In Vitro Culturing of Human Term Placental Explants.

The study of the human placenta has always been appealing, given the importance of this temporal organ capable of sustaining the beginning of life and development of a new human being within the womb. Culturing placental explants has been an easy and reliable method to study some placental morphological, biochemical, and physiological features for a very long time. Besides low time consumption, requirement of few resources, and wide versatility, the placental explant in vitro culture retains cell-cell interaction in a 3D structure resembling the in vivo setting, which is why it is the option of choice for many researchers in the field. This chapter will describe a simplified method for culturing explants from human term placentas.

Topographical Systematization of Human Placenta Model for Training in Microneurosurgery.

BACKGROUND: Neurosurgical training continuously seeks innovative methods to enhance the acquisition of essential technical skills for neurosurgeons worldwide. While various training models have been employed, few truly replicate real-life conditions optimally. Human placenta is a good model for neurosurgical microsurgery training due to its anatomic similarities to neurovascular structures. Placental vessels exhibit a branching pattern and caliber comparable with intracranial vessels, making them suitable for practicing microsurgical techniques. The study aims to delineate the anatomic zones of the placenta and propose a segmented training model, resulting in a reproducible, cost-effective, and realistic neurosurgical microsurgery training environment. METHODS: Twenty human placentas were meticulously prepared, injected with dyes, and categorized into zones on the basis of anatomic features. Measurements of placental vessels were recorded and compared with cerebral vessels. The placenta was divided into 4 quadrants to facilitate specific training techniques. RESULTS: Our results revealed varying vessel diameters across placental zones, closely resembling cerebral vessels. Different microsurgical techniques were applied to specific placental zones, thereby optimizing training scenarios. The applicability section described exercises such as membrane dissection, vessel skeletonization, aneurysm creation, vascular bypass, and tumor dissection within the placental model, providing detailed guidance on the zones suitable for each exercise. CONCLUSIONS: Human placenta serves as an effective microsurgical training model for neurosurgery, enhancing neurosurgeons' skills through anatomic segmentation. Integrating this model into training programs can significantly contribute to skill acquisition and improved surgical outcomes. Further research is warranted to refine and expand its utilization, complemented by clinical experiences and other simulation tools.

Modeling the human placental barrier to understand Toxoplasma gondii´s vertical transmission.

Toxoplasma gondii is a ubiquitous apicomplexan parasite that can infect virtually any warm-blooded animal. Acquired infection during pregnancy and the placental breach, is at the core of the most devastating consequences of toxoplasmosis. T. gondii can severely impact the pregnancy's outcome causing miscarriages, stillbirths, premature births, babies with hydrocephalus, microcephaly or intellectual disability, and other later onset neurological, ophthalmological or auditory diseases. To tackle T. gondii's vertical transmission, it is important to understand the mechanisms underlying host-parasite interactions at the maternal-fetal interface. Nonetheless, the complexity of the human placenta and the ethical concerns associated with its study, have narrowed the modeling of parasite vertical transmission to animal models, encompassing several unavoidable experimental limitations. Some of these difficulties have been overcome by the development of different human cell lines and a variety of primary cultures obtained from human placentas. These cellular models, though extremely valuable, have limited ability to recreate what happens in vivo. During the last decades, the development of new biomaterials and the increase in stem cell knowledge have led to the generation of more physiologically relevant in vitro models. These cell cultures incorporate new dimensions and cellular diversity, emerging as promising tools for unraveling the poorly understood T. gondii´s infection mechanisms during pregnancy. Herein, we review the state of the art of 2D and 3D cultures to approach the biology of T. gondii pertaining to vertical transmission, highlighting the challenges and experimental opportunities of these up-and-coming experimental platforms.

La seguridad de las placentas humanas utilizadas como materia prima farmacéutica desde el enfoque bioético / Human placenta safety used as pharmaceutical raw material based on the bioethical approach

Introducción: El uso de la placenta humana como materia prima farmacéutica se debe al contenido de sustancias biológicamente activas. El Centro de Histoterapia Placentaria -HISPLACEN- investiga, desarrolla, produce y comercializa productos de origen placentario. Objetivo: Analizar el proceso de aseguramiento y control para certificar la calidad de las placentas humanas como materia prima farmacéutica, desde el enfoque bioético en HISPLACEN. Material y Métodos: Se realizó un estudio descriptivo y analítico. Se definió como objeto de investigación: el proceso de aseguramiento y control para certificar la calidad de las placentas humanas en HISPLACEN, y su implantación en el período (2017-2021). Fueron revisados en el estudio, los documentos del Sistema de Gestión de la Calidad institucional, las regulaciones sobre Buenas Prácticas del CECMED, y la literatura científica sobre Bioética. Resultados: Las etapas del proceso de aseguramiento y control de la calidad para la certificación de las placentas se describieron y analizaron, destacándose la aplicación del enfoque bioético en su implantación. Se identificó la correspondencia de una ética humana y ambientalista de interrelación multidisciplinaria y entre los actores del ecosistema empresarial. Todo ello centrado en las dimensiones relativas a la ciencia y la tecnología para la fabricación de medicamentos. Conclusiones: Se evidenció el cumplimiento de los principios bioéticos en la certificación de las placentas humanas lo que potenció el desarrollo de un proceso tipificado por la integralidad, funcionalidad, eficacia y robustez. Este órgano biológico empleado como materia prima se abordó desde la multidimensionalidad -científica, tecnológica y bioética- del proceso descrito en sus tres etapas, lo que impacta positivamente, al focalizarse en un objetivo común: garantizar la salud y el bienestar de las personas, unido a la protección medioambiental.

Introduction: The use of human placenta as pharmaceutical raw material is due to the content of biologically active substances. The Placental Histotherapy Center (HISPLACEN) researches, develops, produces, and markets products of placental origin. Objective: to analyze the assurance and control process to certify the quality of the human placenta as a raw material in the biopharmaceutical industry, based on the bioethical approach. Material and Methods: A descriptive and analytical study was carried out. The process of quality assurance and control of the human placenta in HISPLACEN, and its implementation in the period 2017-2021 was defined as the research object. The documentation of the institutional Quality Management System, Good Practices regulations of the CECMED, and the scientific literature on Bioethics were reviewed. Results: The stages of the quality assurance and control of the placenta process and its derived products were described and analyzed, highlighting the application of the bioethical approach in its implementation. The correspondence of a human and environmental ethics of multidisciplinary interrelation and between the actors involved in the entrepreneurial ecosystem was identified. All this focused on the dimensions related to science and technology in the manufacture of medicines. Conclusions: The compliance of bioethical principles in the certification of human placentas was evidenced, which promoted the development of a process typified by comprehensiveness, functionality, efficacy, and robustness. This biological organ used as raw material was approached from multidimensionality -scientific, technological and bioethical - of the process focused on a common objective: guaranteeing human health and well-being, together with environmental protection.

ANNEXIN A1: Roles in Placenta, Cell Survival, and Nucleus.

The unbiased approaches of the last decade have enabled the collection of new data on the biology of annexin A1 (ANXA1) in a variety of scientific aspects, creating opportunities for new biomarkers and/or therapeutic purposes. ANXA1 is found in the plasma membrane, cytoplasm, and nucleus, being described at low levels in the nuclear and cytoplasmic compartments of placental cells related to gestational diabetic diseases, and its translocation from the cytoplasm to the nucleus has been associated with a response to DNA damage. The approaches presented here open pathways for reflection upon, and intrinsic clarification of, the modulating action of this protein in the response to genetic material damage, as well as its level of expression and cellular localization. The objective of this study is to arouse interest, with an emphasis on the mechanisms of nuclear translocation of ANXA1, which remain underexplored and may be beneficial in new inflammatory therapies.

The art of combining neuroanatomy and microsurgical skills in modern neurosurgery.

Neurosurgical training outside the operating room has become a priority for all neurosurgeons around the world. The exponential increase in the number of publications on training in neurosurgery reflects changes in the environment that future neurosurgeons are expected to work in. In modern practice, patients and medicolegal experts demand objective measures of competence and proficiency in the growing list of techniques available to treat complex neurosurgical conditions. It is important to ensure the myriad of training models available lead to tangible improvements in the operating room. While neuroanatomy textbooks and atlases are continually revised to teach the aspiring surgeon anatomy with a three-dimensional perspective, developing technical skills are integral to the pursuit of excellence in neurosurgery. Parapharsing William Osler, one of the fathers of neurosurgical training, without anatomical knowledge we are lost, but without the experience and skills from practice our journey is yet to begin. It is important to constantly aspire beyond competence to mastery, as we aim to deliver good outcomes for patients in an era of declining case volumes. In this article, we discuss, based on the literature, the most commonly used training models and how they are integrated into the treatment of some surgical brain conditions.

Lactic Acid Transport Mediated by Aquaporin-9: Implications on the Pathophysiology of Preeclampsia.

Aquaporin-9 (AQP9) expression is significantly increased in preeclamptic placentas. Since feto-maternal water transfer is not altered in preeclampsia, the main role of AQP9 in human placenta is unclear. Given that AQP9 is also a metabolite channel, we aimed to evaluate the participation of AQP9 in lactate transfer across the human placenta. Explants from normal term placentas were cultured in low glucose medium with or without L-lactic acid and in the presence and absence of AQP9 blockers (0.3 mM HgCl2 or 0.5 mM Phloretin). Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and lactate dehydrogenase release. Apoptotic indexes were analyzed by Bax/Bcl-2 ratio and Terminal Deoxynucleotidyltransferase-Mediated dUTP Nick-End Labeling assay. Heavy/large and light/small mitochondrial subpopulations were obtained by differential centrifugation, and AQP9 expression was detected by Western blot. We found that apoptosis was induced when placental explants were cultured in low glucose medium while the addition of L-lactic acid prevented cell death. In this condition, AQP9 blocking increased the apoptotic indexes. We also confirmed the presence of two mitochondrial subpopulations which exhibit different morphologic and metabolic states. Western blot revealed AQP9 expression only in the heavy/large mitochondrial subpopulation. This is the first report that shows that AQP9 is expressed in the heavy/large mitochondrial subpopulation of trophoblasts. Thus, AQP9 may mediate not only the lactic acid entrance into the cytosol but also into the mitochondria. Consequently, its lack of functionality in preeclamptic placentas may impair lactic acid utilization by the placenta, adversely affecting the survival of the trophoblast cells and enhancing the systemic endothelial dysfunction.

Central role of the placenta during viral infection: Immuno-competences and miRNA defensive responses.

Pregnancy is a unique immunological condition in which an "immune-diplomatic" dialogue between trophoblasts and maternal immune cells is established to protect the fetus from rejection, to create a privileged environment in the uterus and to simultaneously be alert to any infectious challenge. The maternal-placental-fetal interface (MPFI) performs an essential role in this immunological defense. In this review, we will address the MPFI as an active immuno-mechanical barrier that protects against viral infections. We will describe the main viral infections affecting the placenta and trophoblasts and present their structure, mechanisms of immunocompetence and defensive responses to viral infections in pregnancy. In particular, we will analyze infection routes in the placenta and trophoblasts and the maternal-fetal outcomes in both. Finally, we will focus on the cellular targets of the antiviral microRNAs from the C19MC cluster, and their effects at both the intra- and extracellular level.

Modelo integral de entrenamiento microquirúrgico y neuroendovascular con placenta humana / Comprehensive microsurgical and neuroendovascular training model with human placenta

Introducción: La neurocirugía vascular, tanto la microquirúrgica como endovascular, ha progresado significativamente en el tratamiento de la patología cerebrovascular. Sin embargo, en una considerable proporción de casos este tipo de patología no puede ser resuelta definitivamente mediante un único abordaje. Por lo cual consideramos que el neurocirujano en formación debe capacitarse con ambas técnicas.Se describe un modelo de entrenamiento en microcirugía y en nociones básicas del material y técnica neuroendovascular, utilizando placenta humana y recursos de baja complejidad. Material y método: Se utilizaron 20 placentas humanas, instrumental y sutura de uso habitual en microcirugía, microscopio quirúrgico Newton®XX1, material para procedimientos endovasculares; equipo de radioscopia (arco en C Phillips BV Pulsera®), un cráneo óseo y un cabezal de fijación tipo Sugita® adaptado a su uso en laboratorio. Los ejercicios consistieron en: 1. Disección y exposición de los vasos arteriales y venosos del corion; 2. Anastomosis término-terminal, termino-lateral y latero-lateral; 3. Generación de aneurismas laterales, de bifurcación o trifurcación; 4. Creación de bypass extra-intracraneano; 5. Clipado de los aneurismas en superficie y dentro del cráneo; 6. Control angiográfico pre y post clipado. 7. Embolización con coils de los aneurismas experimentales y de vasos placentarios con partículas de Spongostan®. Resultados: Aunque los vasos tienen una estructura y consistencia diferentes a los habituales para el neurocirujano, la placenta ofrece una variabilidad de calibres y formatos donde practicar los diferentes ejercicios. Conclusión: El entrenamiento en técnicas microquirúrgicas y neurointervencionistas puede ser realizado en modelos placentarios de simulación, que permiten el desarrollo háptico progresivo previo a la realización de un procedimiento in vivo.

Objective: Describe a training model in microsurgery and neuroendovascular surgery, using human placenta and low complexity resources. Material and methods: 20 human placentas, instruments and sutures were used in microsurgery, Newton XX1 surgical microscope, material for endovascular procedures; radioscopy equipment (C-arch Phillips BV Pulsera), a bony skull and a Sugita head adapted for laboratory use. The exercises consisted of: 1. Dissection and exposure of the arterial and venous vessels of the chorion; 2. End-to-end, end-to-side, side-to-side anastomosis; 3. Generation of lateral, bifurcation or trifurcation aneurysms; 4. Creation of extra-intracranial bypass; 5. Clipping of aneurysms on the surface and inside the skull; 6. Pre and post clipping angiographic control. 7. Coil embolization of experimental aneurysms and placental vessels embolization with spongostan particles. Results: Although the vessels have a different structure and consistency than usual for the neurosurgeon, the placenta offers a variability of sizes and formats to practice the different exercises. Conclusion: Training in microsurgical and neurointerventionist techniques can be carried out in placental models, which allow progressive haptic development prior to performing an in vivo procedure.

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.

Lección MOODLE de anatomía e histología de la placenta humana / MOODLE lesson of the anatomy and histology of the human placenta

RESUMEN: La falta de muestras biológicas humanas existentes, debido principalmente a las limitaciones ético-morales relacionadas con su obtención, ponen en relieve la necesidad de buscar otras alternativas de enseñanza y aprendizaje de las ciencias morfológicas. En este sentido, la implementación de lecciones a través de la plataforma MOODLE proporciona la oportunidad al estudiante de interactuar en un entorno que simula una situación de aprendizaje propio del laboratorio tradicional. El objetivo del presente trabajo fue generar una lección MOODLE sobre la anatomía e histología placentaria humana, como complemento a la clase teórica presencial, para estudiantes de la carrera de Obstetricia y Puericultura. Para tal cometido, se realizó búsqueda de información, imágenes y recursos TIC en bibliotecas e internet. Paralelamente, se llevó a cabo un proceso de captura fotográfica de muestras histológicas de placenta, así como también la grabación de un alumbramiento. Posteriormente, se procedió a la articulación y montaje de las actividades en la plataforma MOODLE con un enfoque constructivista. Además, se elaboró una encuesta de satisfacción, la cual fue validada por 3 expertos. La muestra estuvo constituida por 137 estudiantes de la carrera de Obstetricia. Se confeccionó un laboratorio virtual MOODLE de anatomía e histología de la placenta humana, el cual esta constituido por múltiples actividades con orientación clínica, las cuales permiten autoevaluarse. El laboratorio virtual nos ha ayudado ha subsanar la carencia de muestras humanas y los resultados de la encuesta de satisfacción aplicada a los estudiantes señalan una valoración positiva de la iniciativa.

SUMMARY: The lack of existing human biological samples, mainly due to the ethical-moral restrictions related to obtaining these, highlights the need to search for other teaching and learning alternatives in morphological science. In this sense, the implementation of lessons by means of the MOODLE platform provides the students with the opportunity to interact in a setting that simulates a learning situation that belongs to traditional laboratories. The purpose of this work was to generate a MOODLE lesson on the anatomy and histology of the human placenta, as a complement of the traditional theoretical classroom for students of Obstetrics. To that end, TIC information, images, and resources were sought in libraries and in the Internet, and at the same time a set of histological photographs of placenta samples was made, as well as a video recording of a placental delivery. Later, the coordination and set up of activities was made in the MOODLE platform with a constructivist approach. Furthermore, a satisfaction survey was prepared which was validated by three experts. The total sample consisted of 137 students in the 2th year of obstetrics. A virtual MOODLE laboratory of the anatomy and histology of the human placenta was made, which is constituted by multiple activities with a clinical orientation that allow self-evaluation. The virtual laboratory has helped overcome the lack of human samples, and results of the satisfaction survey applied to the students indicate a positive evaluation of this initiative.

Oxygen regulation of aquaporin-4 in human placenta.

RESEARCH QUESTION: We recently reported that blocking of placental aquaporins (AQP) abrogates the apoptotic response of the trophoblast. As trophoblast apoptosis is exacerbated in pre-eclampsia, we hypothesized that changes in AQP in these placentae may trigger programmed cell death. We analysed AQP4 expression in pre-eclamptic placentae and its regulation by oxygen tension. DESIGN: AQP4 expression was studied in placentae from non-pathological and pre-eclamptic pregnancies by reverse transcription polymerase chain reaction (RT-PCR), Western blot, immunofluorescence and immunohistochemistry. Explants from non-pathological placentae were cultured in normoxia, hypoxia, hypoxia-reoxygenation and CoCl2. AQP4 expression was investigated by RT-PCR and Western blot. Hypoxia responsive elements sites on AQP4 promotor were investigated by in-silico analysis. AQP4 degradation was studied in the presence of proteosomal and lysosomal inhibitors. RESULTS: AQP4 protein expression was weakly detectable in pre-eclamptic placentae, but its mRNA was elevated compared with non-pathological placentae. In non-pathological explants cultured in hypoxia, AQP4 mRNA and protein were increased compared with placentae cultured in ambient oxygen but decreased after reoxygenation. Incubation with CoCl2, that stabilizes hypoxia inducible factor (HIF)-1α, also increased AQP4 levels. In-silico analysis showed three putative binding sites for HIF-1α in AQP4 promotor. CONCLUSIONS: Oxygen may regulate AQP4 expression in human placenta, possibly through HIF-1α. Therefore, the decrease in AQP4 throughout pregnancy, previously reported, is consistent with changes in HIF-1α, and suggests that AQP4 might have a crucial role during placentation. Therefore, the abnormal expression of AQP4 may be involved in the cause of pre-eclampsia, but it does not seem to take part in the apoptotic events.

New Insights Into the Role of Placental Aquaporins and the Pathogenesis of Preeclampsia.

Accumulated evidence suggests that an abnormal placentation and an altered expression of a variety of trophoblast transporters are associated to preeclampsia. In this regard, an abnormal expression of AQP3 and AQP9 was reported in these placentas. Recent data suggests that placental AQPs are not only water channel proteins and that may participate in relevant processes required for a normal placental development, such as cell migration and apoptosis. Recently we reported that a normal expression of AQP3 is required for the migration of extravillous trophoblast (EVT) cells. Thus, alterations in this protein might lead to an insufficient transformation of the maternal spiral arteries resulting in fluctuations of oxygen tension, a potent stimulus for oxidative damage and trophoblast apoptosis. In this context, the increase of oxygen and nitrogen reactive species could nitrate AQP9, producing the accumulation of a non-functional protein affecting the survival of the villous trophoblast (VT). This may trigger the exacerbated release of apoptotic VT fragments into maternal circulation producing the systemic endothelial dysfunction underlying the maternal syndrome. Therefore, our hypothesis is that the alteration in the expression of placental AQPs observed at the end of gestation may take place during the trophoblast stem cell differentiation, disturbing both EVT and VT cells development, or during the VT differentiation and turnover. In both situations, VT is affected and at last the maternal vascular system is activated leading to the clinical manifestations of preeclampsia.

Differential infectivity of two Trypanosoma cruzi strains in placental cells and tissue.

Congenital Chagas disease, caused by Trypanosoma cruzi (T. cruzi), has become epidemiologically relevant. The probability of congenital transmission depends on the maternal and developing fetal/newborn immune responses, placental factors and importantly, the virulence of the parasite. It has been proposed, that different genotypes of T. cruzi and their associated pathogenicity, virulence and tissue tropism may play an important role in congenital infection. Since there is no laboratory or animal model that recapitulates the complexities of vertical transmission in humans, here we studied parasite infectivity in human placental explants (HPE) as well as in the human trophoblast-derived cell line BeWo of the Y(DTU II) and the VD (TcVI) T. cruzi strains; the latter was isolated from a human case of congenital infection. Our results show that the VD strain is more infective and pathogenic than the Y strain, as demonstrated by qPCR and cell counting as well as by histopathological analysis. The present study constitutes the first approach to study the relationship between parasite two parasite strains from different genotypes and the infection efficiency in human placenta.

Highly expressed placental miRNAs control key biological processes in human cancer cell lines.

Despite being a healthy tissue, the constituent cells of the placenta, share similar characteristics with tumor cells, such as increased cell growth, migration, and invasion. However, while these processes are stochastic and uncontrolled in cancer cells, in placenta they are precisely controlled. Since miRNAs have been reported to regulate genes that control the molecular mechanisms necessary for the development of both human placenta and cancer, we addressed for miRNAs highly expressed in the placenta that could be involved in tumorigenesis. Here, we assessed the miRNA profile in placenta samples using microarray analysis. The results showed that miR-451 and miR-720, highly expressed placental miRNAs, presented very low or undetectable expression in cancer cell lines compared to the normal placenta and healthy tissues. Additionally, transfection of miR-451 or miR-720 mimics in choriocarcinoma cell line (JEG3) and colorectal adenocarcinoma cell line (HT-29) resulted in impaired cell proliferation, decreased cell migration and invasion and reduced ability of colony formation. These findings provide evidence that placenta may work as an alternative model to identify novel miRNAs involved in pathways controlling tumorigenesis.

Intracellular and extracellular pH dynamics in the human placenta from diabetes mellitus.

The placenta is a vital organ whose function in diseases of pregnancy is altered, resulting in an abnormal supply of nutrients to the foetus. The lack of placental vasculature homeostasis regulation causes endothelial dysfunction and altered vascular reactivity. The proper distribution of acid- (protons (H(+))) and base-equivalents through the placenta is essential to achieve physiological homeostasis. Several membrane transport mechanisms that control H(+) distribution between the extracellular and intracellular spaces are expressed in the human placenta vascular endothelium and syncytiotrophoblast, including sodium (Na(+))/H(+) exchangers (NHEs). One member of the NHEs family is NHE isoform 1 (NHE1), whose activity results in an alkaline intracellular pH (high intracellular pH (pHi)) and an acidic extracellular pH (pHo). Increased NHE1 expression, maximal transport activity, and turnover are reported in human syncytiotrophoblasts and lymphocytes from patients with diabetes mellitus type I (DMT1), and a positive correlation between NHEs activity and plasma factors, such as that between thrombin and platelet factor 3, has been reported in diabetes mellitus type II (DMT2). However, gestational diabetes mellitus (GDM) could result in a higher sensitivity of the human placenta to acidic pHo. We summarized the findings on pHi and pHo modulation in the human placenta with an emphasis on pregnancies in which the mother diagnosed with diabetes mellitus. A potential role of NHEs, particularly NHE1, is proposed regarding placental dysfunction in DMT1, DMT2, and GDM.

Placental programmed cell death: insights into the role of aquaporins.

STUDY HYPOTHESIS: Are the placental aquaporins (AQPs) involved in the apoptosis of human trophoblast? STUDY FINDING: The general blocking of placental AQPs with HgCl2 and, in particular, the blocking of AQP3 activity with CuSO4 abrogated the apoptotic events of human trophoblast cells. WHAT IS KNOWN ALREADY: Although apoptosis of trophoblast cells is a natural event involved in the normal development of the placenta, it is exacerbated in pathological processes, such as pre-eclampsia, where an abnormal expression and functionality of placental AQPs occur without alterations in the feto-maternal water flux. Furthermore, fluctuations in O2 tension are proposed to be a potent inducer of placental apoptotic changes and, in explants exposed to hypoxia/reoxygenation (H/R), transcellular water transport mediated by AQPs was undetectable. This suggests that AQPs might be involved in processes other than water transport, such as apoptosis. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Explants from normal term placentas were maintained in culture under conditions of normoxia, hypoxia and H/R. Cell viability was determined by assessing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide incorporation. For the general or specific inhibition of AQPs, 0.3 mM HgCl2, 5 mM CuSO4, 0.3 mM tetraethylammonium chloride (TEA) or 0.5 mM phloretin were added to the culture medium before explants were exposed to each treatment. Oxidative stress parameters and apoptotic indexes were evaluated in the presence or absence of AQPs blockers. AQP3 expression was confirmed by western blot and immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: First, we observed that in H/R treatments cell viability decreased by 20.16 ± 5.73% compared with those explants cultured in normoxia (P = 0.009; n = 7). Hypoxia did not modify cell viability significantly. Both hypoxia and H/R conditions induced oxidative stress. Spontaneous chemiluminescence and thiobarbituric acid reactive substance levels were significantly increased in explants exposed to hypoxia (n = 6 per group, P = 0.0316 and P = 0.0009, respectively) and H/R conditions (n = 6 per group, P = 0.0281 and P = 0.0001, respectively) compared with those cultured in normoxia. Regarding apoptosis, H/R was a more potent inducer of trophoblast apoptosis than hypoxia alone. Bax expression and the number of apoptotic nuclei were significantly higher in explants cultured in H/R compared with normoxia and hypoxia conditions (n = 12, P = 0.0135 and P = 0.001, respectively). DNA fragmentation was only observed in H/R and, compared with normoxia and hypoxia, the activity of caspase-3 was highest in explants cultured in H/R (n = 12, P = 0.0001). In explants exposed to H/R, steric blocking of AQP activity with HgCl2 showed that DNA degradation was undetectable (n = 12, P = 0.001). Bax expression and caspase-3 activity were drastically reduced (n = 12, P = 0.0146 and P = 0.0001, respectively) compared with explants cultured in H/R but not treated with HgCl2. Similar results were observed in explants exposed to H/R when we blocked AQP3 activity with CuSO4. DNA degradation was undetectable and the number of apoptotic nuclei and caspase-3 activity were significantly decreased compared with explants cultured in H/R but not treated with CuSO4 (n = 12, P = 0.001 and P = 0.0001, respectively). However, TEA and phloretin treatments, to block AQP1/4 or AQP9, respectively, failed in abrogate apoptosis. In addition, we confirmed the expression and localization of AQP3 in explants exposed to H/R. LIMITATIONS, REASONS FOR CAUTION: Our studies are limited by the number of experimental conditions tested, which do not fully capture the variability in oxygen levels, duration of exposure and alternating patterns of oxygen seen in vivo. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest that any alteration in placental AQP expression might disturb the equilibrium of the normal apoptotic events and may be an underlying cause in the pathophysiology of placental gestational disorders such as pre-eclampsia. Furthermore, the dysregulation of placental AQPs may be one of the crucial factors in triggering the clinical manifestations of pre-eclampsia. LARGE SCALE DATA: n/a. STUDY FUNDING AND COMPETING INTERESTS: This study was supported by UBACyT 20020090200025 and 20020110200207 grants and PIP-CONICET 11220110100561 grant, and the authors have no conflict of interest to declare.

Trypanosoma cruzi induces trophoblast differentiation: a potential local antiparasitic mechanism of the human placenta?

INTRODUCTION: The congenital transmission of Trypanosoma cruzi (T. cruzi) is responsible for one-third of new Chagas disease cases each year. During congenital transmission, the parasite breaks down the placental barrier formed by the trophoblast, basal laminae and villous stroma. The observation that only 5% of infected mothers transmit the parasite to the fetus implies that the placenta may impair parasite transmission. The trophoblast undergoes continuous epithelial turnover, which is considered part of innate immunity. Therefore, we propose that T. cruzi induces differentiation in the trophoblast as part of a local antiparasitic mechanism of the placenta. METHODS: We analyzed ß-human chorionic gonadotropin (ß-hCG) and syncytin protein expression in HPCVE and BeWo cells using immunofluorescence and western blotting. Additionally, ß-hCG secretion into the culture medium was measured by ELISA. We assessed the differentiation of trophoblastic cells in BeWo cells using the two-color fusion assay and by determining desmoplakin re-distribution. RESULTS: T. cruzi trypomastigotes induce ß-hCG secretion and protein expression as well as syncytin protein expression in HPCVE and BeWo cells. Additionally, the parasite induces the trophoblast fusion of BeWo cells. DISCUSSION: T. cruzi induces differentiation of the trophoblast, which may contribute to increase the trophoblast turnover. The turnover could be a component of local antiparasitic mechanisms in the human placenta.

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.