declined in severe infection groups (7DPI). This decline in anti-inflammatory cytokines might have contributed to the increased parasite load and adverse pregnancy outcomes observed in early and mid-pregnancy infections. Our findings align with previous studies that have highlighted the significance of IL-5 and IL-10 in providing protection against malaria\u0026nbsp;(Villegas-Mendez et al., 2012; Wunderlich et al., 2014)\u0026nbsp;.\u003c/p\u003e\n\u003cp\u003eIn contrast, pro-inflammatory cytokines TNF-\u0026alpha; and IFN-\u0026gamma; showed elevated levels in infected groups, indicating the activation of a pro-inflammatory response. The strong positive correlation of TNF-\u0026alpha; and IFN-\u0026gamma; with parasitaemia suggests that these cytokines play a crucial role in controlling the infection but may also contribute to the severity of the disease. These observations are consistent with previous studies that have implicated TNF-\u0026alpha; and IFN-\u0026gamma; in the immune response to malaria and the pathogenesis of severe disease\u0026nbsp;(Neres et al., 2008; Villegas-Mendez et al., 2012)\u0026nbsp;.\u003c/p\u003e\n\u003cp\u003eOur study also provided valuable insights into the tissue-specific infections through histopathology analysis. The prominent infiltration of monocytes and macrophages in the placenta, along with the accumulation of infected RBCs and hemozoin in trophoblast cells, mirrors the characteristics of human placental malaria caused by P. falciparum\u0026nbsp;(Poovassery et al., 2009). These similarities indicate that \u003cem\u003ePlasmodium berghei\u003c/em\u003e infection in mice may be a valuable model for studying placental malaria.\u003c/p\u003e\n\u003cp\u003eAs we compare our findings with previous literature, we acknowledge the strengths and limitations of our study. One of the major strengths lies in the comprehensive examination of different stages of pregnancy and the associated immune responses and pregnancy outcomes. By using an animal model, we were able to directly investigate the effects of \u003cem\u003ePlasmodium berghei\u003c/em\u003e infection on pregnancy. The use of histopathology provided detailed insights into tissue-specific infections, and cytokine analysis shed light on the immune response dynamics. However, one limitation of the study is the use of a lethal strain of \u003cem\u003ePlasmodium berghei,\u003c/em\u003e leading to mortality in pregnant mice. Future studies could consider using non-lethal strains to avoid this limitation and allow for long-term observations.\u003c/p\u003e\n\u003cp\u003eAn unexpected finding in our study was the decline in IL-5 levels after infection during early pregnancy, despite its protective role against malaria. This unexpected decline might have contributed to the severity of infection and adverse pregnancy outcomes. Further investigation into the factors influencing this decline could offer valuable insights into potential therapeutic interventions for pregnant women with malaria.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.3. Conclusion\u003c/strong\u003e: The findings of this study shed light on the impact of \u003cem\u003ePlasmodium berghei\u003c/em\u003e infection during pregnancy in mice, uncovering crucial insights into infection severity, immune response, and pregnancy outcomes. The results demonstrate that the timing of infection during pregnancy plays a significant role in determining the severity of outcomes. Early and mid-pregnancy infections resulted in severe anemia, intrauterine fetal deaths, and stillbirths, whereas late pregnancy infections were associated with premature deliveries. Histopathological analysis indicated the accumulation of infected RBCs in various tissues, contributing to mortality in both non-pregnant and pregnant mice.\u003c/p\u003e\n\u003cp\u003eThe cytokine analysis revealed the protective roles of IL-5 and IL-10 in malaria, as their levels remained higher in moderate infection groups but declined in severe infection groups. Conversely, TNF-\u0026alpha; and IFN-\u0026gamma; showed elevated levels in infected groups, indicating pro-inflammatory responses. The study also emphasizes the significance of \u003cem\u003ePlasmodium berghei\u003c/em\u003e as a better model for studying placental malaria compared to other species. It highlights the intricate relationship between cytokine levels, parasitaemia, and pregnancy outcomes, offering valuable implications for understanding malaria during pregnancy.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding information:\u0026nbsp;\u003c/strong\u003eThis work was supported by ICMR and PGIMER, Chandigarh. Prem Lata Manhas was a Ph.D. student at the Post Graduate Institute of Medical Education and Research and had an ICMR-JRF fellowship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing\u003c/p\u003e\n\u003cp\u003efinancial interests or personal relationships that could have appeared to influence the work\u003c/p\u003e\n\u003cp\u003ereported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution:\u0026nbsp;\u003c/strong\u003eStudy concept: PM, RS; Data collection: PLM; Data analysis and curation: PLM, BDR, AM; Writing of manuscript: PLM, PM; Revision of manuscript:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePLM, PM; Study supervision: PM, RS.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCraig, A. G., Grau, G. E., Janse, C., Kazura, J. W., Milner, D., Barnwell, J. 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Liver-inherent immune system: Its role in blood-stage malaria. \u003cem\u003eFrontiers in Microbiology\u003c/em\u003e, \u003cem\u003e5\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fmicb.2014.00559\u003c/span\u003e\u003cspan address=\"10.3389/fmicb.2014.00559\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":" \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cdiv class=\"SimplePara\"\u003eCorrelation of Cytokines levels with parasitaemia\u003c/div\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv 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colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.985\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eMP-5DPI\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e-0.349\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.565\u003c/div\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cdiv class=\"SimplePara\"\u003eLP-5DPI\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.476\u003c/div\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cdiv class=\"SimplePara\"\u003e0.280\u003c/div\u003e \u003c/td\u003e 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\u003cbr/\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Plasmodium berghei, Pregnancy, Parasitaemia, Cytokines, Histopathology, Pregnancy outcomes","lastPublishedDoi":"10.21203/rs.3.rs-3327515/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3327515/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMalaria infection during pregnancy presents notable risks to both maternal and fetal health. Present study examines the impact of \u003cem\u003ePlasmodium berghei NK-65\u003c/em\u003e strain infection during different stages of pregnancy in mice. The study categorized mice into non-pregnant and pregnant groups (early, mid, and late pregnancy) and induced infection on specific gestation days. Parasitaemia, cytokine profiles, histopathological changes, and pregnancy outcomes were evaluated. Major findings include severe anemia in mid-pregnancy infection, fetal deaths in early and mid-pregnancy infections, stillbirths in mid-pregnancy infections, and premature deliveries in late-pregnancy infections. Cytokine profiles differed between pregnant and non-pregnant mice, indicating varied immune responses. \u003cem\u003ePlasmodium berghei\u003c/em\u003e infection in pregnant mice provides valuable insights into understanding placental malaria and its implications for maternal and fetal health.\u003c/p\u003e","manuscriptTitle":"Unraveling the Interplay of Parasitaemia, Cytokines, and Pregnancy Outcomes in Plasmodium bergheiInfection: Insights from a Murine Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-12 14:44:33","doi":"10.21203/rs.3.rs-3327515/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f6016f2-076c-4884-8830-cd84c067aea7","owner":[],"postedDate":"September 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-27T16:59:14+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-12 14:44:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3327515","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3327515","identity":"rs-3327515","version":["v1"]},"buildId":"omnImTCwR2MFx8CMYfrG7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}