The implementation of a hepatitis C testing service in community pharmacies: I-COPTIC consensus statement.

OBJECTIVES: This aimed to develop a blueprint for an effective community pharmacy Hepatitis C virus (HCV) testing service by producing a consensus statement. STUDY DESIGN: This was a modified Delphi process. METHODS: We recruited a heterogenous panel of experts (who had been involved in the setup or delivery of a community pharmacy HCV testing service) by purposive and chain referral methods. We had three rounds of a modified Delphi process. The first was a series of questions with free text responses and was analysed using thematic analysis, and the second and third were statements for the respondents to rate using a 7-point Likert scale. Consensus was predefined in a published protocol, and the results were reviewed by a public and patient involvement panel before the statement was finalised. RESULTS: We had 24 participants, including community and hospital-based pharmacists, local pharmaceutical committee members, charity representatives (Hepatitis C Trust), local clinical service lead, nurse specialists and doctors. The response rate of the first, second and third rounds were 100%, 96% and 88%, respectively. After the third round, we had 60 statements that reached consensus. We discussed the accepted statements with a patient and public involvement group. We used these statements to produce the I-COPTIC statement and a graphical summary. CONCLUSIONS: We developed a blueprint for the design of a gold standard community pharmacy HCV testing service. We believe this will support the successful implementation of community pharmacy testing for HCV. Community pharmacy testing is an important service to help achieve and maintain HCV elimination.

Preimplantation genetic screening outcomes are associated with culture conditions.

BACKGROUND: Although preimplantation genetic screening (PGS) is widely offered, there are contradictory reports on the clinical merit of this procedure. Any gain from embryo selection following aneuploidy screening must significantly outweigh the impact of the procedure. Variability of technical expertise in embryo biopsy, blastomere fixation, fluorescence in situ hybridization analysis, along with suboptimal laboratory quality control and inappropriate patient selection may impact PGS outcomes. METHODS: To investigate such effects, a total of 1508 stimulated in vitro fertilization (IVF) cycles were retrospectively analysed. During 2004, a significant change was made to the embryo culture media used. Clinical outcomes from cycles with PGS were compared prior to and after the change in media and compared with matched controls not utilizing PGS during the same period. RESULTS: Clinical PGS success rates were found to improve following the media change. For patients aged less than 40, clinical outcomes following PGS were significantly lower than those without PGS prior to the change, but became equivalent after the change. For patients >or=40 years and

Towards defining parameters for a successful single embryo transfer in frozen cycles.

BACKGROUND: Twin pregnancies in IVF should be avoided by transferring embryos one at a time, even for frozen cycles. In this study, we investigated the effect of blastomere lysis and cleavage in singleton frozen embryo transfer (sFET) cycles. Outcomes were compared with the transfer of two embryos in frozen transfer cycles (dFET). METHODS: A retrospective analysis was performed on 891 FET cycles, involving 404 sFET and 487 dFET cycles. RESULTS: Overall, in sFET cycles, the pregnancy and implantation rates were 8.9 and 8.7%. When blastomere lysis was more than 25% but no greater than 50%, the pregnancy and implantation rates were 3.2%. If blastomere lysis was greater than 50% there were no pregnancies. If blastomere lysis was less than 25%, but with no cleavage, the pregnancy and implantation rates were 4.1%. The results significantly improved (P = 0.007) in the group with less than 25% lysis, when cleavage occurred. The pregnancy and implantation rates for this group were 17.3 and 16.6%. This was not significantly different from unselected two embryo transfers (22 and 12.7%,P = 0.2 and 0.19, respectively). There were 21 twins with dFET (19.6% of pregnancies) and none in sFET. CONCLUSION: Both blastomere lysis and cleavage affect the outcome in sFET. To avoid the risk of twins, sFET should be considered when the embryo shows less than 25% blastomere lysis and at least one blastomere cleaves.

Recent advances in the design and performance of research assistants.

Every laboratory needs personnel who work 24 hours a day 7 days a week, are efficient, obedient, capable of showing initiative, insightful and affable. Our experience of laboratory workers tells us that there is an immense way to go to achieve these goals. Having tired of the normal, Darwinian approach, a project has been initiated to achieve these ends through genetic technologies.

The selective removal of phosphorus from soil: is event size important?

Data from the Woburn Erosion Reference Experiment (Bedfordshire, UK) were used to test the hypothesis that losses of phosphorus (P) in small erosion events are as great as those in infrequent large events, and to examine the effect of storm characteristics on the selective enrichment of P in eroded sediment. For almost every plot event in the period 1988 to 1994, the clay-sized fraction of the sediment was enriched compared with the soil of the plots. There was more variation in clay enrichment for smaller erosion events than for larger ones. The clay and P contents of the sediment were strongly correlated (p < 0.01), and there was a wider range of P concentrations in the sediment derived from small events than in that from large events. However, individual events resulting in small soil losses (< 100 kg) did not account for greater P losses than larger events (> 100 kg). The greater frequency of smaller events, combined with the likelihood of higher P concentrations in the sediment, therefore accounted for a greater proportion of the P lost over the 6-yr period than the infrequent large events. Phosphorus concentrations generally increased with increasing peak discharge and decreased with increasing event duration. For the same return period, P losses were generally greater from plots cultivated up and down the slope than from those cultivated across the slope. Overall, our results suggest that small erosion events should be controlled to prevent P contamination of surface waters and that the most effective means of doing this are by the introduction of minimal tillage techniques and across-slope cultivations.

Toxic effects of oxygen on human embryo development.

The toxic effects of oxygen on the embryos of various animal species are reviewed. Methodologies for assessing embryonic damage are discussed and possible ways of preventing the damage are explored. Three methods of potentially minimizing oxidative damage to human embryos were tested using gametes, zygotes, and embryos from a clinical IVF programme: (i) decreasing the oxygen tension in the gas phase used for culture during insemination, fertilization, and embryo growth; (ii) changing the formulation of culture media to include some components designed to protect against oxidative damage; and (iii) reducing the duration of insemination to minimize the effect of oxidative damage caused by spermatozoal metabolism. Fertilization, cleavage, embryo utilization, pregnancy, and embryo implantation rates were used to monitor these changes. Although all three methods gave an increase in success rates, there was still a dramatic decrease in success with patient age. It is suggested that, although the system of handling and culturing embryos can be optimized with respect to embryonic mitochondrial function, there are inherent age-related defects in oocytes and embryos that are still more fundamental than the environmental conditions of the embryo.

A prisoner with chronic myeloid leukemia.

The health care received by prisoners in the area of tissue and organ transplantation is not well discussed or documented. We encountered a prisoner with newly diagnosed chronic myelogenous leukemia who was a candidate for allogeneic bone marrow transplantation and had two HLA-identical siblings who were willing to donate bone marrow. Based on humanitarian, constitutional, and ethical considerations, we suggest that the prisoner (patient) should receive the same health care as individuals who are not incarcerated and that the costs of care should play no greater role for prisoners than for other members of society.

Time course of pronuclear formation and fertilisation after insemination in vitro and intracytoplasmic sperm injection of in vitro matured sheep oocytes.

The time course of sperm decondensation, oocyte activation, pronuclear formation and the possible causes of abnormalities after intracytoplasmic sperm injection (ICSI) and in vitro fertilisation (IVF) were examined. Frozen-thawed and pooled fresh semen from three different rams were washed and capacitated for ICSI or IVF. In vitro matured oocytes were cultured after sperm injection for 0.5, 0.75, 1, 2, 3, 4, 5, 6, 8, 18, 21 and 23 h, and oocytes were cultured after in vitro insemination for the same times other than 18 and 23 h. All oocytes were cultured in bicarbonate-buffered synthetic oviduct fluid medium (BSOF) supplemented with 2% oestrous sheep serum. A total of 746 metaphase II oocytes were injected with a single spermatozoon and 986 oocytes were inseminated for IVF. The earliest oocyte activation after ICSI was observed at 0.5 h, when 14.8% of oocytes were in anaphase II; this was earlier than after IVF, when only 6.4% of the oocytes exhibited anaphase II 1 h after insemination. Decondensing spermatozoa were first observed 1 h after ICSI and 3 h after insemination for IVF. The earliest female and male pronuclei after ICSI were observed at 2 and 3 h respectively, while the female and male pronuclei after IVF were observed at 4 h after insemination. The overall fertilisation rate was lower after ICSI (28.6%) than IVF (70.4%) but the percentage of abnormal fertilisation was not different between ICSI (8.7%) and IVF (15.2%). It was concluded that the fertilisation events were more advanced for ICSI than IVF, using injection and insemination time as reference points. The formation of male and female pronuclei were asynchronous after ICSI, in contrast to IVF when they appeared simultaneously at 4 h. Abnormalities found in fertilisation after ICSI may therefore be induced by the injection technique.

Sheep oocyte activation after intracytoplasmic sperm injection (ICSI).

The effect of calcium concentration on fertilization and activation was examined in oocytes injected in vitro with sperm. Oocytes were subjected to sperm injection, to sham injection or remained uninjected, and were then cultured for 19 h in bicarbonate-buffered synthetic oviduct fluid (BSOF) without calcium, or containing either calcium chloride or calcium ionophore. There was no difference in fertilization rates after ICSI when oocytes were cultured in vitro in media containing calcium chloride or calcium ionophore but the rate was lower in calcium-free media. There was also no difference in the fertilization rate after ICSI when oocytes were cultured in vivo compared with that observed in vitro in media containing calcium chloride or calcium ionophore. In calcium chloride-treated oocytes, activation was induced by mechanical injection, and in calcium ionophore-treated oocytes, by the ionophore. In uninjected oocytes, calcium itself did not cause oocyte activation. It is concluded that it is possible to induce activation by the injection process, but that manipulation alone is inadequate to cause proper oocyte activation unless calcium is also present. No difference in oocyte activation between ICSI and sham injection was found, indicating that the sperm may play no role in the early events of oocyte activation.

Do alterations in the sex ratio occur at fertilization? A case report using fluorescent in situ hybridization.

PURPOSE: The mechanisms by which the sex ratio might be altered at fertilization were reviewed, following a case of preimplantation gender analysis revealing a significantly skewed proportion of male-to-female embryos. METHODS: The case of a known carrier of X-linked hydrocephalus with a history of three affected male pregnancies is presented. Her husband's family history consisted of a strong increase in the number of males relative to females. She had four cycles of stimulated in vitro fertilization, with sex chromosome analysis using fluorescent in situ hybridization (FISH) on suitable cleavage-stage embryos. The difference in the sex ratio of normal male-to-female embryos was compared using a significance probabilities test for sex ratio. The sex ratio of sperm from a semen sample from the male partner was determined by FISH. RESULTS: Fifty embryos were suitable for analysis. A significantly higher number of normal male (n = 20) than normal female (n = 8) embryos was obtained (P < 0.05). The FISH assessment of the husband's semen analysis revealed no alteration in the normal X:Y ratio. CONCLUSIONS: As the sperm analysis revealed a normal X:Y ratio, an alteration in the embryo sex ratio might be explained by the preferential binding of Y-bearing sperm to the oocyte, an oocyte-related "discouragement" of binding of X-bearing sperm, or a postfertilization event.

Cleavage, development and competence of sheep embryos fertilized by intracytoplasmic sperm injection and in vitro fertilization.

More abnormal fertilization has been found in sheep oocytes after intracytoplasmic sperm injection (ICSI) than after in vitro fertilization (IVF). Although the birth of a normal lamb has been reported, the efficiency of blastocyst production is low. We therefore evaluated the cleavage, development and viability of sheep embryos obtained from ICSI, IVF and sham injection. In vitro matured oocytes either injected or inseminated with spermatozoa were assessed for cleavage 1 and 4 d after injection or insemination, and for development to blastocyst after 7 d of culture. A total of 699 oocytes was injected (ICSI); 198 (30.6%) were activated and 55 (8.5%) developed to the blastocyst stage. Of the 17 recipient ewes with 1, 2, 3 or 4 embryos, 15 (88.2%) were pregnant on Day 18; of these 17 recipients, 7 (41.1%) and 6 (35.2%) ewes remained pregnant on Days 45 and 110, respectively. Two normal lambs were born, one ewe died on Day 110 with 2 normal male fetuses, another ewe aborted on Day 90 and 4 pregnancies were maintained. A total of 517 oocytes was inseminated (IVF); 296 (62%) were activated and 90 (18.8%) reached the blastocyst stage. A total of 19 ewes received 1, 2, 3 or 4 embryos; of these, 13 (68.4%) were pregnant on Day 18, 8 (42.1%) ewes remained pregnant on each of Days 45 and 110. Three ewes delivered 5 lambs. Five pregnancies were maintained. A total of 156 oocytes was sham injected, 38 (24.3%) were activated and no blatocysts were obtained after culture. The results of this study showed that blastocysts obtained after ICSI are potentially viable and are not a result of parthenogenesis.

Expression of immunoglobulin superfamily cell adhesion molecules on murine embryonic stem cells.

The expression of cell adhesion molecules of the Ig superfamily (Ig-CAM) were examined on embryonic stem (ES) cells during culture in vitro. ES cells maintained an undifferentiated phenotype when cultured in the presence of leukemia inhibitory factor (LIF) or with fibroblast feeder cells; > 90% of cells reacted positively to an antibody (ECMA-7) that marks undifferentiated ES cells. Using flow cytometry, high concentrations of ICAM-1, VCAM-1, and NCAM antigens were detected on undifferentiated ES cells, but their specific receptors, Mac-1, LFA-1, and VLA-4, were not detected. There was also no class I or II major histocompatibility complex (MHC) antigen expression. The ICAM-1 expressed was functional, since anti-ICAM-1 significantly (p < 0.0001) blocked ES cell-lymphocyte binding. Ig-CAM and MHC-1 expression on undifferentiated ES cells was not up-regulated by treatment of cells with interferon-gamma (IFN-gamma), tumor necrosis factor alpha, or flavivirus infection, agents that up-regulate these molecules in other embryonic cell types. Twelve hours after LIF withdrawal, ICAM-1 and NCAM expression decreased significantly, while VCAM-1 was undetectable. However, morphology and ECMA-7 expression remained unchanged. Similar patterns of expression were seen on ES cells maintained on fibroblast feeder cells. This suggests that LIF or other cytokines may maintain the expression of Ig-CAMs on undifferentiated cells. Differentiation was induced by dimethyl sulfoxide treatment for 14 days. Cells changed from a colony-forming to a monolayer morphology, and approximately 60% of the cell population no longer expressed ECMA-7. In these cells, VCAM-1 was undetectable and ICAM-1 and NCAM had declined to low levels. In these differentiated cells, ICAM-1 and MHC-1 were inducible by IFN-gamma. This study suggests that the pattern of expression of the Ig-CAMs in ES cells may have a role in defining the phenotype of differentiated and undifferentiated cells.

Effect of culture, incubation and acrosome reaction of fresh and frozen-thawed ram spermatozoa for in vitro fertilization and intracytoplasmic sperm injection.

This study evaluated different sperm treatments for fertilization of sheep oocytes by intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF). In Experiment 1, fresh and frozen semen was separated by Percoll centrifugation and incubated at 30 degrees C or 39 degrees C in HSOF or BSOF medium for 1 h before use for IVF or ICSI. For IVF, oocytes were inseminated and incubated with sperm for 30 min, 4 h and 19 h. Sperm were assessed for acrosome integrity after Percoll centrifugation and 1 h incubation, and those used for IVF were assessed after each period of exposure to the oocytes. Fertilization rates after ICSI were higher for fresh than for frozen-thawed sperm and were highest 19 h after IVF with fresh or frozen-thawed sperm in the presence of HSOF at 30 degrees C. In Experiment 2, fresh semen was separated by Percoll centrifugation and incubated for 5 h in HSOF, and the acrosome reaction was induced with lysophosphatidylcholine. Acrosome integrity was then assessed. Fertilization rates after ICSI were similar for acrosome-reacted and control spermatozoa. These results suggest that induction of the acrosome reaction in spermatozoa before ICSI is unnecessary, whereas a capacitating treatment of spermatozoa is required before IVF.

Birth of a male lamb derived from an in vitro matured oocyte fertilised by intracytoplasmic injection of a single presumptive male sperm.

The developmental competence of in vitro matured ovine oocytes, cytoplasmically injected with single male or female chromosome-bearing sperm, was investigated. Eighty-five unsorted, 92 'female-sorted' and 74 'male-sorted' ram sperm were injected into in vitro matured sheep oocytes and, two to four hours later, placed into the oviducts of 28 oestrous sheep. The sperm were separated according to sex by analysis of their DNA content with a flow cytometer. One pregnancy was diagnosed by ultrasound after 55 days and a 3 kg male lamb was born after 150 days gestation. This lamb was derived from an oocyte injected with 'male-sorted' sperm.

Manipulation of sperm before intracytoplasmic sperm injection improves fertilization rates.

OBJECTIVE: To determine the effect of sperm manipulation before intracytoplasmic sperm injection (ICSI) on fertilization rates. DESIGN: Three methods of sperm manipulation before direct ICSI were compared in two sibling oocyte trials. SETTING: In vitro fertilization unit within a teaching hospital. PATIENTS: Patients undergoing infertility treatment using ICSI. INTERVENTIONS: Oocytes were inseminated by ICSI. MAIN OUTCOME MEASURE: Fertilization rate. RESULTS: In the first trial, a standard manipulation technique gave a 67% fertilization rate compared with 64% with a minimal manipulation technique. In the second trial the standard technique gave a significantly greater fertilization rate compared with no prior manipulation (67% versus 45%). CONCLUSIONS: Manipulation of sperm before direct ICSI is not mandatory for fertilization but significantly improves the fertilization rate.

Fertilization rates using intracytoplasmic sperm injection are greater than subzonal insemination but are dependent on prior treatment of sperm.

OBJECTIVE: To determine a suitable method of sperm preparation for use in micromanipulation. To compare the fertilization rates of sibling oocytes inseminated by intracytoplasmic sperm injection (ICSI) and subzonal insemination (SUZI). DESIGN: Two methods of gamete micromanipulation to overcome male factor infertility were compared. Within this study, three trials were conducted to determine the most suitable method of sperm preparation. This method then was used to complete the study. SETTING: Procedures were performed in a teaching hospital research environment. PATIENTS: Ninety-six patients were recruited for this study, undergoing 99 stimulation cycles. INTERVENTIONS: Sibling oocytes were inseminated by subzonal sperm or intracytoplasmic injection. MAIN OUTCOME MEASURES: Fertilization, zygote development, and pregnancy rates. RESULTS: In the absence of manipulative pretreatment of sperm (trial 1), there was no difference in normal fertilization rates between ICSI and SUZI (19% and 25%, respectively). In the second trial there again was no sperm pretreatment for SUZI but, for ICSI, a polyvinylpyrrolidone (PVP) solution was used to reduce sperm velocity and the sperm tails were incised before injection. The fertilization rates were significantly different between ICSI (44%) and SUZI (17%). In the third trial, PVP was added to the sperm used for both types of insemination and the sperm tails also were incised for the ICSI insemination. Fertilization was again significantly different (16% for SUZI and 44% for ICSI). The trial 2 method of sperm preparation then was used to complete the study (trial 4) and confirmed the results of trial 2 (21% for SUZI and 42% for ICSI). The results suggest that ICSI can give improved fertilization compared with SUZI provided the sperm are treated before injection. No significant differences were found in the development rates of zygotes suitable for transfer or cryopreservation between the two micromanipulation methods.

In vivo disposition and in vitro metabolism of an anxiolytic compound, BMS-184111, in rats.

Plasma concentrations of BMS-184111, an anxiolytic, were determined as a function of time following single intravenous, intraperitoneal and oral administrations. In order to assess the brain penetration of this compound, concentrations in whole brain samples were also determined in the intravenous leg of the study. Concentrations of BMS-184111 in plasma and brain homogenate samples were determined using an HPLC assay following liquid/liquid extraction. After intravenous administration, BMS-184111 was eliminated from plasma with a half-life of about 3.6 hours. The brain/plasma AUC ratio for BMS-184111 concentration was 5.5, indicating effective penetration of the compound into the brain. Comparison of the plasma AUC values obtained following intravenous and intraperitoneal doses indicated that BMS-184111 was only 33% bioavailable after intraperitoneal administration, suggesting that the compound undergoes significant first-pass hepatic extraction. The oral bioavailability of BMS-184111 was found to be 10% after administration of the free base and 23% after administration of the hydrochloride salt. These results suggest that BMS-184111 undergoes incomplete GI absorption and/or intestinal metabolism in addition to first-pass hepatic extraction. The in vitro metabolism of BMS-184111 was studied using rat liver homogenate preparation (the 9000 g supernatant; S-9). Several of the metabolites thus generated were profiled using LC/MS and LC/MS/MS. Metabolism of BMS-184111 in rat liver S-9 occurs through hydroxylation, O-demethylation, and demethylenation.

Comparative intracytoplasmic sperm injection (ICSI) in human and domestic species.

The current clinical use of intracytoplasmic sperm injection (ICSI) for the alleviation of male factor infertility has prompted a re-investigation of sperm injection techniques in a number of animal species. This report examines sperm injection of in vitro matured oocytes in the major domestic species and compares the results with the human. Ovine, bovine and porcine oocytes can undergo fertilization and at least limited development without exogenous activation either prior to or subsequent to injection. Porcine is temperature sensitive during fertilization and the early stages of embryo development. The oocytes of all three domestic species, particularly ovine, have a tendency to activate after the injection procedure regardless of the presence or absence of sperm. The implications for early development studies and the practical use of direct sperm injection for domestic species are discussed.