The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

The UTX gene escapes X inactivation in mice and humans

We recently have identified a ubiquitously transcribed mouse Y chromosome gene, Uty , which encodes a tetratricopeptide repeat (TPR) protein. A peptide derived from the UTY protein confers H-Y antigenicity on male cells. Here we report the characterization of a widely transcribed X-linked homologue of Uty , called Utx , which maps to the proximal region of the mouse X chromosome and which detects a human X-linked homologue at Xp11.2. Given that Uty is ubiquitously transcribed, we assayed for Utx expression from the inactive X chromosome (Xi) in mice and found that Utx escapes X chromosome inactivation. Only Smcx and the pseudoautosomal Sts gene on the mouse X chromosome have been reported previously to escape inactivation. The human UTX gene was also found to be expressed from Xi. We discuss the significance of these data for our understanding of dosage compensation of X-Y homologous genes in humans and mice.      

High throughput parallel analysis of hundreds of patient samples for more than 100 mutations in multiple disease genes

As more mutations are identified in genes of known sequence, there is a crucial need in the areas of medical genetics and genome analysis for rapid, accurate and cost-effective methods of mutation detection. We have developed a multiplex allele-specific diagnostic assay (MASDA) for analysis of large numbers of samples (> 500) simultaneously for a large number of known mutations (> 100) in a single assay. MASDA utilizes oligonucleotide hybridization to interrogate DNA sequences. Multiplex DNA samples are immobilized on a solid support and a single hybridization is performed with a pool of allele-specific oligonucleotide (ASO) probes. Any probes complementary to specific mutations present in a given sample are in effect affinity purified from the pool by the target DNA. Sequence-specific band patterns (fingerprints), generated by chemical or enzymatic sequencing of the bound ASO(s), easily identify the specific mutation(s). Using this design, in a single diagnostic assay, we tested samples for 66 cystic fibrosis (CF) mutations, 14 beta-thalassemia mutations, two sickle cell anemia (SCA) mutations, three Tay-Sachs mutations, eight Gaucher mutations, four mutations in Canavan disease, four mutations in Fanconi anemia, and five mutations in BRCA1. Each mutation was correctly identified. Finally, in a blinded study of 106 of these mutations in > 500 patients, all mutations were properly identified. There were no false positives or false negatives. The MASDA assay is capable of detecting point mutations as well as small insertion or deletion mutations. This technology is amenable to automation and is suitable for immediate utilization for high-throughput genetic diagnostics in clinical and research laboratories.      

Stimulation of β-adrenoceptors inhibits store-operated channel currents via a cAMP-dependent protein kinase mechanism in rabbit portal vein myocytes

Previously we have described the properties of store-operated channel currents (SOCs) in freshly dispersed rabbit portal vein smooth muscle cells. In addition to Ca²⁺ store depletion these SOCs could also be activated by α-adrenoceptor stimulation and diacylglycerol (DAG) via a protein kinase C (PKC)-dependent mechanism. In the present study we have investigated the effect of β-adrenoceptor stimulation on SOCs in rabbit portal vein myocytes. With whole-cell recording the selective β-adrenoceptor agonist isoprenaline reduced the current evoked by cyclopiazonic acid (CPA, sarcoplasmic/endoplasmic reticulum ATPase inhibitor) by over 85%. With cell-attached patch recording, bath application of isoprenaline produced a pronounced inhibition of SOC activity evoked by either CPA or the acetoxymethyl ester form of BAPTA (BAPTA-AM). SOC activity evoked by CPA, the DAG analogue, 1-oleoyl-acetyl- sn -glycerol (OAG) or the phorbol ester, phorbol-12,13-dibutyrate (PDBu) was also markedly inhibited by the adenylate cyclase activator, forskolin, and the cell-permeable non-hydrolysable analogue of cyclic adenosine monophosphate (cAMP), 8-Br-cAMP. With inside-out patches, bath application of PDBu evoked channel currents with similar properties to SOCs which were inhibited by over 90% by a catalytic subunit of protein kinase A (PKA) and by 8-Br-cAMP. Moreover bath application of PKA inhibitors, H-89, KT5720 and an inhibitory peptide to quiescent cell-attached or inside-out patches, activated channel currents with similar properties to SOCs. These data suggest that in rabbit portal vein myocytes, stimulation of β-adrenoceptors inhibits SOC activity via a cAMP-dependent protein kinase signal transduction cascade. In addition it is concluded that constitutive PKA activity has a profound inhibitory effect on SOC activity in this vascular preparation.     

Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Importance of progesterone and estrogen priming for the induction of maternal behavior by vaginocervical stimulation in sheep: effects of maternal experience

The effects of treating ovariectomised nulliparous and multiparous ewes with progesterone and estrogen on maternal behavior with or without vagino-cervical stimulation were investigated. Following 48 h of estrogen treatment there was neither evidence for stimulation of maternal behavior, nor a reduction in aggressive behavior, towards lambs by either multiparous or nulliparous ewes compared with control treatment (injections of the oil vehicle). Following 5 min of mechanical vaginocervical stimulation, the multiparous estrogen-treated ewes showed both positive maternal responses (low pitch bleats, licking, sniffing and approaching the lamb) and a reduction in aggression (butts) and negative behavior (withdrawal from the lamb) towards the lambs, whereas the nulliparous ewes showed only a reduction in aggression and negative behavior. Neither group showed positive maternal responses following vaginocervical stimulation without steroid priming. When the ewes received two weeks of progesterone priming prior to estrogen treatment, both multiparous and nulliparous ewes showed reduced aggression towards, and withdrawal from, the lambs compared to the control condition, but no positive maternal responses apart from an increased sniffing of the lamb. After vaginocervical stimulation positive maternal responses showed by multiparous ewes were significantly potentiated compared to those shown following estrogen treatment alone. Nulliparous ewes remained unresponsive on positive maternal behaviors. These studies show that hormonal priming has little effect in inducing the onset of maternal behavior in multiparous or nulliparous sheep but is an essential prerequisite for vaginocervical stimulation to do so in multiparous animals.     

Vasoconstrictor Fibre Discharge to Skeletal Muscle,Kidney, Intestine and Skin at Varying Levels of Arterial Baroreceptor Activity in the Cat

Estimations of the average discharge rates in vasoconstrictor fibres to skeletal muscle, kidney, intestine and skin at varying levels of arterial baroreceptor activity were made by comparing quantitatively the reflexly induced vascular responses with those obtained with stimulations of the regional vasoconstrictor fibres at known frequencies. The results indicate that withdrawal of baroreceptor restraint was normally followed by a considerably lower firing rate in renal vasoconstrictor fibres than in e.g. skeletal muscle vasoconstrictor fibres. A complete elimination of the inhibitory influence induced vasoconstrictor fibre discharge rates up to 8 imp/s in skeletal muscle but only at most 4 imp/s in the kidney. Intestinal vasomotor fibres were found to be activated to approximately the same extent as those to skeletal muscle. The vasomotor fibres to the cutaneous arterio-venous anastomoses were evidently not activated when the baroreceptors were unloaded. The reason for this non-uniform activation of the different sets of vasoconstrictor fibres seems to be different levels of spontaneous activity in the respective “neuron pools” in the bulbar vasomotor centre. The renal vascular responses to baroreceptor unloading became augmented when the excitatory level in the vasomotor centre was enhanced, as e.g. when the animal became acidotic.     

Bereavement care in general practice: a survey in South Thames Health Region.

BACKGROUND: Studies have shown that bereaved individuals suffer increased rates of physical and mental ill health. Bereavement support has recently been advocated as an area of prevention in primary care, with suggestions that general practitioners (GPs) should adopt protocols for the active follow-up of their bereaved patients, which relies on the early notification of deaths by hospitals and hospices. Little is known about the routine care currently provided by GPs and primary health care teams (PHCTs) to support their bereaved patients. AIMS: To explore GPs' perceptions of patient death notifications by hospitals and hospices. To describe practice policies relating to patient deaths and the provision of bereavement support. METHOD: Postal questionnaires were sent to senior partners of a random sample of 500 general practices in South Thames Health Region. RESULTS: Three hundred and fifty-three practitioners responded (71%). Hospitals were perceived to be significantly slower than hospices in notifying deaths (P < 0.0001). One hundred and ninety-six practices (56%) kept death registers, 230 (65%) discussed deaths together, and 142 (40%) identified bereaved relatives. One hundred and thirty-seven practices (39%) routinely offered bereaved relatives contact with a PHCT member; while 133 (38%) supported only those who asked for help. Routine support was significantly more likely to be provided by practices that kept a death register, discussed deaths together, identified bereaved relatives, and had a special interest in palliative care. CONCLUSIONS: GPs perceive hospitals to be slower than hospices at notifying deaths, particularly in the first 24 hours. They are divided over whether bereavement support should be proactive or reactive. Keeping a practice death register, discussing deaths together, and identifying newly bereaved relatives are activities related to providing routine bereavement care.     

Limiting factors for cytopathological diagnosis of high-grade squamous intraepithelial lesions: a cytohistological correlation between findings in cervical smears and loop electrical excision procedure.

The present study sought possible factors leading to the cytological diagnosis of atypical squamous cells of uncertain significance (ASCUS) in cases of high-grade squamous intraepithelial lesions (HSIL). Based on retrospective histopathological analysis of loop electrical excision procedure (LEEP) products that diagnosed HSIL, two study groups were randomly selected. The first was consisted of cases with two consecutive Papanicolaou (Pap) smears with the diagnosis of ASCUS. The second (control) group was represented by cases diagnosed as HSIL by cytology. From the Pap smears diagnosed as ASCUS, the sampling limitations was different from control group (P < 0.05). The median size of the largest lesion in each case with ASCUS was 2.66 mm (+/- 1.71 mm). In the control group, the median size of the largest lesion was 5.15 mm (+/-2.58 mm) (P < 0.05). The size of the lesion and sample limitations led patients with cervical intraepithelial neoplasms to be diagnosed as ASCUS for two consecutive times, after a 6-mo period.