Introduction of an efficient method for placenta decellularization with high potential to preserve ultrastructure and support cell attachment.

The placenta, as a large discarded tissue and rich in extracellular matrix (ECM), is an excellent candidate for biological scaffolds in reconstructive medicine. Considering the importance of ECM structure in cell fate, the aim of this study was to achieve human placenta decellularization protocol that preserve the structure of scaffolds. Thus, human placenta was decellularized by four protocols and decellularization efficacy was compared by hematoxylin and eosin (H&E), 4',6-diamidino-2-phenylindole (DAPI) staining, and DNA measurement. Decellularized placenta structure preservation was assessed by Masson's trichrome staining, scanning electron microscopy (SEM), and immunofluorescence (IF) for collagen I, IV, and fibronectin. Finally, liquid displacement measured scaffolds' porosity. After culturing menstrual blood-derived stem cells (MenSCs) on placenta scaffolds, cell adhesion was investigated by SEM imaging, and cell viability and proliferation were assessed by MTT assay. According to H&E and DAPI staining, only protocols 1 and 3 could completely remove cells from the scaffolds. DNA measurements confirmed a significant reduction in the genetic material of decellularized scaffolds compared to native placenta. According to Masson's trichrome, IF, and SEM imaging, scaffold structure is better preserved in P3 than P1 protocol. Liquid displacement showed higher porosity of P3 scaffold than P1. SEM imaging confirmed cells adhesion to the decellularized placenta, and the attached cells showed good viability and maintained their proliferative capacity, indicating the suitability of the scaffolds for cell growth. Results introduced an optimized protocol for placenta decellularization that preserves the scaffold structure and supports cell adhesion and proliferation.

Comparative analysis of trophoblasts and angiogenesis in human placental compartments / Análisis comparativo de trofoblastos y angiogénesis en compartimentos placentarios humanos

SUMMARY: Trophoblasts perform different functions depending on their location. This study aimed to obtain structural clues about the functions of villous and extravillous trophoblasts by using light and electron microscopy. Term placenta samples were obtained from 10 healthy pregnant women following cesarean sections. Frozen sections were stained with hematoxylin-eosin, semi- thin sections were stained with toluidine blue and examined with a light microscope, while thin sections were contrasted using uranyl acetate-lead citrate and evaluated under an electron microscope. Fine structural features of villous trophoblasts overlapped some villous stromal cells. In addition to the usual appearance of mature capillaries in villous stroma, we demonstrated and reported maturational stages of angiogenetic sprouts in term placenta. Extravillous trophoblasts were classified according to their location: fibrinoid, chorion, trophoblastic, column, maternal vascular endothelium, or decidua. All of these trophoblasts shared some ultrastructural features but also were distinct from each other. In decidua, it was noted that the endothelial lining of some vessels was invaded by a few endovascular trophoblasts with irregular microvilli. These cells shared some ultrastructural properties with both villous trophoblasts and stromal cells. Examination showed that angiogenesis was still present in term placentas and that trophoblasts, endothelial and stromal cells have very similar properties ultrastructurally, suggesting they represent transformational forms.

RESUMEN: Los trofoblastos dependiendo de su ubicación realizan diferentes funciones. Este estudio tuvo como objetivo obtener pistas estructurales sobre las funciones de los trofoblastos vellosos y extravellosos mediante el uso de microscopía óptica y electrónica. Se obtuvieron muestras de placenta a término de 10 mujeres embarazadas sanas después de cesáreas. Las secciones congeladas se tiñeron con hematoxilina-eosina, las secciones semidelgadas se tiñeron con azul de toluidina y se examinaron con un microscopio óptico, mientras que las secciones delgadas se contrastaron con acetato de uranilo-citrato de plomo y se evaluaron con un microscopio electrónico. Las finas características estructurales de los trofoblastos vellosos se superponen a algunas células estromales vellosas. Además de la apariencia habitual de capilares maduros en el estroma velloso, demostramos e informamos etapas de maduración de brotes angiogenéticos en la placenta a término. Los trofoblastos extravellosos se clasificaron según su localización: fibrinoide, corion, trofoblástico, columna, endotelio vascular materno o decidua. Todos estos trofoblastos compartían algunas características ultraestructurales, pero también eran distintos entre sí. En decidua se observó que el revestimiento endotelial de algunos vasos estaba invadido por unos pocos trofoblastos endovasculares con microvellosidades irregulares. Estas células compartían algunas propiedades ultraestructurales tanto con los trofoblastos vellosos como con las células del estroma. El examen mostró que la angiogénesis todavía estaba presente en las placentas a término y que los trofoblastos, las células endoteliales y estromales tienen propiedades ultraestructurales muy similares, lo que sugiere que representan formas de transformación.

Suppression of uterine and placental ferroptosis by N-acetylcysteine in a rat model of polycystic ovary syndrome.

The mechanisms that link hyperandrogenism and insulin (INS) resistance (HAIR) to the increased miscarriage rate in women with polycystic ovary syndrome (PCOS) remain elusive. Previous studies demonstrate that increased uterine and placental ferroptosis is associated with oxidative stress-induced fetal loss in a pre-clinical PCOS-like rat model. Here, we investigated the efficacy and molecular mechanism of action of the antioxidant N-acetylcysteine (NAC) in reversing gravid uterine and placental ferroptosis in pregnant rats exposed to 5α-dihydrotestosterone (DHT) and INS. Molecular and histological analyses showed that NAC attenuated DHT and INS-induced uterine ferroptosis, including dose-dependent increases in anti-ferroptosis gene content. Changes in other molecular factors after NAC treatment were also observed in the placenta exposed to DHT and INS, such as increased glutathione peroxidase 4 protein level. Furthermore, increased apoptosis-inducing factor mitochondria-associated 2 mRNA expression was seen in the placenta but not in the uterus. Additionally, NAC was not sufficient to rescue DHT + INS-induced mitochondria-morphological abnormalities in the uterus, whereas the same treatment partially reversed such abnormalities in the placenta. Finally, we demonstrated that NAC selectively normalized uterine leukemia inhibitory factor, osteopontin/secreted phosphoprotein 1, progesterone receptor, homeobox A11 mRNA expression and placental estrogen-related receptor beta and trophoblast-specific protein alpha mRNA expression. Collectively, our data provide insight into how NAC exerts beneficial effects on differentially attenuating gravid uterine and placental ferroptosis in a PCOS-like rat model with fetal loss. These results indicate that exogenous administration of NAC represents a potential therapeutic strategy in the treatment of HAIR-induced uterine and placental dysfunction.

Placental extracellular vesicles retain biological activity after short-term storage (14 days) at 4 °C or room temperature.

INTRODUCTION: To investigate the role of placental extracellular vesicles (EVs), especially in pathological pregnancy, the use of freshly isolated EVs is often limited due to the sporadic and unpredictable availability of placental samples. Therefore, it is important to understand and use optimised storage conditions for placental EVs. In this study, we investigated different conditions for the short-term storage of placental micro- and nano-EVs and examined their biological activity. METHODS: Placental EVs were collected from first trimester placentae. EVs were suspended in PBS and aliquoted, and then stored for up to 14 days at room temperature, 4 °C or -20 °C. Total protein and DNA levels were measured at various time points. The ability of stored placental EVs to alter endothelial cell activation was quantified by monocyte adhesion assays. RESULTS: There was no difference in the concentration of placental micro- or nano-EVs between each time point, when stored at either room temperature or 4 °C. However, there was a significant loss of placental EVs after storage at -20 °C. There was no difference in protein or DNA levels of placental EVs when stored at either room temperature or 4 °C. Biological activity of placental EVs was retained for up to 14 days at either room temperature or 4 °C measured by monocyte adhesion assays. DISCUSSION: We have shown that placental micro- and nano-EVs are stable and retain biological activities following storage in PBS or media for 14 days at either room temperature or 4 °C.

ZIKV Disrupts Placental Ultrastructure and Drug Transporter Expression in Mice.

Congenital Zika virus (ZIKV) infection can induce fetal brain abnormalities. Here, we investigated whether maternal ZIKV infection affects placental physiology and metabolic transport potential and impacts the fetal outcome, regardless of viral presence in the fetus at term. Low (103 PFU-ZIKVPE243; low ZIKV) and high (5x107 PFU-ZIKVPE243; high ZIKV) virus titers were injected into immunocompetent (ICompetent C57BL/6) and immunocompromised (ICompromised A129) mice at gestational day (GD) 12.5 for tissue collection at GD18.5 (term). High ZIKV elicited fetal death rates of 66% and 100%, whereas low ZIKV induced fetal death rates of 0% and 60% in C57BL/6 and A129 dams, respectively. All surviving fetuses exhibited intrauterine growth restriction (IUGR) and decreased placental efficiency. High-ZIKV infection in C57BL/6 and A129 mice resulted in virus detection in maternal spleens and placenta, but only A129 fetuses presented virus RNA in the brain. Nevertheless, pregnancies in both strains produced fetuses with decreased head sizes (p<0.05). Low-ZIKV-A129 dams had higher IL-6 and CXCL1 levels (p<0.05), and their placentas showed increased CCL-2 and CXCL-1 contents (p<0.05). In contrast, low-ZIKV-C57BL/6 dams had an elevated CCL2 serum level and increased type I and II IFN expression in the placenta. Notably, less abundant microvilli and mitochondrial degeneration were evidenced in the placental labyrinth zone (Lz) of ICompromised and high-ZIKV-ICompetent mice but not in low-ZIKV-C57BL/6 mice. In addition, decreased placental expression of the drug transporters P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) and the lipid transporter Abca1 was detected in all ZIKV-infected groups, but Bcrp and Abca1 were only reduced in ICompromised and high-ZIKV ICompetent mice. Our data indicate that gestational ZIKV infection triggers specific proinflammatory responses and affects placental turnover and transporter expression in a manner dependent on virus concentration and maternal immune status. Placental damage may impair proper fetal-maternal exchange function and fetal growth/survival, likely contributing to congenital Zika syndrome.

The Use of Atomic Force Microscopy in Comprehensive Assessment of the "Mother-Placenta-Fetus" System in Obstetric and Endocrine Pathology during Pregnancy.

Atomic force microscopy is not very popular in practical health care, therefore, its potential is not studied enough, for example, in obstetrics when studying the "mother-placenta-fetus" system. Our study summarizes the possibilities of using atomic force microscopy for detection of various circulatory disorders and vascular changes at the microscopic level in the uterus (endometrium and myometrium), placenta, and umbilical cord in the main variants of obstetric and endocrine pathology. For instance, in the case of endocrine pathologies, changes in the form of stasis, sludge, diapedesis, ischemia, destruction and separation of endotheliocytes in villous blood vessels were found in the mother. The oxygen content in erythrocytes also naturally decreased in pathologies; poikilo- and anisocytosis were observed.

Comparison of methods for separating fluorescently labelled placental extracellular vesicles from free stain.

The field of extracellular vesicles (EVs) is relatively new and the methods for EV isolation and quantification are still maturing. For example, there is no consensus on how to separate free stain from labelled EVs. Here we report a comparison of the recovery of labelled EVs following separation from free stain using ultracentrifugation, diafiltration with different devices and a charged size exclusion chromatography column. Of the methods we tested, the charged size exclusion column provided the greatest recovery of labelled EVs.

Confined placental mosaicism involving multiple de novo copy number variants associated with fetal growth restriction: A case report.

The presence of multiple large (>1 Mb) copy number variants (CNVs) in non-malignant tissue is rare in human genetics. We present a liveborn male with a birth weight below the first percentile associated with placental mosaicism involving eight 2.4-3.9 Mb de novo duplications. We found that the duplications likely co-localized to the same cells, were mosaic in the placenta, and impacted maternal and paternal chromosomes. In addition, 27.4 Mb and 240 genes were duplicated in affected cells, including candidate placental genes KISS1 and REN. We ruled out involvement of homologous recombination-based mechanisms or an altered epigenome in generating the CNVs. This case highlights the diversity of genetic abnormalities in the human placenta and the gaps in our knowledge of how such errors arise.

Maternal platelets pass interstices of trophoblast columns and are not activated by HLA-G in early human pregnancy.

In early human gestation, maternal arterial blood flow into the intervillous space of the developing placenta is obstructed by invaded trophoblasts, which form cellular plugs in uterine spiral arteries. These trophoblast plugs have recently been described to be loosely cohesive with clear capillary-sized channels into the intervillous space by 7 weeks of gestation. Here, we analysed localisation of maternal platelets at the maternal-foetal interface of human first trimester pregnancy, and tested the hypothesis whether HLA-G, which is primarily expressed by extravillous trophoblasts, affects aggregation and adhesion of isolated platelets. Immunohistochemistry of first trimester placental sections localised maternal platelets in vessel-like channels and adjacent intercellular gaps of extravillous trophoblasts in distal parts of columns. Furthermore, this localisation was confirmed by transmission electron microscopy. Neither co-incubation of HLA-G overexpressing JAR cells with isolated platelets, nor incubation with cell-derived soluble HLA-G or recombinant HLA-G affected platelet adhesion and aggregation. Our study suggests that maternal platelets flow through vessel-like channels of distal trophoblast columns and spread into adjacent lateral intercellular gaps, where platelet-derived factors could contribute to trophoblast differentiation into the invasive phenotype.

Trophoblast inclusions in the human placenta: Identification, characterization, quantification, and interrelations of subtypes.

We sought to examine placentas enriched for trophoblast inclusions (TIs) in order to characterize, quantify, and examine the interrelations between subtypes of TIs to better understand their underlying biology. We examined a cohort of 600 placentas from deliveries between 200 and 430 weeks of gestation. Forty-five percent of the placentas had at least one TI in the two slides examined. Four percent of the placentas had 10 or more TIs and two placentas had more than 70 TIs. Four distinct TI subtypes were observed: inclusionoids (early forming inclusions), inclusions, calcified inclusions, and calcified bodies. We suggest this reflects a developmental trajectory of TI maturation, the timing of which might be useful when comparing TI expression to clinical outcomes.

Mitochondrial transformations in the aging human placenta.

Mitochondria play a key role in homeostasis and are central to one of the leading hypotheses of aging, the free radical theory. Mitochondria function as a reticulated network, constantly adapting to the cellular environment through fusion (joining), biogenesis (formation of new mitochondria), and fission (separation). This adaptive response is particularly important in response to oxidative stress, cellular damage, and aging, when mitochondria are selectively removed through mitophagy, a mitochondrial equivalent of autophagy. During this complex process, mitochondria influence surrounding cell biology and organelles through the release of signaling molecules. Given that the human placenta is a unique organ having a transient and somewhat defined life span of ∼280 days, any adaption or dysfunction associated with mitochondrial physiology as a result of aging will have a dramatic impact on the health and function of both the placenta and the fetus. Additionally, a defective placenta during gestation, resulting in reduced fetal growth, has been shown to influence the development of chronic disease in later life. In this review we focus on the mitochondrial adaptions and transformations that accompany gestational length and share similarities with age-related diseases. In addition, we discuss the role of such changes in regulating placental function throughout gestation, the etiology of gestational complications, and the development of chronic diseases later in life.

Placental scaffolds have the ability to support adipose-derived cells differentiation into osteogenic and chondrogenic lineages.

Prudent choices of cell sources and biomaterials, as well as meticulous cultivation of the tissue microenvironment, are essential to improving outcomes of tissue engineering treatments. With the goal of providing a high-quality alternative for bone and cartilage tissue engineering, we investigated the capability of bovine placental scaffolds to support adipose-derived cell differentiation into osteogenic and chondrogenic lineages. Decellularized bovine placenta, a high-quality scaffold with practical scalability, was chosen as the biomaterial due to its rich extracellular matrix, well-developed vasculature, high availability, low cost, and simplicity of collection. Adipose-derived cells were chosen as the cell source as they are easy to isolate, nontumorigenic, and flexibly differentiable. The bovine model was chosen for its advantages in translational medicine over the mouse model. When seeded onto the scaffolds, the isolated cells adhered to the scaffolds with cell projections, established cell-scaffold communication and proliferated while maintaining cell-cell communication. Throughout a 21-day culture period, osteogenically differentiated cells secreted mineralized matrix, and calcium deposits were observed throughout the scaffold. Under chondrogenic specific differentiation conditions, the cells modified their morphology from fibroblast-like to round cells and cartilage lacunas were observed as well as the deposit of cartilaginous matrix on the placental scaffolds. This experiment provides evidence, for the first time, that bovine placental scaffolds have the potential to support bovine mesenchymal stem cell adherence and differentiation into osteogenic and chondrogenic lineages. Therefore, the constructed material could be used for bone and cartilage tissue engineering.

Preserved efficiency of sickle cell disease placentas despite altered morphology and function.

INTRODUCTION: Pregnant women with sickle cell disease (SCD) are at high risk for sickle cell-related complications, obstetrical complications, and perinatal morbidity. Chronic inflammation and the proangiogenic environment associated with SCD have been associated with endothelial damage. It is unknown whether SCD complications could be associated with placental dysfunction or abnormal placental morphology. Moreover, circulating angiogenic factors in pregnant women with SCD are unexplored. METHODS: Clinical records, placental and blood samples were collected at term delivery for 21 pregnant patients with SCD and 19 HbAA pregnant controls with adapted to gestational age birth weight newborns. Histological and stereological analyses and scanning electron microscopy (SEM) of the placenta, and PlGF and sFlt1 measurements in blood were performed. RESULTS: In the SCD group, the parenchyma-forming villi of placentas were thinner than in controls, and increased fibrinoid necrosis and an overabundance of syncytial knots were seen. SEM revealed elongated intermediate villous endings with a reduction in the number of terminal villi compared to controls, indicating a significant branching defect in SCD placentas. Finally, SCD patients had an imbalance in the angiogenic ratio of sFlt1/PlGF (p = 0.008) with a drop of PlGF concentrations. DISCUSSION: We evidence for the first time both abnormal placenta morphology and altered sFlt1/PlGF ratio in SCD patients, uncorrelated with maintained placental efficiency and fetal growth.

Maternal high fat diet-induced obesity affects trophoblast differentiation and placental function in mice†.

Evidence suggests that maternal obesity (MO) can aggravate placental function causing severe pathologies during the perinatal window. However, molecular changes and mechanisms of placental dysfunction remain largely unknown. This work aimed to decipher structural and molecular alterations of the placental transfer zone associated with MO. To this end, mice were fed a high fat diet (HFD) to induce obesity before mating, and pregnant dams were sacrificed at E15.5 to receive placentas for molecular, histological, and ultrastructural analysis and to assess unidirectional materno-fetal transfer capacity. Laser-capture microdissection was used to collect specifically placental cells of the labyrinth zone for proteomics profiling. Using BeWo cells, fatty acid-mediated mechanisms of adherens junction stability, cell layer permeability, and lipid accumulation were deciphered. Proteomics profiling revealed downregulation of cell adhesion markers in the labyrinth zone of obese dams, and disturbed syncytial fusion and detachment of the basement membrane (BM) within this zone was observed, next to an increase in materno-fetal transfer in vivo across the placenta. We found that fetuses of obese dams develop a growth restriction and in those placentas, labyrinth zone volume-fraction was significantly reduced. Linoleic acid was shown to mediate beta-catenin level and increase cell layer permeability in vitro. Thus, MO causes fetal growth restriction, molecular and structural changes in the transfer zone leading to impaired trophoblast differentiation, BM disruption, and placental dysfunction despite increased materno-fetal transfer capacity. These adverse effects are probably mediated by fatty acids found in HFD demonstrating the need for obesity treatment to mitigate placental dysfunction and prevent offspring pathologies.

Visualizing ultrastructural details of placental tissue with super-resolution structured illumination microscopy.

Super-resolution fluorescence microscopy is a widely employed technique in cell biology research, yet remains relatively unexplored in the field of histopathology. Here, we describe the sample preparation steps and acquisition parameters necessary to obtain fluorescent multicolor super-resolution structured illumination microscopy (SIM) images of both formalin-fixed paraffin-embedded and cryo-preserved placental tissue sections. We compare super-resolved images of chorionic villi against diffraction-limited deconvolution microscopy and show the significant contrast and resolution enhancement attainable with SIM, demonstrating the applicability of this imaging technique for both clinical diagnosis and biological research.

Term placenta of the southern elephant seal (Mirounga leonina).

INTRODUCTION: The pinnipeds' placenta has been described as zonary, annular, labyrinthic and endotheliochorial, like that of the terrestrial carnivores. This article describes the placenta of Mirounga leonina, a phocid pinniped, focusing on some morphological features related to fetal nutrition. METHODS: Placental samples from three elephant seals were collected and conditioned after natural delivery at the Antarctic Specially Protected Area 132. Histological and ultrastructural studies were conducted; cytokeratins, vimentin, α-smooth muscle actin, and desmin proteins were detected using immunohistochemistry. RESULTS: The placentas were zonary, lobed, belt-shaped, and showed multiple vivid orange areas, which corresponded to bilirubin crystalline pigment found among chorionic villi and inside trophoblast cells. In the labyrinth, cytotrophoblast cells were isolated and there was a scant syncytium interposed between maternal and fetal vessels. Fetal vessels were small, round, and frequently intratrophoblastic, while maternal vessels were large, irregular, sinuous, and thin-walled. Vimentin and actin were detected in some scattered non-vascular cells throughout the labyrinth. Broad areas of degenerated and necrotic maternal components were also observed. DISCUSSION: The placentas of pinniped and fissiped carnivores share several traits. However, some remarkable features might maximize respiratory efficiency, collaborating to endure deep-diving hypoxia. Some of them, as the notably large sinuous maternal capillaries and fetal capillary indentation into the syncytium, are shared, e.g., by Phocidae and Mustelidae. Besides hemotropic nutrition taking place through an extremely narrow barrier, the abundant necrotic material and hematic products might allow substantial endocytosis of detritus even in term placentas, in this species giving birth to precocious offspring.

Characteristics of the equine placenta at first trimester / Características de la placenta equina en el primer trimestre

The equine placenta is a simple apposition of fetal and maternal tissues, becoming more complex with the formation of microcotyledons around days 75 and 100 of gestation. The present study aimed to describe the gross and microscopic morphology of early equine placenta. Embryonic/fetal membranes from thirty-seven mares were submitted to macroscopic description, light, scanning and transmission microscopy. Overall the gross characteristics of membranes were similar with already described for older stages. However, transmission electron microscopy evidenced high metabolic rate in chorion and allantois, and high secretion profile in amnion and even higher in yolk sac. Gene ontologies enrichment, using published data, pointed several common ontologies in allantoic and amniotic fluids, related to oxygen and iron transport, extracellular space and high-density lipoprotein receptor binding. Overall, the morphological and ontology enrichment could indicate allantois and amnion crosstalk.

La placenta equina es una simple aposición de tejidos fetales y maternos, que se vuelve más compleja con la formación de microcotiledones alrededor de los días 75 y 100 de gestación. El presente estudio tuvo como objetivo describir la morfología macroscópica y microscópica de la placenta equina temprana. Las membranas embrionarias / fetales de treinta y siete yeguas fueron sometidas a descripción macroscópica, luz, escaneo y microscopía de transmisión. En general, las características generales de las membranas fueron similares a las ya descritas para las etapas más antiguas. Sin embargo, la microscopía electrónica de transmisión mostró una alta tasa metabólica en corion y alantoides, y un alto perfil de secreción en amnios e incluso mayor en el saco vitelino. El enriquecimiento de ontologías génicas, utilizando datos publicados, señaló varias ontologías comunes en fluidos alantoideos y amnióticos, relacionados con el transporte de oxígeno y hierro, espacio extracelular y unión a receptores de lipoproteínas de alta densidad. En general, el enriquecimiento morfológico y ontológico podría indicar alantoides y diafonía de amnios.

Preservation of Mesenchymal Stem Cell-Derived Extracellular Vesicles after Abdominal Delivery in the Experiment.

Light luminescent microscopy was used to study the distribution of extracellular microvesicles with PKH26-stained membranes secreted by placenta-derived mesenchymal stromal cells in the uterine tissues at different terms after injections to intact rats and after abdominal delivery (a model of cesarian section). Microvesicles migrated through the uterine tissues and were detected for at least 8 days after injection. In some cases, microvesicles were more numerous in the uterus after cesarian section modeling, which can be related to blockade of microcirculation and lymph flow due to inflammation accompanying surgical intervention. The content of microvesicles in the uterine tissues gradually declined due to macrophage phagocytosis and, probably, due to their migration into the vascular bed. Despite their size, properly stained extracellular microvesicles can be detected by light microscopy in tissues after injections.

Cryo-EM structures and functional properties of CALHM channels of the human placenta.

The transport of substances across the placenta is essential for the development of the fetus. Here, we were interested in the role of channels of the calcium homeostasis modulator (CALHM) family in the human placenta. By transcript analysis, we found the paralogs CALHM2, 4, and 6 to be highly expressed in this organ and upregulated during trophoblast differentiation. Based on electrophysiology, we observed that activation of these paralogs differs from the voltage- and calcium-gated channel CALHM1. Cryo-EM structures of CALHM4 display decameric and undecameric assemblies with large cylindrical pore, while in CALHM6 a conformational change has converted the pore shape into a conus that narrows at the intracellular side, thus describing distinct functional states of the channel. The pore geometry alters the distribution of lipids, which occupy the cylindrical pore of CALHM4 in a bilayer-like arrangement whereas they have redistributed in the conical pore of CALHM6 with potential functional consequences.