Case reports to suggest an algorithm for management of total fertilisation failure prior to use of donor gametes.

PURPOSE: Total fertilisation failure (TFF), even with intracytoplasmic sperm injection (ICSI), occurs in approximately 3 % of cycles, can be recurrent and the exact cause is difficult to elucidate. Differentiation between oocyte and sperm-related cause of TFF is possible using mouse oocyte-activation techniques, but is not an option within most clinical settings. Therefore, the management of these couples is clinically driven, and the endpoint, if recurrent, is often the use of donor gametes. However, with the invariable lack of a definitive cause of TFF, any decision between the use of donor sperm or oocytes remains an emotive one. We present two case reports demonstrating the importance of appropriate investigation, activation techniques (mechanical and chemical) and clinical management options to develop a clinical algorithm prior to the use of donor gametes. METHODS: This study is composed of two case reports of assisted reproduction investigation and treatment within an assisted conception unit for couples with recurrent total fertilisation failure. RESULTS: Using appropriate investigation (endocrine, urological and embryological) and treatments (ICSI, IMSI, oocyte-activation techniques), a fertilisation rate of 48 % was achieved in two cycles in couples following a total of nine previous cycles (and 200 previously collected eggs) with TFF. CONCLUSIONS: Oocyte activation requires the triggering of intracellular calcium oscillations by the release of a sperm-specific factor (phospholipase C zeta (PLCζ)) into the oocyte cytoplasm. Although, PLCζ deficiencies have been demonstrated as putative causes of failed activation, impaired oocyte responsiveness may also be a factor. The use of donor gametes is often recommended and is often the required endpoint of treatment. However, these reports outline a clinical algorithm that potentially offers success without donation, and also offers a systematic approach to help decide whether donor oocytes or sperm should be recommended.

Management of arterial lines and blood sampling in intensive care: a threat to patient safety.

In 2008, the UK National Patient Safety Agency (NPSA) made recommendations for safe arterial line management. Following a patient safety incident in our intensive care unit (ICU), we surveyed current practice in arterial line management and determined whether these recommendations had been adopted. We contacted all 241 adult ICUs in the UK; 228 (94.6%) completed the survey. Some NPSA recommendations have been widely implemented - use of sodium chloride 0.9% as flush fluid, two-person checking of fluids before use - and their practice was consistent. Others have been incompletely implemented and many areas of practice (prescription of fluids, two-person checking at shift changes, use of opaque pressure bags, arterial sampling technique) were highly variable. More importantly, the use of the wrong fluid as an arterial flush was reported by 30% of respondents for ICU practice, and a further 30% for practice elsewhere in the hospital. Our survey provides evidence of continuing risk to patients.

Patient selection criteria for blastocyst culture in IVF/ICSI treatment.

BACKGROUND: The development and refinement of blastocyst media in recent years has allowed embryos to be cultured in-vitro for 5 or 6 days post oocyte retrieval and has been established as an effective selection tool to aid embryo selection for IVF treatment. It is generally accepted that blastocyst culture is not an appropriate option for all patients but the criteria for patient selection varies between clinics. Our blastocyst culture programme started in February 2005; the patient criteria was set at a minimum of 4 oocytes retrieved, a minimum of 4 2PN pronuclear embryos and at least 4 8-cell embryos of any quality on Day 3 where the female patient was 34 years and under. In the female age group of 35 years and over the criteria was at least 6 oocytes retrieved, a minimum of 6 2PN pronuclear embryos and at least 6 8-cell embryos of any quality on day 3. Improvements in pregnancy rates demonstrated the effectiveness of blastocyst transfer and clinical opinion was that the criteria should be adjusted to allow this option to be available to an increased patient population. From February 2007 the blastocyst patient selection criteria was changed to at least 4 oocytes retrieved, at least 4 2PN pronuclear embryos and at least 2 8-cell and 2 6-cell or 7-cell embryos of top quality on Day 3 in women 38 years and under. For women 39 years and over the criteria was lowered to at least 5 eggs retrieved, at least 5 2PN and at least 3 8-cell embryos and 2 6-cell embryos of top quality on Day 3. METHODS: Retrospective statistical analysis was carried out to determine the pregnancy rates, live birth rates and twin rate for the period under the initial criteria and to examine the impact that lowering the criteria for patient selection for blastocyst culture had on these parameters. RESULTS: There was an overall fall in the ongoing pregnancy/live birth rate from 50.9% under the old criteria to 45.0% under the new criteria. However, the patients who had blastocyst culture under the new criteria but would have had day-3 embryo transfer under the initial criteria had a significantly increased live birth/ongoing pregnancy rate from 22.7% to 40.7%. There is an increase in the number of blastocyst culture cycles from 26.4% under the old criteria to 39.1% with the refined criteria. The twin pregnancy rate was reduced from 25.2% to 17.5%. CONCLUSION: The result of this cohort study revealed that lowering the blastocyst selection criteria may lead to a lower overall clinical live birth rate from blastocyst culture; however, it will benefit a specific group of patients to achieve a better pregnancy and live birth rate. Furthermore, it increases the number of patients who will benefit from the blastocyst culture programme and also reduces multiple pregnancy rate.

Primer-dependent synthesis by poliovirus RNA-dependent RNA polymerase (3D(pol)).

Properties of poliovirus RNA-dependent RNA polymerase (3D(pol)) including optimal conditions for primer extension, processivity and the rate of dissociation from primer-template (k(off)) were examined in the presence and absence of viral protein 3AB. Primer-dependent polymerization was examined on templates of 407 or 1499 nt primed such that fully extended products would be 296 or 1388 nt, respectively. Maximal primer extension was achieved with low rNTP concentrations (50-100 microM) using pH 7 and low (<1 mM) MgCl(2) and KCl (<20 mM) concentrations. However, high activity (about half maximal) was also observed with 500 microM rNTPs providing that higher MgCl(2) levels (3-5 mM) were used. The enhancement observed with the former conditions appeared to result from a large increase in the initial level or active enzyme that associated with the primer. 3AB increased the number of extended primers at all conditions with no apparent change in processivity. The k(off) values for the polymerase bound to primer-template were 0.011 +/- 0.005 and 0.037 +/- 0.006 min(-1) (average of four or more experiments +/- SD) in the presence or absence of 3AB, respectively. The decrease in the presence of 3AB suggested an enhancement of polymerase binding or stability. However, binding was tight even without 3AB, consistent with the highly processive (at least several hundred nucleotides) nature of 3D(pol). The results support a mechanism whereby 3AB enhances the ability of 3D(pol) to form a productive complex with the primer-template. Once formed, this complex is very stable resulting in highly processive synthesis.

Molecular expression of the negative growth factor murine beta-galactoside binding protein (mGBP).

Characterisation of the negative growth factor mGBP at molecular and biological levels indicates that the protein has no lectin nature and suggests instead a participation in the cytokine network. The protein is shown to be expressed as a monomer in two forms, one of which is non-covalently linked to a glycan complex. This confers greater efficiency to the inhibitor and may favour a paracrine role. The two monomeric forms may oxidise into tetramers which retain biological activity, but lack ability to link to specific saccharide residues.

Structure and expression of the negative growth factor mouse beta-galactoside binding protein gene.

Following the identification of murine beta-galactoside binding protein (mGBP) as an autocrine negative growth factor we have now isolated and characterized the genomic region spanning the mGBP gene and have determined the 5' end of the transcript by primer extension, S1 mapping and mRNA sequence. The gene is found to be contained within 4 kilobases and composed of four exons of 79, 80, 171 and 197 nucleotides separated by three introns of 1200, 1600 and 193 nucleotides. The DNA region upstream of the 5' end of the transcript contains canonical sequences for eukaryotic promoter elements including CAT and TATA boxes and several DNA motifs for potential transcription regulation. The gene is differentially expressed in a variety of normal tissues.

Characterisation and antiproliferative activity of an alpha-type murine interferon from embryonic fibroblasts.

Interferons play a part in the negative control of cell proliferation of mammalian cells. Here a natural interferon has been isolated from soluble proteins secreted by secondary murine embryonic fibroblasts using Blue Sepharose chromatography, immunoaffinity exclusion and Q Sepharose ion exchange fractionation. Partial amino acid sequencing assigns it to the interferon alpha family. Its biological and physico-chemical properties single it out from all other murine alpha interferons. The embryonic interferon has stronger antiproliferative activity, is acid labile, has stronger affinity for Blue Sepharose and weak affinity for antibodies which recognise other murine interferon alpha subtypes.

Cell cycle arrest and induction of apoptosis by beta galactoside binding protein (beta GBP) in human mammary cancer cells. A potential new approach to cancer control.

Conflict between mitogenic pressure, as is the case in tumour cells and an imposed inability to proceed through the cell cycle may result in cell death. In the present study we examined the effect of beta galactoside binding protein (beta GBP), a negative growth factor which controls cell cycle transition from S phase into G2, on three human mammary cell lines which differ for oncogenic potential, oestrogen receptor expression and expression of the EGF receptor family. We found that in all cases beta GBP induced a cell cycle block prior to the cells' entry into G2 and that this was followed by progressive apoptotic death. This evidence on epithelial cancer cells parallels previous data on tumour cells of mesenchymal origin and suggests that beta GBP has potential therapeutic implications in the treatment of cancers.

Determination of the mutation rate of poliovirus RNA-dependent RNA polymerase.

The fidelity of poliovirus RNA-dependent RNA polymerase (3D(pol)) was determined using a system based on the fidelity of synthesis of the alpha-lac gene which codes for a subunit of beta-galactosidase. Synthesis products are screened for mutations by an alpha-complementation assay, in which the protein product from alpha-lac is used in trans to complement beta-galactosidase activity in bacteria that do not express alpha-Lac. Several polymerases have been analyzed by this approach allowing comparisons to be drawn. The assay included RNA synthesis by 3D(pol) on an RNA template that coded for the N-terminal region of alpha-Lac. The product of this reaction was used as a template for a second round of 3D(pol) synthesis and the resulting RNA was reverse transcribed to DNA by MMLV-RT. The DNA was amplified by PCR and inserted into a vector used to transform Escherichia coli. The bacteria were screened for beta-galactosidase activity by blue-white phenotype analysis with white or faint blue colonies scored as errors made during synthesis on alpha-lac. Results showed a mutation rate for 3D(pol) corresponding to approximately 4.5x10(-4) errors per base (one error in approximately 2200 bases). Analysis of mutations showed that base substitutions occurred with greater frequency than deletions and insertions.

Infectivity of a glucan synthesis-defective mutant of Streptococcus gordonii (Challis) in a rat endocarditis model.

Streptococcus gordonii, a member of the human indigenous oral microflora, colonizes smooth tooth surfaces and contributes to dental plaque formation. Although it is not recognized as being a cariogenic pathogen, it may cause endocarditis following invasion of the bloodstream. Using allelic exchange mutagenesis, we have constructed a mutant of S. gordonii (Challis) which is defective in its single functional glucosyltransferase gene and, hence, is unable to synthesize glucan exopolymers from sucrose. When examined in a rat endocarditis model, the sucrose-grown mutant did not differ significantly from S. gordonii wild-type, suggesting that glucan polymers did not contribute to infectivity. This result was in striking contrast to that previously observed with a polymer-defective S. mutans mutant.

NIOSH selection of chemicals and study publications: setting priorities for reproductive research.

The National Institute for Occupational Safety and Health (NIOSH) prioritizes topics for reproductive research in order to allocate limited resources to the most important issues. In the past, we have relied on an informal network including our own project officers, toxicologists, and the public to nominate chemicals to study. Setting priorities has consisted of the evaluation of research ideas by criteria such as the chemical's probable toxicity and the extent of exposure to the chemical in the workplace. NIOSH researchers are developing a system for evaluating large databases of toxicologic and occupational exposure information to identify potential reproductive toxins. When this system is operational it will provide a systematic way to nominate chemicals for study.

Methods of monitoring menstrual function in field studies: attitudes of working women.

This study was designed to determine the attitudes and compliance of working women toward methods being evaluated for use in the assessment of the effects of toxicants on reproductive potential. Women such as the highly motivated fertility patients and nurses, who are typically familiar with the methods and procedures of fertility assessment and the value of medical research, have been used to validate such methods in a clinical setting. However, the attitudes of a general working female population toward these methods are unknown. Nine participants were selected on the bases, in part, of not seeking fertility assistance, working full-time but not in the medical field, and having less than one year of college education. Attitudes were also evaluated for 193 non-participating women to whom the procedures had been verbally described. Participants measured basal body temperature and salivary and vaginal mucous electrical resistance, evaluated cervical mucus manually (CME), and collected the first morning urine for two menstrual cycles. Blood, saliva, and transvaginal ultrasonograms (US) were obtained at a fertility clinic 6 to 9 days per cycle. Participants brought urine to the laboratory every 3 days. All participants performed all methods. Participants were paid $400; nonparticipants were not compensated. Only 3% of the respondents objected to the proposed methods: principally to CME, US, and giving blood samples. No respondent perceived the study as unimportant.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional inhibition of PI3K by the betaGBP molecule suppresses Ras-MAPK signalling to block cell proliferation.

The mechanisms of signal transduction from cell surface receptors to the interior of the cell are fundamental to the understanding of the role that positive and negative growth factors play in cell physiology and in human diseases. Here, we show that a functional link between phosphatidylinositol-3-OH kinase (PI3K) and Ras is suppressed by the beta-galactoside binding protein (betaGBP) molecule, a cytokine and a negative cell-cycle regulator. Ras-mitogen-activated protein kinase (MAPK) signalling is blocked by betaGBP owing to its ability to inhibit the p110 catalytic subunit of PI3K, whose basal activity is required for Ras activation. Functional inhibition of p110 by betaGBP results in downregulation of PI3K activity, suppression of Ras-GTP loading, consequent loss of MAPK activation and block of cell proliferation. This study sheds light on the molecular mechanisms whereby betaGBP can control cell proliferation and, by extension, may potentially control tumorigenesis by controlling PI3K.

Expression of the 2-5A system during the cell cycle.

To determine whether the 2-5A system has a role in the regulation of cell growth we have examined all constituents of the 2-5A pathway in mouse embryo fibroblasts undergoing one cycle of division at the tertiary stage under conditions where a high degree of uniformity is maintained within each stage of the cycle. Levels of the 2-5A synthetase increased up to tenfold late in S phase and declined as cells moved through G2. A similar but smaller increase in the 2-5A-dependent ribonuclease was observed, whereas activity of the 2'5' phosphodiesterase was highest in quiescent cells. At the time of maximum synthetase levels no phosphorylated 2-5A could be detected in the intact cell. Endogenous interferon (IFN) was found in the culture supernatants in increasing concentration with cell cycle progression and addition of antibodies to IFN reduced the increase in synthetase seen in late S. Treatment of cells with a growth inhibitor that cells produce also affected synthetase activity.