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.

Human placental villi contain stromal macrovesicles associated with networks of stellate cells.

Placental function is essential for fetal development and establishing the foundations for lifelong health. The placental villous stroma is a connective tissue layer that supports the fetal capillaries and villous trophoblast. All the nutrients that cross the placenta must also cross the stroma, and yet little is known about this region. This study uses high-resolution three-dimensional imaging to explore the structural complexity of this region within the placental villi. Serial block-face scanning electron microscopy and confocal microscopy were used to image the placental villous stroma in three-dimensions. Transmission electron microscopy (TEM) was used to generate high resolution two-dimensional images. Stereological approaches were used to quantify volumes of stromal constituents. Three-dimensional imaging identified stromal extracellular vesicles, which constituted 3.9% of the villous stromal volume. These stromal extracellular vesicles were ovoid in shape, had a median length of 2750 nm (range 350-7730 nm) and TEM imaging confirmed that they were bounded by a lipid bilayer. Fifty-nine per cent of extracellular vesicles were in contact with a fibroblast-like stellate cell and these vesicles were significantly larger than those where no contact was observed. These stellate cells formed local networks with adherent junctions observed at contact points. This study demonstrates that the villous stroma contains extracellular macrovesicles which are considerably larger than any previously described in tissue or plasma. The size and abundance of these macrovesicles in the villous stroma highlight the diversity of extracellular vesicle biology and their roles within connective tissues.

Serial block-face scanning electron microscopy reveals novel intercellular connections in human term placental microvasculature.

The placental microvasculature is a conduit for fetal blood allowing solute exchange between the mother and the fetus. Serial block-face scanning electron microscopy (SBF SEM) allows ultrastructure to be viewed in three dimensions and provides a new perspective on placental anatomy. This study used SBF SEM to study endothelial cells within the human placental microvasculature from uncomplicated pregnancies. Term human placental villi were aldehyde-fixed and processed for imaging by SBF SEM. Manual segmentation was carried out on a terminal villous capillary and an intermediate villous arteriole and venule. Twenty-seven SBF SEM stacks from terminal villi were analysed using stereological approaches to determine the volumes of microvascular components and the proportions of pericyte coverage. SBF SEM analysis of capillary endothelial cells revealed the presence of interendothelial protrusions (IEPs) originating from the donor cell at the endothelial junction and forming deep thin projections up to 7 µm into the adjacent endothelial cells. IEP density was estimated to be in the order of 35 million cm-3 placental tissue. Pericytes cover 15% of the fetal capillary surface area in terminal villi. In comparison, the cytotrophoblast covered 24% of the syncytiotrophoblast basal membrane. A trans-endothelial channel was observed in a region of the vasculo-syncytial capillary. Pericyte coverage was extensive in both arteriole and venule. Three-dimensional imaging of the placental microvasculature identified novel ultrastructural features and provided an insight into factors that may influence capillary permeability and placental function. We hypothesise that the IEPs may allow mechanosensing between adjacent endothelial cells to assist in the maintenance of vessel integrity. The numbers of endothelial junctions, the presence of trans-endothelial channels and the extent of pericyte coverage all provide an insight into the factors determining capillary permeability.

Spatial organization of chromosome territories in the interphase nucleus of trisomy 21 cells.

In the interphase cell nucleus, chromosomes adopt a conserved and non-random arrangement in subnuclear domains called chromosome territories (CTs). Whereas chromosome translocation can affect CT organization in tumor cell nuclei, little is known about how aneuploidies can impact CT organization. Here, we performed 3D-FISH on control and trisomic 21 nuclei to track the patterning of chromosome territories, focusing on the radial distribution of trisomic HSA21 as well as 11 disomic chromosomes. We have established an experimental design based on cultured chorionic villus cells which keep their original mesenchymal features including a characteristic ellipsoid nuclear morphology and a radial CT distribution that correlates with chromosome size. Our study suggests that in trisomy 21 nuclei, the extra HSA21 induces a shift of HSA1 and HSA3 CTs out toward a more peripheral position in nuclear space and a higher compaction of HSA1 and HSA17 CTs. We posit that the presence of a supernumerary chromosome 21 alters chromosome compaction and results in displacement of other chromosome territories from their usual nuclear position.

Impact of chlorpyrifos on human villous trophoblasts and chorionic villi.

Placental barrier regulates maternal-fetal interchange protecting the baby from damage caused by substances found in the uterine environment or circulating in the vascular system. Organophosphate (OP) pesticides are a paramount group of environmental pollutants used in intensive agriculture for protection against diseases and pests. While many studies have reported an increased risk of pregnancy alterations in pregnant women exposed to OPs, few have analyzed the effects caused by these pesticides in the placenta. Herein, we evaluated the effects of chlorpyrifos (CPF), one of the most widely used OP insecticides, on human placenta using in vitro and ex vivo exposure models. Villous cytotrophoblast cells isolated from normal human term placentas maintained their cell viability, differentiated into syncytiotrophoblast-like structures, and increased the expression of ß-hCG, ABCG2, and P-gp in the presence of CPF at concentrations of 10 to 100µM. The same doses of CPF induced marked changes in chorionic villi samples. Indeed, CPF exposure increased stroma cell apoptosis, altered villi matrix composition, basement membrane thickness, and trophoblastic layer integrity. Histomorphological and ultrastructural alterations are compatible with those found in placentas where maternal-placenta injury is chronic and able to impair the placental barrier function and nutrient transport from mother to the fetus. Our study shows that placental ex vivo exposure to CPF produces tissue alterations and suggest that human placenta is a potential target of CPF toxicity. In addition, it highlights the importance of using different models to assess the effects of a toxic on human placenta.

Conditions for Collection of Placental Tissue Samples for Culturing of Multipotent Mesenchymal Stromal Cells.

Placentas from women aged 25-32 years with normal course of gestation were studied. It is essential to stick to certain methodological approaches for preparing viable multipotent mesenchymal stromal cell culture and to carry out morphological (macro and micro) evaluation of the chorionic villi, umbilical cords, and placentas. At stage I of the study, patients' histories, labor course, and examinations of the newborns should be analyzed to exclude women with genital and extragenital diseases. At stage II, it is essential to stick to special regulations and methods for collection of specimens of the cord, amnion, and placental tissue proper. Histological control of the placental structures collected for multipotent mesenchymal stromal cell culturing is obligatory.

Quantitative assessment of placental perfusion by contrast-enhanced ultrasound in macaques and human subjects.

BACKGROUND: The uteroplacental vascular supply is a critical determinant of placental function and fetal growth. Current methods for the in vivo assessment of placental blood flow are limited. OBJECTIVE: We demonstrate the feasibility of the use of contrast-enhanced ultrasound imaging to visualize and quantify perfusion kinetics in the intervillous space of the primate placenta. STUDY DESIGN: Pregnant Japanese macaques were studied at mid second trimester and in the early third trimester. Markers of injury were assessed in placenta samples from animals with or without contrast-enhanced ultrasound exposure (n = 6/group). Human subjects were recruited immediately before scheduled first-trimester pregnancy termination. All studies were performed with maternal intravenous infusion of lipid-shelled octofluoropropane microbubbles with image acquisition with a multipulse contrast-specific algorithm with destruction-replenishment analysis of signal intensity for assessment of perfusion. RESULTS: In macaques, the rate of perfusion in the intervillous space was increased with advancing gestation. No evidence of microvascular hemorrhage or acute inflammation was found in placental villous tissue and expression levels of caspase-3, nitrotyrosine and heat shock protein 70 as markers of apoptosis, nitrative, and oxidative stress, respectively, were unchanged by contrast-enhanced ultrasound exposure. In humans, placental perfusion was visualized at 11 weeks gestation, and preliminary data reveal regional differences in intervillous space perfusion within an individual placenta. By electron microscopy, we demonstrate no evidence of ultrastructure damage to the microvilli on the syncytiotrophoblast after first-trimester ultrasound studies. CONCLUSIONS: Use of contrast-enhanced ultrasound did not result in placental structural damage and was able to identify intervillous space perfusion rate differences within a placenta. Contrast-enhanced ultrasound imaging may offer a safe clinical tool for the identification of pregnancies that are at risk for vascular insufficiency; early recognition may facilitate intervention and improved pregnancy outcomes.

[HISTOLOGYCAL AND HISTOCHEMICAL CHARACTERISTICS OF AREOLAE IN THE PIG PLACENTA].

Rounded white lustreless dome-shaped wheels are detected by visually from allantochorion side in the in fetal areas epiteliochorial pig's placenta on day 30 of pregnancy. These structures are located over the opening of the uterine glands. Areolaes consist from maternal and foetal parts. Areola include glandular epithelium, chorial and endometrial stroma at the mouth of the uterine glands, areolar cavity-enhanced formed by endometrial and chorial invaginations. Chorion gives in cavity radial folds lining differences high epithelium. Glycogen, neutral and acid sulfated glycoproteins, proteoglycans, glycosaminoglycans, hyaluronates, total and cationic protein, RNA, arginine, gistidine, lysin were founded in structural components of areoles during gestation period. Numerous areolas serve as specialized sites for absorption the secrets of uterine glands; they are form a powerful functional system of histotrophic nutrition.

Establishment of an in vitro model of the human placental barrier by placenta slice culture and ussing chamber.

Our purpose was to establish an in vitro model of the human placental barrier based on placenta slice culture and Ussing chamber. The villous morphology, beta-human chorionic gonadotropin (ß-hCG), mRNA and efflux function of P-glycoprotein (P-gp), and the permeability of the fluorescent marker were confirmed. The results showed that syncytiotrophoblast cells with abundant endoplasmic reticulum and mitochondria were covered with a dense microvillus in the placenta slice. The ß-hCG secretion levels in the Ussing chamber were 274.13 ± 13.52 mIU/mL at 5 h, significantly higher than that in the incubator 95.2 ± 13.14 mIU/mL, and ß-hCG continued to secrete for 48 h. P-gp mRNA was expressed in the placenta slice. The Rho123 apparent permeability coefficient (Papp) value from maternal side to the fetal side was 26.34 ± 1.87 nm/s, but it was significantly increased, to 289.55 ± 6.02 nm/s after adding verapamil. The Rho123 efflux value was >2. The fluorescein Papp value was (3.42 ± 0.24) × 10(-3) nm/s. In contrast, the fluorescein isothiocyanate-dextran (FD70) Papp value was (3.93 ± 0.08) × 10(-5) nm/s. This indicates that the placenta slice in the Ussing chamber had the activity of a placenta, and can act as a valuable in vitro model of placental barrier.

Placental expression of vimentin, desmin and ultrastructural changes in the villi in patients with HELLP syndrome.

OBJECTIVES: To examine placental expression of vimentin and desmin in relation to ultrastructural changes within the placental villi in cases of HELLP syndrome. STUDY DESIGN: Formaldehyde-fixed and paraffin-embedded specimens of 15 healthy pregnant and 13 Hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome, placentas were used for Harris hematoxylin staining, vimentin and desmin immunohistochemistry, and transmission electron microscopy (TEM). RESULTS: Increased of fibrinoid necrosis in vascular wall and the periphery of villi were observed in sections of the placentas with HELLP syndrome. Increased expression of vimentin in the intravillous area and increased expression of desmin on blood vessel wall, were seen in placentas of patients with HELLP syndrome when compared to placentas of healthy pregnant. CONCLUSIONS: Augmentation of intermediate filaments, desmin, vimentin may disturb normal movements of endothelial cells, and may display placental dysfunction that is unable to compensate the endothelial instability and the related hypertension in HELLP syndrome. Further studies are needed to get more definit results and also to compare HELLP syndrome with preeclampsia.

Angiogenesis in villous chorangiosis observed by ultrastructural studies.

Chorangiosis is microscopically designated as more than ten terminal capillaries within the villous stroma of the placenta and is mostly related to chronic fetal hypoxia. However, the histogenetic relationship between increased number of terminal villous capillaries and chronic hypoxia has not yet been clarified. Of 665 placentas histologically examined at Saitama Medical University from 2003 to 2010, chorangiosis was found in 58 cases (8.7 %), which were mostly more than 35 gestational weeks. In addition, low birth weight (less than 2,500 g) infants (74.1 %) and those who suffered from cardiac anomalies, chromosome anomalies, and single umbilical artery comprised 32.7 % of cases. Placental lesions were associated with chorangiosis involved in infarct (46.6 %), intervillous thrombosis (20.7 %), and marginal hemorrhages (22.4 %). Scanning electron microscopic studies showed narrowing of vessel ostium and disorders of endothelium in the umbilical cord vessel complicated by chorangiosis. Furthermore, in transmission electron microscopic observation, not only the chorionic villi had multiple enlarged vessels within the villous stroma, but we also found that new capillaries were formed by angiogenesis with endothelial cells derived from fibroblasts under the chronic hypoxic state.

CD34 expression of chorionic villous in pre-eclamptic placenta: an immunohistochemical and ultrastructural study.

In this study, pre-eclampsia, proteinuria, and edema associated with hypertension in pregnancy were assessed at the Dicle University School of Medicine Department of Obstetrics and Gynecology Clinic. One group included 20 pre-eclamptic pregnant women with gestational age 20-35 weeks of pregnancy and the same in the control group that included; however, 20 normotensive pregnant women. Histochemistry, immunohistochemistry, and electron microscopy techniques were used. Histopathological examination of syncytial nodes and stromal cells were observed in the increase in hyperplasia and hyalinization. The evaluation immunohistochemical of chorionic villi, placenta, and hematopoietic stem cell markers showed a positive reaction with CD34. Ultrastructural examination showed endoplasmic reticulum dilatation, degeneration of mitochondria in endothelial cells, and capillary vessel edema.

The first trimester human placenta is a site for terminal maturation of primitive erythroid cells.

Embryonic hematopoiesis starts via the generation of primitive red blood cells (RBCs) that satisfy the embryo's immediate oxygen needs. Although primitive RBCs were thought to retain their nuclei, recent studies have shown that primitive RBCs in mice enucleate in the fetal liver. It has been unknown whether human primitive RBCs enucleate, and what hematopoietic site might support this process. Our data indicate that the terminal maturation and enucleation of human primitive RBCs occurs in first trimester placental villi. Extravascular ζ-globin(+) primitive erythroid cells were found in placental villi between 5-7 weeks of development, at which time the frequency of enucleated RBCs was higher in the villous stroma than in circulation. RBC enucleation was further evidenced by the presence of primitive reticulocytes and pyrenocytes (ejected RBC nuclei) in the placenta. Extravascular RBCs were found to associate with placental macrophages, which contained ingested nuclei. Clonogenic macrophage progenitors of fetal origin were present in the chorionic plate of the placenta before the onset of fetoplacental circulation, after which macrophages had migrated to the villi. These findings indicate that placental macrophages may assist the enucleation process of primitive RBCs in placental villi, implying an unexpectedly broad role for the placenta in embryonic hematopoiesis.

[Human placenta structure in II and III trimesters of physiological pregnancy].

The placenta of 20 women with normal pregnancy was studied during II and III trimesters to obtain the complex characteristic of the structures participating in the formation of syncytio-capillary membranes. Immunocytochemical endothelial cell marker CD34 and morphometry were used for the assessment of some villous parameters: villous area, villous stromal area, villous epithelial area and vascular endothelium area. The main attention was given to the remodeling of the villous epithelium and capillary network. The significant reduction of the epithelial area and that of the villous stroma was detected simultaneously with intensive their vascularization. Morpho-functionally, most of the terminal villi were specialized, containing sinusoid-type capillaries which were in close contact with attenuated, anuclea regions of syncytiotrophoblast. The proportion of terminal villous endothelium in respect to the stroma was significantly increased. Thus, in II and III trimesters of physiological pregnancy the structural changes take place in the placenta, in particular, the capillaries are transformed into thin-walled sinusoids, that approach and closely interact with syncytiotrophoblast resulting in the formation of the syncytio-capillary membranes necessary for an adequate diffusion and meeting the growing needs of the fetus.

A new possible function for placental pericytes.

The pericyte is a multifunctional cell closely associated with endothelial cells and may play a role in angiogenesis and vessel stabilisation. Re-examination of over 1,100 micrographs from archival material used to investigate ultrastructural changes in placental development and pathology has identified previously undescribed structures associated with the pericyte of the human placental terminal villus. These structures take the form of outgrowths from the main body of the cell, with a narrow neck rich in cytoplasmic filaments, terminating in swollen tips which appear to bleb off the pericyte and form electron lucent stromal vesicles. Semi-quantitative analysis indicated that these features are present in some placentae from normal, term pregnancies but are increasingly found where capillaries show abnormalities such as a failure to form sinusoids, as in pregnancies complicated by diabetes, postmaturity, rhesus incompatibility and pre-eclampsia. This blebbing is compared with similar phenomena associated with apoptosis and zeiosis and it is suggested that it may contribute to fluid homeostasis where normal mechanisms are impaired by thickening or damage to endothelial cells.

Chorionic villi ultrastructure in the prenatal diagnosis of glycogenosis type II.

OBJECTIVE: To perform the ultrastructural examination of a chorionic villi biopsy as a predictor of foetal involvement in the infantile form of glycogenosis type II (Pompe disease). METHODS: Ultrastructural, biochemical and genetic analyses were performed on chorionic villi biopsies of three consecutive pregnancies in a woman with a previous child affected by Pompe disease. RESULTS: In the only affected foetus, glycogen storage was observed in fibrocytes and endothelial cells of a chorionic villi sample at 11 week's gestation. Severe multi-organ involvement was demonstrated in the tissues of the aborted foetus. No abnormal material was found in the chorionic samples of two subsequent pregnancies, and a healthy boy and girl were born at term and remain unaffected. Both exhibited a partial reduction in acid maltase and were carriers of the maternal mutation. CONCLUSIONS: Ultrastructural findings correlated with biochemical and genetic results, providing a clear and early indicator of the definite diagnosis for future pregnancy management or an early therapeutic approach.

Nanobacteria and psammoma bodies: ultrastructural observations in a case of pathological placental calcification.

Nanobacteria are controversial infectious agents with nanometric size, the capacity to nucleate hydroxyapatite and grow in culture, and present in human diseases associated with calcification and psammoma bodies. The authors report a case of pathological placental calcifications associated with nanobacteria. Electron microscopy and electron energy loss spectroscopy imaging were used to recognize 160-nm-sized calcium-free bodies mainly presenting as extracellular fibrillary tangles and 500-nm-sized calcified bodies; they encrusted the syncito-trophoblast basal membrane and aggregated into miniaturized psammoma bodies. Nanobacteria may be composed of a prionoid protein with self-assembling and self-propagating abilities whose growth is associated with the formation of psammoma bodies.

Placenta and umbilical cord blood deserve attention.

OBJECTIVES: This work follows up with our already published results concerning consequences of lead on prenatal and postnatal development of child in connection with the rise of hyperkinetic syndrome (ADHD). This disease has in children increasing tendency all over the world. METHODS: In our work we used a set of histological and histochemical methods, method of scanning electron microscopy, infrared spectroscopy and statistical evaluation. RESULTS: Our new method for proof of lead in placenta enabled us to show how lead is cumulated in syncytiotrophoblast. We have found release of lead from mother's erythrocytes in the intervillous space and receipt of lead by erythrocytes of fetus in the vessels of the villi of placenta. This finding enriches knowledge about relation between mother's erythrocytes, lead, calcium that is lead carrier, syncytiotrophoblast, and erythrocytes of fetus in the vessels of placental villi. We have proved that syncytiotrophoblast is the most frequent place for cumulation of lead deposits. We verified our ecomorphologic results by means of infrared spectroscopy in cooperation with physicists and statistically evaluated occurrence of ADHD in particular age categories what helps to fill gaps in knowledge of ADHD etiology. CONCLUSIONS: Our finding of lead in umbilical cord blood immediately after the child birth is forewarning against the possible rise of the ADHD. This finding facilitates early diagnostics and means preventing step against the rise, development and consequences of this disease. The obtained results give evidence about the serious influence of mother's dwelling in environment polluted with neurotoxic metal - lead on the prenatal and postnatal development of child.

Prenatal diagnosis of 18q-syndrome: a case of fetal mosaicism with a normal karyotype in chorionic villi.

We report a case of fetoplacental discrepancy with normal karyotype on chorionic villi and deletion of the long arm of chromosome 18 on amniotic fluid. Cytogenetic tests were repeated because of a short corpus callosum on ultrasound examination. This 18q-syndrome has been reported to be associated with poor neurodevelopmental outcome.