Defining infertility: a qualitative interview study of patients and physicians.

PURPOSE: To investigate if infertility patients and physicians apply a traditional biomedical model of disease in their conceptualisation of infertility, examine any contradictions and conflicts in conceptualisations, and examine areas of concordance and discordance between physicians and patients. METHODS: Semi-structured interviews were conducted with 20 infertility patients and 18 infertility physicians between September 2010 and April 2012. Interviews were analysed qualitatively to determine physician and patient conceptualisations of infertility, reactions to the definition of infertility as a disease, and potential benefits and concerns related to application of a disease label to the condition. RESULTS: Most physicians (n = 14/18) and a minority of patients (n = 6/20) were supportive of defining infertility as a disease. Many of the patients who agreed with classifying infertility as a disease expressed that they had not personally defined it as such previously. Physicians (n = 14) and patients (n = 13) described potential benefits of a disease label, including increases in research funding, insurance coverage, and social acceptability. Some patients (n = 10) described potential stigma as a negative consequence. When describing appraisals of infertility, both physicians (n = 7) and patients (n = 8) invoked religious/spiritual concepts. The potential for religious/spiritual appraisal to contribute to stigmatising or de-stigmatising infertility was discussed. CONCLUSION: Our findings contradict the assumption that infertility physicians and patients are fully supportive of defining infertility as a disease. While potential benefits of the disease label were recognised by both groups, caution against potential for stigmatisation and unsolicited invocation of religion/spirituality suggest a more holistic model may be appropriate.

Embryo blastulation and quality between days 5 and 6 of extended embryo culture.

PURPOSE: This study aims to know what proportion of culture day 5 pre-blastocyst-stage embryos develop into blastocysts by culture day 6 and what patient and cycle characteristics are associated with delayed blastocyst formation. METHODS: A retrospective observational cohort analysis was performed including a total of 9886 embryos from 1008 IVF cycles in 835 patients, who underwent treatment between January 1, 2016, and December 31, 2018. Autologous fresh in vitro fertilization (IVF) cycles at a single academic center were included in the analysis. Embryos were group-cultured using single-step culture media. Blastulation was defined as the presence of a new blastocyst. Usable blastulation was defined as the presence of a new good or excellent quality, expanded, hatching, or hatched blastocysts. RESULTS: The mean blastulation rate between days 5 and 6 of extended embryo culture was 30.9%. The mean percentage of embryos developing into usable blastocyst-stage embryos was 19.8%. The factors associated with blastulation on day 6 included the total number of embryos and the number of pre-blastocysts on day 5, as well as the use of ICSI. Age, the number of total embryos, those remained in culture and pre-blastocysts, as well as the blastulation rate on day 5 were associated with usable blastulation. CONCLUSION: It is important to know the usable blastocyst development rate between culture days 5 and 6 in order to adequately counsel patients debating whether to proceed with fresh ET on day 5 or forego ET with the expectation that embryos will be biopsied for PGT and/or cryopreserved on culture day 6. Our findings provide evidence to help guide patients in this difficult decision.

Comparative transcriptomic profiling of myxomatous mitral valve disease in the cavalier King Charles spaniel.

BACKGROUND: Almost all elderly dogs develop myxomatous mitral valve disease by the end of their life, but the cavalier King Charles spaniel (CKCS) has a heightened susceptibility, frequently resulting in death at a young age and suggesting that there is a genetic component to the condition in this breed. Transcriptional profiling can reveal the impact of genetic variation through differences in gene expression levels. The aim of this study was to determine whether expression patterns were different in mitral valves showing myxomatous degeneration from CKCS dogs compared to valves from non-CKCS dogs. RESULTS: Gene expression patterns in three groups of canine valves resulted in distinct separation of normal valves, diseased valves from CKCS and diseased valves from other breeds; the latter were more similar to the normal valves than were the valves from CKCS. Gene expression patterns in diseased valves from CKCS dogs were quite different from those in the valves from other dogs, both affected and normal. Patterns in all diseased valves (from CKCS and other breeds) were also somewhat different from normal non-diseased samples. Analysis of differentially expressed genes showed enrichment in GO terms relating to cardiac development and function and to calcium signalling canonical pathway in the genes down-regulated in the diseased valves from CKCS, compared to normal valves and to diseased valves from other breeds. F2 (prothrombin) (CKCS diseased valves compared to normal) and MEF2C pathway activation (CKCS diseased valves compared to non-CKCS diseased valves) had the strongest association with the gene changes. A large number of genes that were differentially expressed in the CKCS diseased valves compared with normal valves and diseased valves from other breeds were associated with cardiomyocytes including CASQ2, TNNI3 and RYR2. CONCLUSION: Transcriptomic profiling identified gene expression changes in CKCS diseased valves that were not present in age and disease severity-matched non-CKCS valves. These genes are associated with cardiomyocytes, coagulation and extra-cellular matrix remodelling. Identification of genes that vary in the CKCS will allow exploration of genetic variation to understand the aetiology of the disease in this breed, and ultimately development of breeding strategies to eliminate this disease from the breed.

Environmentally enriched pigs have transcriptional profiles consistent with neuroprotective effects and reduced microglial activity.

Environmental enrichment (EE) is widely used to study the effects of external factors on brain development, function and health in rodent models, but very little is known of the effects of EE on the brain in a large animal model such as the pig. Twenty-four young pigs (aged 5 weeks at start of study, 1:1 male: female ratio) were housed in environmentally enriched (EE) pens and provided with additional enrichment stimulation (a bag filled with straw) once daily. Litter, weight and sex matched controls n= (24) were housed in barren (B) conditions. Behaviour was recorded on alternate days from study day 10. After 21 days, RNA-sequencing of the frontal cortex of male piglets culled one hour after the enrichment stimulation, but not those at 4 h after stimulation, showed upregulation of genes involved in neuronal activity and synaptic plasticity in the EE compared to the B condition. This result is mirrored in the behavioural response to the stimulation which showed a peak in activity around the 1 h time-point. By contrast, EE piglets displayed a signature consistent with a relative decrease in microglial activity compared to those in the B condition. These results confirm those from rodents, suggesting that EE may also confer neuronal health benefits in large mammal models, through a potential relative reduction in neuroinflammatory process and increase in neuroprotection driven by an enrichment-induced increase in behavioural activity.

Myxomatous Degeneration of the Canine Mitral Valve: From Gross Changes to Molecular Events.

Myxomatous mitral valve disease (MMVD) is the single most common acquired heart disease of the dog, but is also of emerging importance in human medicine, with some features of the disease shared between both species. There has been increased understanding of this disease in recent years, with most research aiming to elucidate the cellular and molecular events of disease pathogenesis. For gross and histological changes, much of our understanding is based on historical studies and there has been no comprehensive reappraisal of the pathology of MMVD. This paper reviews the gross, histological, ultrastructural, cellular and molecular changes in canine MMVD.

Comparative transcriptome analysis of equine alveolar macrophages.

REASONS FOR PERFORMING STUDY: Alveolar macrophages (AMs) are the first line of defence against pathogens in the lungs of all mammalian species and thus may constitute appropriate therapeutic target cells in the treatment and prevention of opportunistic airway infections. Therefore, acquiring a better understanding of equine macrophage biology is of paramount importance in addressing this issue in relation to the horse. OBJECTIVES: To compare the transcriptome of equine AMs with that of equine peritoneal macrophages (PMs) and to investigate the effect of lipopolysaccharide (LPS) on equine AM. STUDY DESIGN: Gene expression study of equine AMs. METHODS: Cells from both bronchoalveolar and peritoneal lavage fluid were isolated from systemically healthy horses that had been submitted to euthanasia. Cells were cryopreserved. RNA was extracted and comparative microarray analyses were performed in AMs and PMs, and in AMs treated and untreated with LPS. Comparisons with published data derived from human AM studies were made, with particular focus on LPS-induced inflammatory status. RESULTS: The comparison between AMs and PMs revealed the differential basal expression of 451 genes. Gene expression analysis revealed an alternative (M2) macrophage polarisation profile in AMs and a hybrid macrophage activation profile in PMs, a phenomenon potentially attributable to a degree of induced endotoxin tolerance. The gene expression profile of equine AMs following LPS stimulation revealed significant changes in the expression of 240 genes, including well-known upregulated inflammatory genes. This LPS-induced gene expression profile of equine AMs more closely resembles that of human rather than murine macrophages. CONCLUSIONS: This study improves current understanding of equine macrophage biology. These data suggest that the horse may represent a suitable animal model for the study of human macrophage-associated lung inflammation and data derived from human macrophage studies may have significant relevance to the horse.

Large animal models of cardiovascular disease.

The human cardiovascular system is a complex arrangement of specialized structures with distinct functions. The molecular landscape, including the genome, transcriptome and proteome, is pivotal to the biological complexity of both normal and abnormal mammalian processes. Despite our advancing knowledge and understanding of cardiovascular disease (CVD) through the principal use of rodent models, this continues to be an increasing issue in today's world. For instance, as the ageing population increases, so does the incidence of heart valve dysfunction. This may be because of changes in molecular composition and structure of the extracellular matrix, or from the pathological process of vascular calcification in which bone-formation related factors cause ectopic mineralization. However, significant differences between mice and men exist in terms of cardiovascular anatomy, physiology and pathology. In contrast, large animal models can show considerably greater similarity to humans. Furthermore, precise and efficient genome editing techniques enable the generation of tailored models for translational research. These novel systems provide a huge potential for large animal models to investigate the regulatory factors and molecular pathways that contribute to CVD in vivo. In turn, this will help bridge the gap between basic science and clinical applications by facilitating the refinement of therapies for cardiovascular disease.

Dogslife: A cohort study of Labrador Retrievers in the UK.

Studies of animals that visit primary and secondary veterinary centres dominate companion animal epidemiology. Dogslife is a research initiative that collects data directly from owners about the health and lifestyle of Kennel Club (KC) registered Labrador Retrievers (LR) in the UK. The ultimate aim is to seek associations between canine lifestyle and health. A selection of data from Dogslife regarding the height, weight and lifestyle of 4307 LR up to four years of age is reported here. The majority of the dogs were household pets, living with at least one other pet, in families or households with more than one adult. The dogs typically ate diets of dried food and daily meal frequency decreased as the dogs aged. Working dogs spent more time exercising than pets, and dogs in Wales and Scotland were exercised more than their counterparts in England. Dogs in households with children spent less time exercising than dogs in other types of households. There was considerable variation in height and weight measurements indicative of a highly heterogeneous population. The average male height at the shoulders was 2-3cm taller than the UK breed standard. Dog weights continued to increase between one and four years of age. Those with chocolate coloured coats were heavier than their yellow and black counterparts. Greater dog weight was also associated with dogs whose owners reported restricting their dog's exercise due to where they lived. These findings highlight the utility of wide public engagement in the collation of phenotypic measures, providing a unique insight into the physical development and lifestyle of a cohort of LRs. In combination with concurrently collected data on the health of the cohort, phenotypic data from the Dogslife Project will contribute to understanding the relationship between dog lifestyle and health.

Histopathology and fibrillin-1 distribution in severe early onset Marfan syndrome.

Marfan syndrome (MFS) is an autosomal dominant condition which may involve the cardiovascular, ocular, skeletal, and other systems. Mutations causing MFS are found in the FBN1 gene, encoding fibrillin-1, an extracellular matrix protein involved in microfibril formation. In the most severe cases, mutations are generally found in exons 24-32, and children with these mutations usually die in the first years of life, of cardiopulmonary failure. We present clinical, molecular and histopathological studies on a patient with severe early onset MFS. He has a mutation in exon 25 of FBN1, a G>A transition at nucleotide position 3131 that converts the codon TGC, coding for cysteine at position 1044, to TAC, coding for tyrosine (C1044Y). This has resulted in abnormalities of the extracellular matrix and a severe clinical phenotype, although he has survived to the age of 14 years.

The murine chaperonin 10 gene family contains an intronless, putative gene for early pregnancy factor, Cpn10-rs1.

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. Human platelet-derived EPF shares amino acid sequence identity with chaperonin 10 (Cpn10), a mitochondrial matrix protein which functions as a molecular chaperone. The striking differences in cellular localization and function of the two proteins suggest differential regulation of production reflecting either alternative transcription of the same gene or transcription from different genes. In mammals and more distantly related genera, there is a large gene family with homology to CPN10 cDNA, which includes intronless copies of the coding sequence. To determine whether this could represent the gene for EPF, we have screened a mouse genomic library and sequenced representative Cpn10 family members, looking for a functional gene distinct from that of Cpn10, which could encode EPF. Eight distinct genes were identified. Cpn10 contains introns, while other members are intronless. Six of these appear to be pseudogenes, and the remaining member, Cpn10-rs1, would encode a full-length protein. The 309-bp open reading frame (ORF) is identical to that of mouse Cpn10 cDNA with the exception of three single-base changes, two resulting in amino acid changes. Only one further single nucleotide difference between the Cpn10-rs1 and Cpn10 cDNAs is observed, located in the 3' UTR. Single nucleotide primer extension was applied to discriminate between Cpn10-rs1 and Cpn10 expression. Cpn10, which is ubiquitous, was detected in all tissue samples tested, whereas Cpn10-rs1 was expressed selectively. The pattern was completely coincident with known patterns of EPF activity, strongly suggesting that Cpn10-rs1 does encode EPF. The complete ORF of Cpn10-rs1 was expressed in E. coli. The purified recombinant protein was found to be equipotent with native human platelet-derived EPF in the bioassay for EPF, the rosette inhibition test.

Production of a recombinant form of early pregnancy factor that can prolong allogeneic skin graft survival time in rats.

Early pregnancy factor (EPF), an extracellular chaperonin 10 homologue, has immunosuppressive and growth factor properties. In order to carry out more extensive studies on the in vivo characteristics of EPF, a recombinant form of the molecule has been prepared. Recombinant human EPF (rEPF) was expressed in Escherichia coli using the plasmid pGEX-2T expression system. Potency of rEPF in vitro in the rosette inhibition test, the bioassay for EPF, was equivalent to that of native EPF (nEPF), purified from human platelets, and synthetic EPF (sEPF). However, the half-life of activity (50% decrease in the log value) in serum, following i.p. injection, was significantly decreased (3.2 h, compared with nEPF 6.2 days, sEPF 5.8 days). This was thought to be due to modification of the N-terminus of the recombinant molecule inhibiting binding to serum carrier proteins. Because EPF can modify Th1 responses, the ability of the recombinant molecule to suppress allogeneic graft rejection was investigated. Following skin grafts from Lewis rats to DA rats and vice versa, rEPF was delivered locally at the graft site and the effect on survival time of the allografts noted. Results demonstrated that rEPF treatment significantly prolonged skin graft survival time by as much as 55% in stringent models of transplantation across major histocompatibility barriers.

Autosomal dominant cataracts and Peters anomaly in a large Australian family.

Peters anomaly is a congenital corneal opacity with underlying defects in the posterior stroma, Descemets membrane and corneal endothelium. It is a disorder resulting from abnormal migration or function of neural crest cells and may include abnormalities of other anterior segment structures, such as the lens and iris. We report a family in which anterior segment abnormalities, including Peters anomaly and cataracts, were inherited in an autosomal dominant fashion. Although the PAX6 gene on chromosome 11 has been shown to be involved in some cases of anterior segment developmental defects, we found no evidence that the condition in this family is linked to the PAX6 gene. Identification of this gene will indicate another gene with major involvement in the development of the anterior segment of the eye.

Mapping and characterization of the eukaryotic early pregnancy factor/chaperonin 10 gene family.

Early pregnancy factor and mitochondrial chaperonin 10 have very different functions within mammals but the mature peptides have identical amino acid sequences. In order to understand the mechanisms by which identical proteins can have different functions and sites of activity, we have examined genomic DNA which could encode the protein. In most species studied, there is a large gene family of at least ten members with homology to the DNA sequence for this protein. Using a monochromosomal somatic cell hybrid panel, we have mapped the gene for human chaperonin 10 to chromosome 2. Other members of the human gene family map to several chromosomes. Chromosomes 1, 2 and 9 contain pseudogenes with Alu insertions while chromosome 16 has a pseudogene containing a short direct repeat flanking an insert. Chromosomes 1 and 16 may also carry a functional intronless copy of the EPF/Cpn10 sequence.

Association of the brain natriuretic peptide gene with blood pressure and heart weight in the rat.

1. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) are important in the control of body fluid homeostasis, blood pressure (BP) regulation and vascular remodelling. The genes for these peptides may, therefore, be involved in the pathogenesis of genetic hypertension. We have previously described a quantitative trait locus (QTL) for BP in the ANP gene region on rat chromosome 5. We have now assessed the possibility that this QTL lies at the closely linked BNP locus. 2. Intra-arterial BP and heart weight were measured in 12-week-old (n = 207) and 24-week-old (n = 88) F2 rats derived from crosses between Wistar-Kyoto normotensive rats and spontaneously hypertensive rats. We designed polymerase chain reaction primers to amplify a microsatellite in the BNP gene from genomic DNA. Analysis of variance was used for cosegregation analysis. Linkage mapping and localization of QTL was performed using the Mapmaker computer package. 3. A significant correlation was found between genotype for the BNP gene and systolic BP (P < 0.001) in 12-week-old rats. The ANP gene, but not the BNP gene, was associated with systolic BP in 24 week rats. There was no segregation of heart weight with BNP genotype at 12 or 24 weeks of age. The BNP gene mapped approximately 20 cM from the ANP gene in our rat hybrids, away from the previously described QTL. There was evidence for a second BP locus near to but distinct from the BNP gene. 4. These results suggest that BP QTL are present in the natriuretic peptide gene region but that the ANP and BNP genes themselves have no major effect on BP in this cross.

Association analysis of six candidate genes in a sample of Australian hypertensive patients.

1. Essential hypertension is characterized by increased vascular resistance due to narrowing of the small arterioles. This may be influenced by vasoactive substances, cell growth and vascular remodelling. 2. A sample of Australian hypertensive and normotensive subjects was investigated for association with genetic markers which are candidates for a role in blood pressure (BP) regulation due to potential effects on vascular diameter. 3. The six markers used were for genes encoding vasoconstrictors, growth factors and a structural protein of the extracellular matrix. 4. No significant association of any of the markers used was found with BP status in this sample of patients.

Renin-angiotensin system gene polymorphisms and left ventricular hypertrophy. The case against an association.

There is accumulating evidence for association between genetic polymorphisms of components of the renin angiotensin system (RAS), especially angiotensin-converting enzyme (ACE), and cardiovascular disease. However, there is lack of agreement that the ACE polymorphism is associated with left ventricular hypertrophy (LVH) in hypertension. A possible paradigm for the development of LVH involves the ACE gene polymorphism influencing cardiac mass by an action on plasma and/or tissue levels of angiotensin II. Such a model has experimental support and provides the basis for examining the lack of agreement between studies. The finding of lack of association between RAS gene polymorphism and LVH may be due to methodological problems, differences in genetic background between populations, interactions between genetic variants of RAS components or to the model being inappropriate. Low predictability of ACE genotype markers for LVH together with conflicting reports on the influence of RAS genetic variants on angiotensin II production suggests that the simple RAS paradigm may not apply for hypertension. Further information on the nature of the link between the ACE polymorphism and ACE regulation as well as the relation between the RAS and pathophysiology of LVH is needed. At present there is insufficient evidence to accept ACE gene polymorphism as a susceptibility marker for LVH.

Cosegregation of genes on chromosome 5 with heart weight and blood pressure in genetic hypertension.

Genetic factors may be involved in both essential hypertension and cardiac hypertrophy. To identify genes contributing to elevated for blood pressure and cardiac hypertrophy in the spontaneously hypertensive rat (SHR), we performed a cosegregation analysis between blood pressure and heart weight and microsatellite markers for the candidate gene ANF on chromosome 5 in F2 animals obtained by mating SHR with Wistar-Kyoto (WKY) rats. We found evidence for a quantitative trait locus (QTL) determining mean blood pressure on chromosome 5 between atrial natriuretic factor (ANF) and MITR-3893 loci. No evidence for a QTL influencing heart weight was found. We propose that in SHR, blood pressure and heart weight may be independently controlled by different genetic mechanisms and that a gene close to ANF locus on chromosome 5 contributes towards hypertension in these animals.

Angiotensin I converting enzyme gene cosegregates with blood pressure and heart weight in F2 progeny derived from spontaneously hypertensive and normotensive Wistar-Kyoto rats.

The present investigation examines the association of angiotesin I converting enzyme (ACE) genotypes with blood pressure and heart weight in an F2 population of rats derived from a cross between spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. ACE genotype of rats in the F2 population was determined using a microsatellite polymorphism. Our investigation revealed that cardiac mass was not correlated with blood pressure at 12 weeks of age within the SHR, WKY, F1 or F2 groups of rats. In male rats, ACE genotype accounted for approximately 20% of the difference in mean blood pressure between SHR and WKY rats. There was no effect in females. It was also responsible for 21%-29% of the difference in heart weight both in female and male animals. The allele derived from the SHR parent appeared recessive to the allele from WKY parent for both heart weight and blood pressure. These results suggest that a gene in the region of the ACE locus is one of the genetic factor influencing blood pressure and heart weight in SHR.