Alteration of Trophoblast Syncytialization by Plasmodium falciparum-Infected Erythrocytes.

Malaria during pregnancy has been associated with significant risks to both the mother and the fetus, leading to complications such as anemia, low birth weight, and increased infant mortality. The trophoblast cells, a key component of the placenta, are crucial for nutrient and oxygen exchange between mother and fetus. The differentiation of cytotrophoblasts (CTBs) into syncytiotrophoblasts (STBs) is critical for proper pregnancy development. These cells form the bi-stratified epithelium surrounding the placental villi. While previous studies have described an inflammatory activation of STB cells exposed to Plasmodium falciparum-infected erythrocytes (P. falciparum-IE) or components such as hemozoin (HZ), little is known about the direct effect this parasite may have on the epithelial turnover and function of trophoblast cells. This study aims to contribute to understanding mechanisms leading to placental damage during placental malaria using a BeWo cell line as a differentiation model. It was found that P. falciparum-IE interferes with the fusion of BeWo cells, affecting the differentiation process of trophoblast. A reduction in syncytialization could be associated with the adverse effects of infection in fetal health, altering the remodeling of the trophoblast epithelial barrier and reducing their capacity to exchange substances. However, further studies are necessary to assess alterations in the functionality of this epithelium.

Novel 3D human trophoblast culture to explore T. cruzi infection in the placenta.

Introduction: Human trophoblastic cell lines, such as BeWo, are commonly used in 2D models to study placental Trypanosoma cruzi infections. However, these models do not accurately represent natural infections. Three-dimensional (3D) microtissue cultures offer a more physiologically relevant in vitro model, mimicking tissue microarchitecture and providing an environment closer to natural infections. These 3D cultures exhibit functions such as cell proliferation, differentiation, morphogenesis, and gene expression that resemble in vivo conditions. Methods: We developed a 3D culture model using the human trophoblastic cell line BeWo and nonadherent agarose molds from the MicroTissues® 3D Petri Dish® system. Both small (12-256) and large (12-81) models were tested with varying initial cell numbers. We measured the diameter of the 3D cultures and evaluated cell viability using Trypan Blue dye. Trophoblast functionality was assessed by measuring ß-hCG production via ELISA. Cell fusion was evaluated using confocal microscopy, with Phalloidin or ZO-1 marking cell edges and DAPI staining nuclei. T. cruzi infection was assessed by microscopy and quantitative PCR, targeting the EF1-α gene for T. cruzi and GAPDH for BeWo cells, using three parasite strains: VD (isolated from a congenital Chagas disease infant and classified as Tc VI), and K98 and Pan4 (unrelated to congenital infection and classified as Tc I). Results: Seeding 1000 BeWo cells per microwell in the large model resulted in comparable cellular viability to 2D cultures, with a theoretical diameter of 408.68 ± 12.65 µm observed at 5 days. Functionality, assessed through ß-hCG production, exceeded levels in 2D cultures at both 3 and 5 days. T. cruzi infection was confirmed by qPCR and microscopy, showing parasite presence inside the cells for all three tested strains. The distribution and progression of the infection varied with each strain. Discussion: This innovative 3D model offers a simple yet effective approach for generating viable and functional cultures susceptible to T. cruzi infection, presenting significant potential for studying the placental microenvironment.

ERK1/2 phosphorylation and IL-6 production are involved in the differential susceptibility to Toxoplasma gondii infection in three types of human (cyto/ syncytio/ extravillous) trophoblast cells.

During pregnancy, Toxoplasma gondii can triggers serious manifestations and potentially affect the fetal development. In this scenario, differences in susceptibility of trophoblast cells to T. gondii infection might be evaluated in order to establish new therapeutic approaches capable of interfering in the control of fetal infection by T. gondii. This study aimed to evaluate the susceptibility of cytotrophoblast, syncytiotrophoblast and extravillous trophoblast cells to T. gondii infection. Our data demonstrate that HTR-8/SVneo cells (extravillous trophoblast cells) present higher susceptibility to T. gondii infection when compared to syncytiotrophoblast and cytotrophoblast cells, whereas syncytiotrophoblast was the cell type more resistant to the parasite infection. Also, cytotrophoblast and syncytiotrophoblast cells produced significantly more IL-6 than HTR-8/SVneo cells. On the other hand, HTR-8/SVneo cells showed higher ERK1/2 phosphorylation than cytotrophoblast and syncytiotrophoblast cells. ERK1/2 inhibition reduced T. gondii infection and increased IL-6 production in HTR-8/SVneo cells. Thus, it is plausible to conclude that the greater susceptibility of HTR-8/SVneo cells to infection by T. gondii is related to a higher ERK1/2 phosphorylation and lower levels of IL-6 in these cells compared to other cells, suggesting that these mediators may be important to favor the parasite infection in this type of trophoblastic population.

The involvement of annexin A1 in human placental response to maternal Zika virus infection.

The association of Zika virus infection (ZIKV) with congenital malformation and neurological sequelae brought a significant global concern. Recent studies have shown that maternal viral infection leads to inflammation in the placental tissue. In this context, the antiinflammatory protein annexin 1 (ANXA1) has a major determination of the resolution of inflammation and it has been positively associated with antiparasitic activity in infected placental explants. Although these effects have been explored to some degree, ANXA1 expression and potential properties have not yet been fully elucidated in placentas infected with ZIKV. This study was conducted to evaluate the histopathology, inflammatory process and elucidate if ANXA1 were differently expressed in placentas of ZIKV-infected mothers. Three classification groups were used in this study: Neg/Neg (mother and placenta negative for the virus), Pos/Neg (infected mother, but no virus detected in placenta) and Pos/Pos (mother and placenta infected with ZIKV). ANXA1 was expressed in syncytiotrophoblast cells of all studied groups, and its expression was decreased in Pos/Neg group, which displayed also an increase of the inflammatory response, as evinced from the recruitment of inflammatory cells, increased levels of placenta cytokines, and evidence of impaired tissue repair. The presence of ZIKV in placentas of Pos/Pos group shows structural alterations, including detachment and disorganization of the trophoblastic epithelium. In summary, our results suggest that maternal infection with ZIKV, even without direct tissue infection, leads to a placental inflammatory response probably related to the modulation of ANXA1. After placental infection, structural changes - including inflammatory cells influx - are observed leading to placental dysfunction and reduced fetal weight. Our study sheds additional light on the outcomes of ZIKV infection in trophoblast and reveals a potential involvement of ANXA1 in the placental biology.

Isocitrate dehydrogenase type 2 (IDH2) is part of a multiprotein complex for placental steroidogenesis.

BACKGROUND: Human syncytiotrophoblast mitochondria require the activity of the isocitrate dehydrogenase type 2 (IDH2) to obtain reduced coenzymes for progesterone (P4) synthesis. Data from the literature indicate that mitochondrial steroidogenic contact sites transform efficiently cholesterol into P4. In this research, we identified the IDH2 as a member of the steroidogenic contact site and analyzed the steroidogenic role of its activity. METHOD: Human syncytiotrophoblast mitochondria were isolated by differential centrifugation, and steroidogenic contact sites were obtained by osmotic shock and sucrose gradient ultracentrifugation. In-gel native activity assay, mass spectroscopy, and western blot were used to identify the association of proteins and their activities. P4 was determined by immunofluorescence. RESULTS: The IDH2 was mainly identified in steroidogenic contact sites, and its activity was associated with a complex of proteins with an apparent molecular mass of ~590 kDa. Mass spectroscopy showed many groups of proteins with several metabolic functions, including steroidogenesis and ATP synthesis. The IDH2 activity was coupled to P4 synthesis since in the presence of Ca2+ or Na2SeO3, inhibitors of the IDH2, the P4 production decreased. CONCLUSIONS: The human syncytiotrophoblast mitochondria build contact sites for steroidogenesis. The IDH2, a non-membrane protein, supplies the NADPH required for the synthesis of P4 in a complex (steroidosome) that associate the proteins required to transform efficiently cholesterol into P4, which is necessary in pregnancy to maintain the relationship between mother and fetus. GENERAL SIGNIFICANCE: The IDH2 is proposed as a check point in the regulation of placental steroidogenesis.

Subplacental development in Galea spixii / Desenvolvimento subplacentário do preá (Galea spixii)

Animal models are essential to understand healthy human placentation. Guinea pig related rodents became on focus for such purposes. In particular, processes of trophoblast invasion are similar. The latter is associated with a specialized area, the subplacenta. Since previous results showed differences between the guinea pig and its close relative Galea spixii, we aimed to study subplacental development with more detail. We investigated 16 pregnant females of 14 to 55 days of gestation by means of histology, morphometrics, immunohistochemistry and electron microscopy. The overlap between the fetomaternal blood systems resulted as intimate, suggesting some exchange processes. Proliferation was revealed by three independent methods, being most active in early and mid-gestation, which was in accordance to former results. Though degeneration of tissues took place, the subplacenta was maintained towards term with access to the fetal vascularization, supporting a hypothesis about the release of substances to the fetal unit in advanced gestation. In contrast to other species, the extraplacental trophoblast showed a shift from syncytial streamers to giant cells during mid-gestation. Views on placentation in caviomorphs were influenced by the guinea pig, but our data supported recent studies that the subplacenta had a much greater placidity. In regard to subplacental grow, degeneration and likely also exchange processes, Galea and other species showed a more basal pattern of caviomorphs than the guinea pig. Such differences should be considered, when choosing most adequate animal models for special purposes in comparison to human placentation.(AU)

Modelos animais são essenciais para entender a placenta humana sadia. Neste sentido os roedores relacionados ao porquinho da índia tornaram-se foco para tal entendimento. Em particular, os processos de invasão trofoblástica são semelhantes. O último está associado a uma área especializada, a subplacenta. Uma vez que os resultados anteriores mostraram diferenças entre o porquinho da índia e seu relativo o preá, buscamos estudar o desenvolvimento subplacentário com mais detalhes. Pesquisamos 16 fêmeas gestantes de 14 a 55 dias de gestação por meio de histologia, morfometria, imuno-histoquímica e microscopia eletrônica. A sobreposição entre os sistemas sanguíneos materno e fetal apresentou-se com íntima relação, sugerindo alguns processos de troca. A proliferação foi revelada por três métodos independentes, sendo mais ativos no início e metade da gestação, o que corroborou com os resultados anteriores. Embora a degeneração dos tecidos tenha ocorrido, a subplacenta foi mantida até o termo gestacional com acesso à vascularização fetal, apoiando uma hipótese sobre a liberação de substâncias para a unidade fetal em gestação avançada. Em contraste com outras espécies, o trofoblasto extraplacentário mostrou uma mudança de flâmulas sinciciais para células gigantes durante a metade da gestação. As visualizações sobre a placentação em caviomorfos foram influenciadas pelo porquinho da índia, mas nossos dados apoiaram estudos recentes de que a subplacenta apresentava uma plasticidade muito maior. Em relação ao crescimento subplacentário, a degeneração e provavelmente também os processos de troca, o preá e outras espécies apresentaram um padrão mais basal de caviomorfos do que o porquinho da índia. Tais diferenças devem ser consideradas, ao escolher os modelos animais mais adequados para fins especiais em comparação com a placentação humana.(AU)

Subplacental development in Galea spixii / Desenvolvimento subplacentário do preá (Galea spixii)

Animal models are essential to understand healthy human placentation. Guinea pig related rodents became on focus for such purposes. In particular, processes of trophoblast invasion are similar. The latter is associated with a specialized area, the subplacenta. Since previous results showed differences between the guinea pig and its close relative Galea spixii, we aimed to study subplacental development with more detail. We investigated 16 pregnant females of 14 to 55 days of gestation by means of histology, morphometrics, immunohistochemistry and electron microscopy. The overlap between the fetomaternal blood systems resulted as intimate, suggesting some exchange processes. Proliferation was revealed by three independent methods, being most active in early and mid-gestation, which was in accordance to former results. Though degeneration of tissues took place, the subplacenta was maintained towards term with access to the fetal vascularization, supporting a hypothesis about the release of substances to the fetal unit in advanced gestation. In contrast to other species, the extraplacental trophoblast showed a shift from syncytial streamers to giant cells during mid-gestation. Views on placentation in caviomorphs were influenced by the guinea pig, but our data supported recent studies that the subplacenta had a much greater placidity. In regard to subplacental grow, degeneration and likely also exchange processes, Galea and other species showed a more basal pattern of caviomorphs than the guinea pig. Such differences should be considered, when choosing most adequate animal models for special purposes in comparison to human placentation.(AU)

Modelos animais são essenciais para entender a placenta humana sadia. Neste sentido os roedores relacionados ao porquinho da índia tornaram-se foco para tal entendimento. Em particular, os processos de invasão trofoblástica são semelhantes. O último está associado a uma área especializada, a subplacenta. Uma vez que os resultados anteriores mostraram diferenças entre o porquinho da índia e seu relativo o preá, buscamos estudar o desenvolvimento subplacentário com mais detalhes. Pesquisamos 16 fêmeas gestantes de 14 a 55 dias de gestação por meio de histologia, morfometria, imuno-histoquímica e microscopia eletrônica. A sobreposição entre os sistemas sanguíneos materno e fetal apresentou-se com íntima relação, sugerindo alguns processos de troca. A proliferação foi revelada por três métodos independentes, sendo mais ativos no início e metade da gestação, o que corroborou com os resultados anteriores. Embora a degeneração dos tecidos tenha ocorrido, a subplacenta foi mantida até o termo gestacional com acesso à vascularização fetal, apoiando uma hipótese sobre a liberação de substâncias para a unidade fetal em gestação avançada. Em contraste com outras espécies, o trofoblasto extraplacentário mostrou uma mudança de flâmulas sinciciais para células gigantes durante a metade da gestação. As visualizações sobre a placentação em caviomorfos foram influenciadas pelo porquinho da índia, mas nossos dados apoiaram estudos recentes de que a subplacenta apresentava uma plasticidade muito maior. Em relação ao crescimento subplacentário, a degeneração e provavelmente também os processos de troca, o preá e outras espécies apresentaram um padrão mais basal de caviomorfos do que o porquinho da índia. Tais diferenças devem ser consideradas, ao escolher os modelos animais mais adequados para fins especiais em comparação com a placentação humana.(AU)

Immunohistological Techniques.

Preeclampsia is associated with histological alterations in the placenta. These alterations can be described by means of histological techniques. More specifically, immunohistochemistry could be used to detect proteins, and these in turn may be used to identify a specific cell type, to differentiate it from other cell types and to detect the expression of some markers deregulated in preeclampsia.This chapter focuses on the detection of specific cellular and molecular markers that evidence the alterations in the human placenta in preeclampsia.

Alteration of syncytiotrophoblast mitochondria function and endothelial nitric oxide synthase expression in the placenta of rural residents.

The impact of environmental organophosphate (OP) pesticide exposure on respiratory complexes, enzymatic antioxidant defense activities, and oxidative damage markers in the syncytiotrophoblast and cytotrophoblast mitochondria was evaluated. Placental progesterone (PG) levels and endothelial nitric oxide synthase (eNOS) expression were studied. Samples from women non-exposed (control group-CG) and women living in a rural area (rural group-RG) were collected during pesticide spraying season (RG-SS) and non-spraying season (RG-NSS). In RG-SS, the exposure biomarker placental carboxylesterase decreased and syncytiotrophoblast cytochrome c oxidase activity increased, while 4-hydroxynonenal levels decreased. PG levels decreased in RG-SS and in the RG. Nitric oxide synthase expression decreased in RG, RG-SS and RG-NSS. No significant changes in mitochondrial antioxidant enzyme activities were found. These results suggest that the alteration of syncytiotrophoblast mitochondrial complex IV activity and steroidogenic function may be associated to pesticide exposure. Reduction in placental PG and eNOS expression may account for low newborn weight in RG.

Expression of human chorionic gonadotropin (ßhCG) in pre-eclamptic placenta / Expresión de gonadotropina coriónica humana (ßhCG) en placenta preeclámptica

This study aimed to assess association between preeclampsia with trophoblast cells and serum level of b-human chorionic gonadotropin (ß-hCG). Were compared 20 patients with preeclampsia and 20 control patients with respect to demographics, hematological parameters and the presence of trophopblast in placental samples. Patchy necrosis with loss of microvilli and gross thinning of the syncytium with distorted microvilli were seen in terminal villi of placentae of women with pre-eclampsia Syncytial cells at the molecular level crossings, especially at the level of ßhCG in conjunction with the changes in the preeclampsia was made on the histopathological changes to clarify the villi.

El objetivo fue evaluar la asociación entre la preeclampsia con células trofoblásticas y concentración sérica de la gonadotropina coriónica humana b (ß-hCG). Se compararon 20 pacientes con preeclampsia y 20 pacientes de control con respecto a datos demográficos, parámetros hematológicos y la presencia de trofoblasto en muestras de placenta. Se observaron áreas dispersadas de necrosis, con pérdida de microvellosidades y adelgazamiento del sincitio con microvellosidades distorsionadas en las vellosidades terminales de placentas en mujeres con células sincitiales preeclámticas a nivel molecular, junto a altos niveles de ßhCG asociados a los cambios generados por la preeclampsia sobre los parámetros histopatológicos.

Mitochondrial proteases act on STARD3 to activate progesterone synthesis in human syncytiotrophoblast.

BACKGROUND: STARD1 transports cholesterol into mitochondria of acutely regulated steroidogenic tissue. It has been suggested that STARD3 transports cholesterol in the human placenta, which does not express STARD1. STARD1 is proteolytically activated into a 30-kDa protein. However, the role of proteases in STARD3 modification in the human placenta has not been studied. METHODS: Progesterone determination and Western blot using anti-STARD3 antibodies showed that mitochondrial proteases cleave STARD3 into a 28-kDa fragment that stimulates progesterone synthesis in isolated syncytiotrophoblast mitochondria. Protease inhibitors decrease STARD3 transformation and steroidogenesis. RESULTS: STARD3 remained tightly bound to isolated syncytiotrophoblast mitochondria. Simultaneous to the increase in progesterone synthesis, STARD3 was proteolytically processed into four proteins, of which a 28-kDa protein was the most abundant. This protein stimulated mitochondrial progesterone production similarly to truncated-STARD3. Maximum levels of protease activity were observed at pH7.5 and were sensitive to 1,10-phenanthroline, which inhibited steroidogenesis and STARD3 proteolytic cleavage. Addition of 22(R)-hydroxycholesterol increased progesterone synthesis, even in the presence of 1,10-phenanthroline, suggesting that proteolytic products might be involved in mitochondrial cholesterol transport. CONCLUSION: Metalloproteases from human placental mitochondria are involved in steroidogenesis through the proteolytic activation of STARD3. 1,10-Phenanthroline inhibits STARD3 proteolytic cleavage. The 28-kDa protein and the amino terminal truncated-STARD3 stimulate steroidogenesis in a comparable rate, suggesting that both proteins share similar properties, probably the START domain that is involved in cholesterol binding. GENERAL SIGNIFICANCE: Mitochondrial proteases are involved in syncytiotrophoblast-cell steroidogenesis regulation. Understanding STARD3 activation and its role in progesterone synthesis is crucial to getting insight into its action mechanism in healthy and diseased syncytiotrophoblast cells.

PKA tightly bound to human placental mitochondria participates in steroidogenesis and is not modified by cAMP.

INTRODUCTION: Protein phosphorylation plays an important role in the modulation of steroidogenesis and it depends on the activation of different signaling cascades. Previous data showed that PKA activity is related to steroidogenesis in mitochondria from syncytiotrophoblast of human placenta (HPM). PKA localization and contribution in progesterone synthesis and protein phosphorylation of HPM was assessed in this work. METHODS: Placental mitochondria and submitochondrial fractions were used. Catalytic and regulatory PKA subunits were identified by Western blot. PKA activity was determined by the incorporation of (32)P into proteins in the presence or absence of specific inhibitors. The effect of PKA activators and inhibitors on steroidogenesis and protein phosphorylation in HPM was tested by radioimmunoassay and autoradiography. RESULTS: The PKAα catalytic subunit was distributed in all the submitochondrial fractions whereas ßII regulatory subunit was the main isoform observed in both the outer and inner membranes of HPM. PKA located in the inner membrane showed the highest activity. Progesterone synthesis and mitochondrial protein phosphorylation are modified by inhibitors of PKA catalytic subunit but are neither sensitive to inhibitors of the regulatory subunit nor to activators of the holoenzyme. DISCUSSION: The lack of response in the presence of PKA activators and inhibitors of the regulatory subunit suggests that the activation of intramitochondrial PKA cannot be prevented or further activated. CONCLUSIONS: The phosphorylating activity of PKA inside HPM could be an important component of the steroidogenesis transduction cascade, probably exerting its effects by direct phosphorylation of its substrates or by modulating other kinases and phosphatases.

Efecto de los retinoides en la diferenciación del trofoblasto humano en cultivo / Effect of retinoids on the differentiation of human trophoblast culture

Objetivo: El presente estudio analiza el efecto de diferentes retinoides sobre la función y diferenciación celular del trofoblasto humano en cultivo. Material y métodos: Se realizó un estudio experimental in vitro, en donde se utilizaron vellosidades coriónicas y células de citotrofoblasto obtenidas de placentas de término de embarazos normales, que fueron cultivadas y estimuladas con concentraciones fisiológicas diferenciales de retinol (ROL), ácido 9-cis retinoico (CRET) y ácido all-trans retinoico (TRET). Se analizaron los cambios en la actividad de la metaloproteinasa-9 (MMP-9) y la metaloproteinasa-2 (MMP-2), además de documentar las condiciones óptimas para el cultivo. En una segunda fase, se estimularon células de citotrofoblasto (CTB) aisladas y se documentaron los cambios morfológicos de formación de sincicio in vitro al ser estimulados con los retinoides mencionados. Resultados: Los explantes de placenta respondieron en forma dosis dependiente, en la actividad de MMP-2 y MMP-9 cuando fueron incubados en presencia de ROL y en menor proporción cuando se incubaron con CRET y TRET. El CTB aislado y estimulado con ROL y CRET formó sincicio en un lapso de 48 horas, en tanto que los estimulados con TRET lo hicieron hasta el 4o. día de incubación. Conclusiones: La placenta y el trofoblasto aislado responden a los diferentes retinoides. La respuesta del CTB al ROL indica que el CTB cuenta con la maquinaria metabólica para transformar el retinol en CRET y TRET que tienen actividad biológica. El efecto de estos compuestos se pudiera expresar en la función de implantación y en la placenta en desarrollo.

Objective: In the current study we investigate the effect of different retinoids on the function and cellular differentiation of human cytotrophoblast (CTB) cells in culture. Material and Methods: Experimental in vitro study that analyzed explants of placenta from term pregnancy were cultured and stimulated with different physiological concentrations of retinol (ROL), 9-cis retinoic acid (CRET) and all-trans retinoic acid (TRET). The optimal conditions of CTB culture and changes in metalloproteinase-9 (MMP9) and metalloproteinase-2 (MMP2) secretion stimulated by retinoids were analyzed by zymography. Isolated CTB cells were stimulated with retinoids and morphological changes of in vitro syncytio formation were observed by light microscopy. Results: The explants of placenta had a dose-dependent increment in the activity of MMP-2 and MMP-9 when they were incubated in presence of ROL and in less proportion when they were incubated with CRET and TRET. For isolated CTB cells, CRET stimulates the differentiation to syncytio within 48 h of incubation, whereas in the absence of TRET differentiation started after the fourth day incubation. Conclusions: The placenta and isolated CTB cells have the capacity to respond to ROL, this fact suggest the presence of a metabolic pathway necessary to transform retinol to the biological active retinoids like CRET and TRET. The effect of these retinoids might be expressed in the function of implantation and developing placenta.