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.

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.

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.

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.

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.

Diagnosis of primary ciliary dyskinesia: potential options for resource-limited countries.

Primary ciliary dyskinesia is a genetic disease of ciliary function leading to chronic upper and lower respiratory tract symptoms. The diagnosis is frequently overlooked because the symptoms are nonspecific and the knowledge about the disease in the primary care setting is poor. Additionally, none of the available tests is accurate enough to be used in isolation. These tests are expensive, and need sophisticated equipment and expertise to analyse and interpret results; diagnosis is therefore only available at highly specialised centres. The diagnosis is particularly challenging in countries with limited resources due to the lack of such costly equipment and expertise.In this review, we discuss the importance of early and accurate diagnosis especially for countries where the disease is clinically prevalent but diagnostic tests are lacking. We review the diagnostic tests available in specialised centres (nasal nitric oxide, high-speed video microscopy, transmission electron microscopy, immunofluorescence and genetics). We then consider modifications that might be considered in less well-resourced countries whilst maintaining acceptable accuracy.

Culture of primary ciliary dyskinesia epithelial cells at air-liquid interface can alter ciliary phenotype but remains a robust and informative diagnostic aid.

BACKGROUND: The diagnosis of primary ciliary dyskinesia (PCD) requires the analysis of ciliary function and ultrastructure. Diagnosis can be complicated by secondary effects on cilia such as damage during sampling, local inflammation or recent infection. To differentiate primary from secondary abnormalities, re-analysis of cilia following culture and re-differentiation of epithelial cells at an air-liquid interface (ALI) aids the diagnosis of PCD. However changes in ciliary beat pattern of cilia following epithelial cell culture has previously been described, which has brought the robustness of this method into question. This is the first systematic study to evaluate ALI culture as an aid to diagnosis of PCD in the light of these concerns. METHODS: We retrospectively studied changes associated with ALI-culture in 158 subjects referred for diagnostic testing at two PCD centres. Ciliated nasal epithelium (PCD n = 54; non-PCD n  111) was analysed by high-speed digital video microscopy and transmission electron microscopy before and after culture. RESULTS: Ciliary function was abnormal before and after culture in all subjects with PCD; 21 PCD subjects had a combination of static and uncoordinated twitching cilia, which became completely static following culture, a further 9 demonstrated a decreased ciliary beat frequency after culture. In subjects without PCD, secondary ciliary dyskinesia was reduced. CONCLUSIONS: The change to ciliary phenotype in PCD samples following cell culture does not affect the diagnosis, and in certain cases can assist the ability to identify PCD cilia.

Mutations in CCDC39 and CCDC40 are the major cause of primary ciliary dyskinesia with axonemal disorganization and absent inner dynein arms.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by cilia and sperm dysmotility. About 12% of cases show perturbed 9+2 microtubule cilia structure and inner dynein arm (IDA) loss, historically termed "radial spoke defect." We sequenced CCDC39 and CCDC40 in 54 "radial spoke defect" families, as these are the two genes identified so far to cause this defect. We discovered biallelic mutations in a remarkable 69% (37/54) of families, including identification of 25 (19 novel) mutant alleles (12 in CCDC39 and 13 in CCDC40). All the mutations were nonsense, splice, and frameshift predicting early protein truncation, which suggests this defect is caused by "null" alleles conferring complete protein loss. Most families (73%; 27/37) had homozygous mutations, including families from outbred populations. A major putative hotspot mutation was identified, CCDC40 c.248delC, as well as several other possible hotspot mutations. Together, these findings highlight the key role of CCDC39 and CCDC40 in PCD with axonemal disorganization and IDA loss, and these genes represent major candidates for genetic testing in families affected by this ciliary phenotype. We show that radial spoke structures are largely intact in these patients and propose this ciliary ultrastructural abnormality be referred to as "IDA and microtubular disorganisation defect," rather than "radial spoke defect."

Generic microtiter plate assay for triaging clinical specimens prior to genotyping of human papillomavirus DNA via consensus PCR.

A generic human papillomavirus (HPV) probe assay was compared to the Linear Array to detect HPV DNA in 1,013 clinical specimens. The sensitivity, specificity, and negative predictive value of the assay were 99.5% (95% confidence interval [CI], 98.4% to 99.9%), 58.6% (95% CI, 53.9% to 63.1%), and 98.9% (95% CI, 96.5% to 99.8%), respectively. This assay conveniently identifies HPV-positive specimens.

Cost-effectiveness of high-risk human papillomavirus testing for cervical cancer screening in Québec, Canada.

OBJECTIVES: Human papillomavirus (HPV) testing is not widely used for triage of equivocal Pap smears or primary screening in Québec, Canada. Our objective was to evaluate the cost-effectiveness of cervical cancer screening strategies utilizing HPV testing. METHODS: We used a lifetime Markov model to estimate costs, quality of life, and survival associated with the following strategies: 1) cytology; 2) cytology with HPV testing to triage equivocal Pap smears; 3) HPV testing followed by colposcopy for HPV-positive women; 4) HPV testing with cytology to triage HPV-positive women; and 5) simultaneous HPV testing and cytology. Cytology was used in all strategies prior to age 30. Outcome measures included disease incidence, quality-adjusted life-years saved (QALYs), lifetime risk of cervical cancer, and incremental cost-effectiveness ratios. RESULTS: All strategies incorporating HPV testing as a primary screening test were more effective and less expensive than annual cytology alone, while HPV testing to triage equivocal Pap smears annually was very cost-effective ($2,991 per QALY gained compared to annual cytology alone). When compared to cytology every three years, HPV-based strategies cost an additional $8,200 to $13,400 per QALY gained. CONCLUSION: Strategies incorporating HPV testing are not only more effective than screening based on cytology alone but are also highly cost-effective. Provincial policy-makers should evaluate incorporating HPV-based strategies into current cervical cancer screening guidelines.

Process of care failures in invasive cervical cancer: systematic review and meta-analysis.

OBJECTIVE: As invasive cervical cancer is preventable when screening and treatment of pre-invasive lesions are timely and appropriate, several past studies attempted to enumerate the quality of preventive care invasive cervical cancer subjects received before diagnosis. Objectives of the present study were to review and to summarize the findings of these studies in a meta-analysis. METHOD: Data from 42 studies were used to estimate DerSimonian and Laird random effects models for the various failures in care along the cancer care continuum. Analyses were also conducted within strata characterized by variables deemed to account for heterogeneity in meta-regression analyses. RESULTS: Poor Pap screening frequency was the primary factor attributable to development of invasive cervical cancer. On average, 53.8% (95% confidence interval: 43.6-66.3) of invasive cervical cancer subjects had inadequate screening histories and 41.5% (95% confidence interval: 35.4-48.7) were never screened. There was significant temporal improvement in the proportion of women screened at least once over a lifetime but not in the proportion with overall deficient histories. An estimated 29.3% (95% confidence interval: 21.2-40.4) of failures to prevent invasive cervical cancer can be attributed to false-negative Pap smears and 11.9% (95% confidence interval: 9.0-15.6) to poor follow-up of abnormal results. CONCLUSION: Appropriate assessment of the effect of combined failures in the process of care must be done in comprehensive audit studies.

Trends in cancer prevalence in Quebec.

Cancer prevalence is of prime interest in public health because of its use in estimating the disease's burden on the heath care system. This study's objective was to estimate five-year prevalence of tumours from 1989 to 1999 and ten-year prevalence of tumours from 1994 to 1999 in the Province of Quebec (Canada). Five-year prevalence was used to represent tumours for which people are more likely to obtain primary treatment; ten-year prevalence included those tumours in addition to tumours that can be considered cured but still need follow-up. Information was extracted from the Quebec Cancer Registry. Prostate cancer was the most prevalent malignancy among males (25 percent, five-year prevalent tumours), while breast cancer was most prevalent among females (38 percent, five-year prevalent tumours). For both sexes, the greatest observed prevalence increase was for endocrine glands. On average, five-year prevalence proportions were 16 percent higher in men than in women; those of ten year were 14 percent higher in men. Furthermore, the largest differences were observed for bladder and lung cancer. The change in cancer prevalence in Quebec was dependent on the tumour site.