Convergently evolved placental villi show multiscale structural adaptations to differential placental invasiveness.

Despite having a single evolutionary origin and conserved function, the mammalian placenta exhibits radical structural diversity. The evolutionary drivers and functional consequences of placental structural diversity are poorly understood. Humans and equids both display treelike placental villi, however these villi evolved independently and exhibit starkly different levels of invasiveness into maternal tissue (i.e. the number of maternal tissue layers between placental tissue and maternal blood). The villi in these species therefore serve as a compelling evolutionary case study to explore whether placentas have developed structural adaptations to respond to the challenge of reduced nutrient availability in less invasive placentas. Here, we use three-dimensional X-ray microfocus computed tomography and electron microscopy to quantitatively evaluate key structures involved in exchange in human and equid placental villi. We find that equid villi have a higher surface area to volume ratio and deeper trophoblastic vessel indentation than human villi. Using illustrative computational models, we propose that these structural adaptations have evolved in equids to boost nutrient transfer to compensate for reduced invasiveness into maternal tissue. We discuss these findings in relation to the 'maternal-fetal conflict hypothesis' of placental evolution.

Ultrastructural cilia defects in multi-ciliated uterine glandular epithelial cells from women with reproductive failure.

In brief: The causes of subfertility and recurrent pregnancy loss are often unclear. This study shows that endometrial gland cilia from women with subfertility have ultrastructural defects. Abstract: Endometrial glands secrete products into the endometrium and are necessary for embryo implantation and successful pregnancy. However, structural and functional abnormalities in endometrial gland cilia from women with reproductive failure remain poorly understood. This was a cross-sectional study where endometrial biopsies were collected at days 19-23 of the menstrual cycle from women with unexplained recurrent pregnancy loss (n = 15), unexplained subfertility (n = 11) or from egg donor control participants (n = 10). Endometrial gland cilia ultrastructure was imaged by transmission electron microscopy and cilia defects assessed by an electron-microscopist from a national primary ciliary dyskinesia diagnostic centre. Endometrial glands were isolated, and the cilia beat frequency recorded by high speed video. Subfertile women have proportionately lower ultrastructurally normal cilia (P < 0.05); higher frequency of absent dynamin arms (P < 0.01) or inner arm defects (P < 0.01) and lower cilia beat frequency (P < 0.05). The mechanisms underlying these obversions have yet to be determined. Recent studies have identified cilia related gene expression changes associated with reproductive failure and this study adds to the growing body of literature revealing structural and functional changes. The observation that cilia defects occurred at a higher frequency in endometrial glands of subfertile women raises the question of its mechanistic role in implantation.

A Novel Sandwich Method for Serial Block Face SEM Imaging of Airway Multiciliated Epithelium.

Volume electron microscopy technologies such as serial block face scanning electron microscopy (SBF-SEM) allow the characterization of tissue organization and cellular content in three dimensions at nanoscale resolution. Here, we describe the procedure to process and image an air-liquid interface culture of human or mouse airway epithelial cells for visualization of the multiciliated epithelium by SBF-SEM in vertical or horizontal cross section.

Subconjunctival Ocular Argyrosis following Treatment with Ruthenium 106 Brachytherapy for Choroidal Melanoma.

Introduction: Ruthenium-106 (Ru-106) brachytherapy is one of the commonest eye-sparing treatments for choroidal melanoma. These patients require long-term surveillance of the treated tumour remnant to ensure there is no local recurrence. New or progressive pigmented lesions in treated eyes are often regarded as suspicious - especially if there are concerns of extra-scleral extension. Case Presentations: We present two cases of posterior choroidal melanoma treated five and 10 years previously with Ru-106. Both cases developed subconjunctival dark/black lesions on the anterior surface of the eye in the quadrant of the conjunctival peritomy during Ru-106 treatment. Both had similar findings on histopathology: black, non-organic, particulate foreign material of varying confluence deposited on elastin and collagen fibres. Energy dispersive X-ray microanalysis confirmed the material contained silver. Discussion: The Ru-106 applicator consists of a radioactive core of Ru-106 encapsulated within pure silver as a radiation shield. During surgical insertion, stainless steel suture needles and forceps can occasionally scratch the applicator's silver eyelets and scatter microscopic particles of elemental silver into the operative field. These particles were likely deposited within the subconjunctival tissues of these patients during brachytherapy administration, leading to localised ocular argyrosis. Iatrogenic ocular argyrosis should be considered in the differential diagnosis of new pigmented lesions in patients treated with Ru-106 brachytherapy. This study is the first to unequivocally identify the cause of some post-brachytherapy ocular surface pigmentation as caused by silver.

A high-throughput 3D X-ray histology facility for biomedical research and preclinical applications.

Background: The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (µCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a µCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies. Methods: The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients. The facility employs specialised µCT equipment explicitly designed for histology applications, allowing whole-block XRH imaging of formalin-fixed and paraffin-embedded tissue specimens. It also enables correlative imaging by combining µCT imaging with other microscopy techniques, such as immunohistochemistry (IHC) and serial block-face scanning electron microscopy, as well as data visualisation, image quantification, and bespoke analysis. Results: Over the past seven years, the XRH facility has successfully completed over 120 projects in collaboration with researchers from 60 affiliations, resulting in numerous published manuscripts and conference proceedings. The facility has streamlined the µCT imaging process, improving productivity and enabling efficient acquisition of 3D datasets. Discussion & Conclusions: The 3D X-ray Histology (XRH) facility at UHS is a pioneering platform in the field of histology and biomedical imaging. To the best of our knowledge, it stands out as the world's first dedicated XRH facility, encompassing every aspect of the imaging process, from user support to data generation, analysis, training, archiving, and metadata generation. This article serves as a comprehensive guide for establishing similar XRH facilities, covering key aspects of facility setup and operation. Researchers and institutions interested in developing state-of-the-art histology and imaging facilities can utilise this resource to explore new frontiers in their research and discoveries.

Defective airway intraflagellar transport underlies a combined motile and primary ciliopathy syndrome caused by IFT74 mutations.

Ciliopathies are inherited disorders caused by defective cilia. Mutations affecting motile cilia usually cause the chronic muco-obstructive sinopulmonary disease primary ciliary dyskinesia (PCD) and are associated with laterality defects, while a broad spectrum of early developmental as well as degenerative syndromes arise from mutations affecting signalling of primary (non-motile) cilia. Cilia assembly and functioning requires intraflagellar transport (IFT) of cargos assisted by IFT-B and IFT-A adaptor complexes. Within IFT-B, the N-termini of partner proteins IFT74 and IFT81 govern tubulin transport to build the ciliary microtubular cytoskeleton. We detected a homozygous 3-kb intragenic IFT74 deletion removing the exon 2 initiation codon and 40 N-terminal amino acids in two affected siblings. Both had clinical features of PCD with bronchiectasis, but no laterality defects. They also had retinal dysplasia and abnormal bone growth, with a narrowed thorax and short ribs, shortened long bones and digits, and abnormal skull shape. This resembles short-rib thoracic dysplasia, a skeletal ciliopathy previously linked to IFT defects in primary cilia, not motile cilia. Ciliated nasal epithelial cells collected from affected individuals had reduced numbers of shortened motile cilia with disarranged microtubules, some misorientation of the basal feet, and disrupted cilia structural and IFT protein distributions. No full-length IFT74 was expressed, only truncated forms that were consistent with N-terminal deletion and inframe translation from downstream initiation codons. In affinity purification mass spectrometry, exon 2-deleted IFT74 initiated from the nearest inframe downstream methionine 41 still interacts as part of the IFT-B complex, but only with reduced interaction levels and not with all its usual IFT-B partners. We propose that this is a hypomorphic mutation with some residual protein function retained, which gives rise to a primary skeletal ciliopathy combined with defective motile cilia and PCD.

Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue.

Three-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities.

The Palestinian primary ciliary dyskinesia population: first results of the diagnostic and genetic spectrum.

Background: Diagnostic testing for primary ciliary dyskinesia (PCD) started in 2013 in Palestine. We aimed to describe the diagnostic, genetic and clinical spectrum of the Palestinian PCD population. Methods: Individuals with symptoms suggestive of PCD were opportunistically considered for diagnostic testing: nasal nitric oxide (nNO) measurement, transmission electron microscopy (TEM) and/or PCD genetic panel or whole-exome testing. Clinical characteristics of those with a positive diagnosis were collected close to testing including forced expiratory volume in 1 s (FEV1) Global Lung Index z-scores and body mass index z-scores. Results: 68 individuals had a definite positive PCD diagnosis, 31 confirmed by genetic and TEM results, 23 by TEM results alone, and 14 by genetic variants alone. 45 individuals from 40 families had 17 clinically actionable variants and four had variants of unknown significance in 14 PCD genes. CCDC39, DNAH11 and DNAAF11 were the most commonly mutated genes. 100% of variants were homozygous. Patients had a median age of 10.0 years at diagnosis, were highly consanguineous (93%) and 100% were of Arabic descent. Clinical features included persistent wet cough (99%), neonatal respiratory distress (84%) and situs inversus (43%). Lung function at diagnosis was already impaired (FEV1 z-score median -1.90 (-5.0-1.32)) and growth was mostly within the normal range (z-score mean -0.36 (-3.03-2.57). 19% individuals had finger clubbing. Conclusions: Despite limited local resources in Palestine, detailed geno- and phenotyping forms the basis of one of the largest national PCD populations globally. There was notable familial homozygosity within the context of significant population heterogeneity.

3D visualization of trans-syncytial nanopores provides a pathway for paracellular diffusion across the human placental syncytiotrophoblast.

The placental syncytiotrophoblast, a syncytium without cell-cell junctions, is the primary barrier between the mother and the fetus. Despite no apparent anatomical pathway for paracellular diffusion of solutes across the syncytiotrophoblast, size-dependent paracellular diffusion is observed. Here we report data demonstrating that the syncytiotrophoblast is punctuated by trans-syncytial nanopores (TSNs). These membrane-bound TSNs directly connect the maternal and fetal facing sides of the syncytiotrophoblast, providing a pathway for paracellular diffusion between the mother and fetus. Mathematical modeling of TSN permeability based on their 3D geometry suggests that 10-37 million TSNs per cm3 of placental tissue could explain experimentally observed placental paracellular diffusion. TSNs may mediate physiological hydrostatic and osmotic pressure homeostasis between the maternal and fetal circulations but also expose the fetus to pharmaceuticals, environmental pollutants, and nanoparticles.

Endometrial gland specific progestagen-associated endometrial protein and cilia gene splicing changes in recurrent pregnancy loss.

Endometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and PAEP gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.

Microvillous tip vesicles may be an origin of placental extracellular vesicles.

INTRODUCTION: Extracellular vesicles are now believed to be important mediators of placental-maternal communication. However, little is known about the formation of extracellular vesicles by human placenta. This study uses nanoscale three-dimensional imaging to investigate how and where placental extracellular vesicles form. METHODS: Term and first trimester human placental villi were imaged by serial block face scanning electron microscopy. These images were analysed to quantify vesicle surface density. Segmentation was performed to reconstruct three-dimensional images of extracellular vesicles. Live imaging light microscopy of first trimester villous explants was performed. RESULTS: Vesicles were observed on the tips of placental microvilli in term and first trimester placenta. In term placenta these microvillous tip vesicles had a median size of 0.55 µm and their surface area density exceeded 22000 per mm2. Microvillous tip vesicle membranes had a lower electron density than the microvillous plasma membrane. Thirty seven percent of vesicles had a complex membrane structure including double membranes, internal vesicles and vesicle chains. Budding of smaller secondary vesicles from microvillous tip vesicle membranes was observed. Live imaging of a first trimester villus explant observed formation of vesicles which were larger but visually similar to the secondary vesicles observed by electron microscopy. DISCUSSION: These observations suggest that extracellular vesicles are forming on the tips of placental microvilli prior to release into maternal blood. However, it cannot be discounted that there are maternal extracellular vesicles that have bound to microvilli. In either case, the high surface area density of microvillous tip vesicles is consistent with an important role in placental-maternal signalling.

Folding of the syncytiotrophoblast basal plasma membrane increases the surface area available for exchange in human placenta.

INTRODUCTION: The placental syncytiotrophoblast is the primary barrier between the mother and the fetus. To cross the placenta, nutrients and wastes must be transported across the apical microvillous and basal plasma membranes. While the syncytiotrophoblast basal plasma membrane is typically represented as relatively smooth, it has been shown to have invaginations that may increase its surface area. This study aimed to quantify how folding of the syncytiotrophoblast basal membrane contributes to its surface area and to visualise three-dimensional structures of the basal membrane and cytotrophoblast cell structures. METHODS: Transmission electron microscope images of human term placenta were analysed using stereological approaches to quantify how folding of the syncytiotrophoblast basal plasma membrane affected surface area. Serial block-face scanning electron microscopy was used to visualise the three-dimensional structure of the syncytiotrophoblast basal membrane and cytotrophoblast cells. RESULTS: Syncytiotrophoblast basal membrane covered 69.1% of the basal lamina, with cytotrophoblast cells covering the remaining 30.9%. In basal lamina adjacent to syncytiotrophoblast, 34% was adjacent to smooth basal membrane and 66% to folded basal membrane. Syncytiotrophoblast basal membrane folds increased the surface area adjacent to basal lamina by 305%. Including regions overlying the cytotrophoblast cells, basal membrane folds increased syncytiotrophoblast basal membrane surface area by 4.4-fold relative to the basal lamina in terminal villi. Terminal and intermediate villi were similar in terms of trophoblast coverage of the basal lamina and basal membrane folding. The three-dimensional structures of the syncytiotrophoblast basal plasma membrane and cytotrophoblast cells were generated from serial block-face scanning electron microscopy image stacks. DISCUSSION: These findings indicate that the surface area of the syncytiotrophoblast basal plasma membrane is far larger than had been appreciated. We suggest that these folds increase the surface area available for transport to and from the fetus. Changes in the extent of basal membrane folding could affect nutrient transfer capacity and underlie pathological fetal growth, including fetal growth restriction and macrosomia.

Whole genome sequencing in the diagnosis of primary ciliary dyskinesia.

BACKGROUND: It is estimated that 1-13% of cases of bronchiectasis in adults globally are attributable to primary ciliary dyskinesia (PCD) but many adult patients with bronchiectasis have not been investigated for PCD. PCD is a disorder caused by mutations in genes required for motile cilium structure or function, resulting in impaired mucociliary clearance. Symptoms appear in infancy but diagnosis is often late or missed, often due to the lack of a "gold standard" diagnostic tool and non-specific symptoms. Mutations in > 50 genes account for around 70% of cases, with additional genes, and non-coding, synonymous, missense changes or structural variants (SVs) in known genes presumed to account for the missing heritability. METHODS: UK patients with no identified genetic confirmation for the cause of their PCD or bronchiectasis were eligible for whole genome sequencing (WGS) in the Genomics England Ltd 100,000 Genomes Project. 21 PCD probands and 52 non-cystic fibrosis (CF) bronchiectasis probands were recruited in Wessex Genome Medicine Centre (GMC). We carried out analysis of single nucleotide variants (SNVs) and SVs in all families recruited in Wessex GMC. RESULTS: 16/21 probands in the PCD cohort received confirmed (n = 9), probable (n = 4) or possible (n = 3) diagnosis from WGS, although 13/16 of these could have been picked up by current standard of care gene panel testing. In the other cases, SVs were identified which were missed by panel testing. We identified variants in novel PCD candidate genes (IFT140 and PLK4) in 2 probands in the PCD cohort. 3/52 probands in the non-CF bronchiectasis cohort received a confirmed (n = 2) or possible (n = 1) diagnosis of PCD. We identified variants in novel PCD candidate genes (CFAP53 and CEP164) in 2 further probands in the non-CF bronchiectasis cohort. CONCLUSIONS: Genetic testing is an important component of diagnosing PCD, especially in cases of atypical disease history. WGS is effective in cases where prior gene panel testing has found no variants or only heterozygous variants. In these cases it may detect SVs and is a powerful tool for novel gene discovery.

Occupational Localized Cutaneous Argyria With Pseudo-Ochronosis in a Jeweler.

ABSTRACT: A case of localized argyria in a 36-year-old female jeweler is described who presented with 2 discrete and asymptomatic bluish-black pigmented macules on the pulp of her left middle finger. A skin biopsy from both lesions demonstrated deposition of brown/black pigmented granules along the basement membrane zone of eccrine glands, blood vessels, nerves, and the dermo-epidermal junction fully in keeping with silver deposition. In addition, there was yellow-brown deposition seen within the interstitial dermis mimicking an early form of ochronosis, so called "pseudo-ochronosis." This latter feature is rarely described in cases of argyria. Transmission electron microscopy and energy dispersive x-ray spectroscopy confirmed the presence of electron dense particles up to 150 nm in diameter and the presence of silver, respectively. On further questioning, the patient had a history of localized and chronic exposure to silver, which specifically involved holding and manipulating silver wires and rings over the left middle finger. This case highlights an unusual and rare presentation of localized argyria in a jeweler. In addition, our case showed preferential silver deposition on dermal elastic fibers which has not been previously described in the literature.

Quantitative assessment of dentine mineralization and tubule occlusion by NovaMin and stannous fluoride using serial block face scanning electron microscopy.

Dentine hypersensitivity (DH) is one of the most common dental conditions affecting most adults during their lifetime. Tubule occlusion is a widely accepted method for treating DH. Current in-vitro techniques such as focused ion beam, scanning electron microscopy (SEM), or hydraulic conductance that are used to determine tubule occlusion do not provide the depth of occlusion, are time-consuming, expensive and the volume of dentine tested is limited. The presented study aimed to assess the ability of serial block-face SEM (SBF-SEM) to section dentine, to quantify the number of occluded tubules including the depth of penetration by NovaMin and stannous fluoride (SnF2 ) and to compare mineral density between the control and treated dentine. Results demonstrated that NovaMin provided a better occlusion with 100% of the tubules blocked at the surface compared to 83% for SnF2 . The grayscale value (230.42) was significantly higher (p ≤ 0.05) after treatment with NovaMin compared to SnF2 (222.06) and the control (196.37), indicating increased mineral density and dentine mineralization. SBF-SEM has the potential to be used for large volume analysis of bone-like materials at high resolution with minimal sample preparation over a short period. It can be significantly useful in the development and research of new biomaterials.

Pericytes on placental capillaries in terminal villi preferentially cover endothelial junctions in regions furthest away from the trophoblast.

INTRODUCTION: Pericytes are a common feature in the placental microvasculature but their roles are not well understood. Pericytes may provide physical or endocrine support for endothelium and in some tissues mediate vasoconstriction. METHODS: This study uses serial block-face scanning electron microscopy (SBFSEM) to generate three-dimensional (3D) reconstructions of placental pericytes of the terminal villi and transmission electron microscopy (TEM) to study pericyte endothelial cell interactions. The proportion of endothelial cell junctions covered by pericytes was determined. RESULTS: The detailed 3D models of placental pericytes show pericyte structure at a new level of detail. Placental pericytes have many fingers extending from the cell body which can span multiple capillary branches. The proportion of endothelial cell-cell junctions covered by pericytes was significantly higher than pericyte coverage of capillary endothelium as a whole (endothelium: 14%, junctions: 43%, p < 0.0001). However, the proportion of endothelial cell-cell junctions covered by pericytes in regions adjacent to trophoblast was reduced compared to regions >3 µm away from trophoblast (27% vs 62% respectively, p < 0.001). No junctional complexes were observed connecting pericytes and endothelial cells but there were regions of cell membrane with features suggestive of intercellular adhesions. DISCUSSION: These data suggest that the localisation of pericytes on the villous capillary is not random but organised in relation to both endothelial junctions and the location of adjacent trophoblast. This further suggests that pericyte coverage may favour capillary permeability in regions that are most important for exchange, but limit capillary permeability in other regions.

3D-Reconstructed Retinal Pigment Epithelial Cells Provide Insights into the Anatomy of the Outer Retina.

The retinal pigment epithelium (RPE) is located between the neuroretina and the choroid, and plays a critical role in vision. RPE cells internalise outer segments (OS) from overlying photoreceptors in the daily photoreceptor renewal. Changes to RPE structure are linked with age and retinopathy, which has been described in the past by conventional 2D electron microscopy. We used serial block face scanning electron microscopy (SBF-SEM) to reconstruct RPE cells from the central mouse retina. Three-dimensional-reconstructed OS revealed the RPE to support large numbers of photoreceptors (90-216 per RPE cell). Larger bi-nucleate RPE maintained more photoreceptors, although their cytoplasmic volume was comparable to smaller mono-nucleate RPE supporting fewer photoreceptors. Scrutiny of RPE microvilli and interdigitating OS revealed the angle and surface area of contact between RPE and photoreceptors. Bi-nucleate RPE contained more mitochondria compared to mono-nucleate RPE. Furthermore, bi-nucleate cells contained larger sub-RPE spaces, supporting a likely association with disease. Use of perfusion-fixed tissues ensured the highest possible standard of preservation, providing novel insights into the 3D RPE architecture and changes linked with retinopathy. This study serves as a benchmark for comparing retinal tissues from donor eyes with age-related macular degeneration (AMD) and other retinopathies.

A Revised Protocol for Culture of Airway Epithelial Cells as a Diagnostic Tool for Primary Ciliary Dyskinesia.

Air-liquid interface (ALI) culture of nasal epithelial cells is a valuable tool in the diagnosis and research of primary ciliary dyskinesia (PCD). Ex vivo samples often display secondary dyskinesia from cell damage during sampling, infection or inflammation confounding PCD diagnostic results. ALI culture enables regeneration of healthy cilia facilitating differentiation of primary from secondary ciliary dyskinesia. We describe a revised ALI culture method adopted from April 2018 across three collaborating PCD diagnostic sites, including current University Hospital Southampton COVID-19 risk mitigation measures, and present results. Two hundred and forty nasal epithelial cell samples were seeded for ALI culture and 199 (82.9%) were ciliated. Fifty-four of 83 (63.9%) ex vivo samples which were originally equivocal or insufficient provided diagnostic information following in vitro culture. Surplus basal epithelial cells from 181 nasal brushing samples were frozen in liquid nitrogen; 39 samples were ALI-cultured after cryostorage and all ciliated. The ciliary beat patterns of ex vivo samples (by high-speed video microscopy) were recapitulated, scanning electron microscopy demonstrated excellent ciliation, and cilia could be immuno-fluorescently labelled (anti-alpha-tubulin and anti-RSPH4a) in representative cases that were ALI-cultured after cryostorage. In summary, our ALI culture protocol provides high ciliation rates across three centres, minimising patient recall for repeat brushing biopsies and improving diagnostic certainty. Cryostorage of surplus diagnostic samples was successful, facilitating PCD research.

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.

Clinical features and management of children with primary ciliary dyskinesia in England.

OBJECTIVE: In England, the National Health Service commissioned a National Management Service for children with primary ciliary dyskinesia (PCD). The aims of this study were to describe the health of children seen in this Service and compare lung function to children with cystic fibrosis (CF). DESIGN: Multi-centre service evaluation of the English National Management PCD Service. SETTING: Four nationally commissioned PCD centres in England. PATIENTS: 333 children with PCD reviewed in the Service in 2015; lung function data were also compared with 2970 children with CF. RESULTS: Median age at diagnosis for PCD was 2.6 years, significantly lower in children with situs inversus (1.0 vs 6.0 years, p<0.001). Compared with national data from the CF Registry, mean (SD) %predicted forced expiratory volume in one second (FEV1) was 76.8% in PCD (n=240) and 85.0% in CF, and FEV1 was lower in children with PCD up to the age of 15 years. Approximately half of children had some hearing impairment, with 26% requiring hearing aids. Children with a lower body mass index (BMI) had lower FEV1 (p<0.001). One-third of children had positive respiratory cultures at review, 54% of these grew Haemophilus influenzae. CONCLUSIONS: We provide evidence that children with PCD in England have worse lung function than those with CF. Nutritional status should be considered in PCD management, as those with a lower BMI have significantly lower FEV1. Hearing impairment is common but seems to improve with age. Well-designed and powered randomised controlled trials on management of PCD are needed to inform best clinical practice.