Identifying and quantifying secondhand smoke in source and receptor rooms: logistic regression and chemical mass balance approaches.

Identifying and quantifying secondhand tobacco smoke (SHS) that drifts between multiunit homes is critical to assessing exposure. Twenty-three different gaseous and particulate measurements were taken during controlled emissions from smoked cigarettes and six other common indoor source types in 60 single-room and 13 two-room experiments. We used measurements from the 60 single-room experiments for (i) the fitting of logistic regression models to predict the likelihood of SHS and (ii) the creation of source profiles for chemical mass balance (CMB) analysis to estimate source apportionment. We then applied these regression models and source profiles to the independent data set of 13 two-room experiments. Several logistic regression models correctly predicted the presence of cigarette smoke more than 80% of the time in both source and receptor rooms, with one model correct in 100% of applicable cases. CMB analysis of the source room provided significant PM2.5 concentration estimates of all true sources in 9 of 13 experiments and was half-correct (i.e., included an erroneous source or missed a true source) in the remaining four. In the receptor room, CMB provided significant estimates of all true sources in 9 of 13 experiments and was half-correct in another two.

Controlled experiments measuring personal exposure to PM2.5 in close proximity to cigarette smoking.

Few measurements of exposure to secondhand smoke (SHS) in close proximity to a smoker are available. Recent health studies have demonstrated an association between acute (<2 h) exposures to high concentrations of SHS and increased risk of cardiovascular and respiratory disease. We performed 15 experiments inside naturally ventilated homes and 16 in outdoor locations, each with 2-4 non-smokers sitting near a cigarette smoker. The smoker's and non-smokers' real-time exposures to PM2.5 from SHS were measured by using TSI SidePak monitors to sample their breathing zones. In 87% of the residential indoor experiments, the smoker received the highest average exposure to SHS, with PM2.5 concentrations ranging from 50-630 µg/m(3) . During the active smoking period, individual non-smokers sitting within approximately 1 m of a smoker had average SHS exposures ranging from negligible up to >160 µg/m(3) of PM2.5 . The average incremental exposure of the non-smokers was higher indoors (42 µg/m(3) , n = 35) than outdoors (29 µg/m(3) , n = 47), but the overall indoor and outdoor frequency distributions were similar. The 10-s PM2.5 averages during the smoking periods showed great variability, with multiple high concentrations of short duration (microplumes) both indoors and outdoors.

Delayed blastocyst development does not influence the outcome of frozen-thawed transfer cycles.

OBJECTIVE: To compare the outcome of transfer of thawed blastocysts frozen on either day 5 or day 6 after in vitro fertilisation. DESIGN: Cohort observational study. SETTING: Tertiary assisted conception unit in London, UK. POPULATION: Six hundred and forty-two consecutive nondonor programmed thawed blastocyst transfer (TBT) cycles. METHODS: High-grade blastocysts were frozen on day 5 (n = 314) or day 6 (n = 328) after fertilisation using a slow-freezing protocol. Endometrial preparation was performed using estradiol valerate. Progesterone supplementation was commenced when the endometrial thickness had reached 7 mm or more. Frozen blastocysts were thawed on day 6 of progesterone supplementation and assessed immediately after thawing for survival, and after 3-4 hours for blastocoele re-expansion. Main outcome measures Thawed blastocyst survival and re-expansion rates, and pregnancy and live birth rates, per TBT. RESULTS: Thawed blastocyst survival and re-expansion rates were comparable between the day 5 and day 6 groups (87% versus 87%, P = 0.50 and 73% versus 71%, P = 0.35, respectively). The live birth rate was similar between the two groups (29% versus 28.5%, P = 0.93, respectively). After adjusting for confounding variables, the odds ratio (OR) of a live birth in cycles in which the thawed blastocysts were frozen on day 6 compared with day 5 was 1.23 [95% confidence interval (CI), 0.81-1.86, P = 0.34]. CONCLUSION: The pregnancy potential of high-grade blastocysts frozen on day 5 and day 6 after in vitro fertilisation and replaced in programmed TBT cycles is comparable.

GSK962040: a small molecule, selective motilin receptor agonist, effective as a stimulant of human and rabbit gastrointestinal motility.

There is an urgent clinical need for a safe, efficacious stimulant of gastric emptying; current therapies include erythromycin (an antibiotic with additional properties which preclude chronic use) and metoclopramide (a 5-hydroxytryptamine type 4 receptor agonist and an antagonist at brain D2 receptors, associated with movement disorders). To move away from the complex motilide structure of erythromycin, a small molecule motilin receptor agonist, GSK962040, was identified and characterized. The compound was evaluated using recombinant human receptors, rabbit and human isolated stomach preparations known to respond to motilin and in vivo, by measuring its ability to increase defecation in conscious rabbits. At the human motilin receptor, the pEC50 (the negative logarithm to base 10 of the EC50 value, the concentration of agonist that produces 50% of the maximal response) values for GSK962040 and erythromycin as agonists were, respectively, 7.9 and 7.3; GSK962040 had no significant activity at a range of other receptors (including ghrelin), ion channels and enzymes. In rabbit gastric antrum, GSK962040 300 nmol L(-1)-10 micromol L(-1) caused a prolonged facilitation of the amplitude of cholinergically mediated contractions, to a maximum of 248 +/- 47% at 3 micromol L(-1). In human-isolated stomach, GSK962040 10 micromol L(-1), erythromycin 10 micromol L(-1) and [Nle13]-motilin 100 nmol L(-1), each caused muscle contraction of similar amplitude. In conscious rabbits, intravenous doses of 5 mg kg(-1) GSK962040 or 10 mg kg(-1) erythromycin significantly increased faecal output over a 2-h period. Together, these data show that GSK962040, a non-motilide structure, selectively activates the motilin receptor. Simplification of the structural requirements to activate this receptor greatly facilitates the design of potentially new medicines for gastroparesis.

Inhibition of colonic motility and defecation by RS-127445 suggests an involvement of the 5-HT2B receptor in rodent large bowel physiology.

BACKGROUND: 5-HT(2B) receptors are localized within the myenteric nervous system, but their functions on motor/sensory neurons are unclear. To explore the role of these receptors, we further characterized the 5-HT(2B) receptor antagonist RS-127445 and studied its effects on peristalsis and defecation. EXPERIMENTAL APPROACH: Although reported as a selective 5-HT(2B) receptor antagonist, any interactions of RS-127445 with 5-HT(4) receptors are unknown; this was examined using the recombinant receptor and Biomolecular Interaction Detection technology. Mouse isolated colon was mounted in tissue baths for isometric recording of neuronal contractions evoked by electrical field stimulation (EFS), or under an intraluminal pressure gradient to induce peristalsis; the effects of RS-127445 on EFS-induced and on peristaltic contractions were measured. Faecal output of rats in grid-bottom cages was measured over 3 h following i.p. RS-127445 and separately, validation of the effective doses was achieved by determining the free, unbound fraction of RS-127445 in blood and brain. KEY RESULTS: RS-127445 (up to 1 micromol x L(-1)) did not interact with the 5-HT(4) receptor. RS-127445 (0.001-1 micromol x L(-1)) did not affect EFS-induced contractions of the colon, although at 10 micromol x L(-1) the contractions were reduced (to 36 +/- 8% of control, n= 4). RS-127445 (0.1-10 micromol x L(-1)) concentration-dependently reduced peristaltic frequency (n= 4). RS-127445 (1-30 mg x kg(-1)), dose-dependently reduced faecal output, reaching significance at 10 and 30 mg x kg(-1) (n= 6-11). In blood and brain, >98% of RS-127445 was protein-bound. CONCLUSIONS AND IMPLICATIONS: High-protein binding of RS-127445 indicates that relatively high doses are required for efficacy. The results suggest that 5-HT(2B) receptors tonically regulate colonic motility.

Selective single blastocyst transfer reduces the multiple pregnancy rate and increases pregnancy rates: a pre- and postintervention study.

OBJECTIVE: To examine the clinical pregnancy rate (CPR) and multiple pregnancy rate (MPR) in a large in vitro fertilisation (IVF) programme before and after the introduction of single blastocyst transfer (SBT) strategy in a selected group of women. DESIGN: A 3-year pre- and postintervention study. SETTING: A tertiary reproductive medicine and assisted conception unit in a London teaching hospital. POPULATION: Two thousand four hundred and fifty-one fresh IVF cycles performed between July 2004 and June 2007 at the Assisted Conception Unit at Guy's and St Thomas' Hospital NHS Foundation Trust were included in the study. METHODS: In January 2006, we implemented a multidisciplinary intervention involving the introduction of a selective day 5 SBT service together with an educational programme on the risks of multiple pregnancy and potential advantages of blastocyst transfer aimed at couples at high risk of multiple pregnancy. MAIN OUTCOME MEASURES: The CPR per cycle started and MPR per clinical pregnancy achieved. RESULTS: A statistically significant increase in the CPR from 27% (324/1198) to 32% (395/1253) (risk difference [RD] 5%, risk ratio [RR] 1.17, 95% CI 1.03-1.32, P= 0.015) and reduction in the MPR per clinical pregnancy from 32% (103/272) to 17% (69/395) (RD 15%, RR 0.46, 95% CI 0.35-0.60, P < 0.001) were observed after introduction of the SBT service. CONCLUSION: Selective SBT in women with good prognosis can reduce the MPR after IVF while maintaining the overall success rate of the IVF programme.

Assisted hatching is more effective when embryo quality was optimal in previous failed IVF/ICSI cycles.

Assisted hatching (AH) was developed as a possible solution to repeated implantation failure. The aim of this analysis was to examine the relationship between the morphology of embryos in a previous cycle on outcome in a subsequent cycle with AH. A total of 175 AH cycles performed after previous failed ART without hatching were divided into group A with optimal and group B with suboptimal embryos transferred previously. The groups were similar in terms of demographic and cycle characteristics. In group A, there was a significant improvement (p<0.001) in implantation (28.8 vs 5.1%), clinical pregnancy (41.9 vs 12.1%) and live birth rate (38.5 vs 8.6%) compared with group B. The data suggest that the prognosis for treatment is better if AH is performed after failure despite optimal embryos compared with failure associated with suboptimal embryos and embryo quality is the most significant factor affecting outcome.

Live births after intracytoplasmic sperm injection in the management of oligospermia and azoospermia in Nigeria.

Intracytoplasmic sperm injection has revolutionised the management of male infertility. We report two cases that demonstrate the successful application of this technology in Nigeria in the management of both oligospermia and azoospermia. The first case relates to the treatment of a 31-year-old woman who required intracytoplasmic sperm injection of her husband's sperm for the treatment of both tubal fertility and male infertility. She had three embryos transferred on 9th June 1999 and was delivered of healthy male and female infants by caesarean section in January 2000 at 33 weeks gestation. The second case describes a 38-year-old woman who required intracytoplasmic sperm injection of the husband's surgically collected sperm for the management of azoospermia. She had two embryos transferred on 16th December 1999 and was delivered of a healthy male infant by caesarean section on 19th July 2001.

The use of amplified cDNA to investigate the expression of seven imprinted genes in human oocytes and preimplantation embryos.

Imprinted genes are characterized by expression of only one of the two alleles according to its inheritance from the mother or the father. This mono-allelic expression must arise from primary differential epigenetic modification of the parental alleles of the imprinted gene in the spermatozoon and the oocyte. Most of the information on the onset of imprinted gene expression, and on the molecular mechanisms regulating mono-allelic expression, have been derived from studies in the mouse. In this paper, we investigate the expression of seven imprinted genes in human preimplantation development. Due to limitations imposed by the rarity of human embryos available for research, our approach has been to screen amplified cDNA preparations prepared from human unfertilized oocytes and individual embryos at each of the 4-cell, 8-cell and blastocyst stages. Gene-specific primers were used to investigate expression of the imprinted genes by polymerase chain reaction (PCR) analysis of these amplified cDNA. We found that expression is inherently variable in the amplified cDNA from embryo to embryo but the use of several samples at each stage showed that the SNRPN, UBE3A and PEG1 genes are expressed throughout human preimplantation development. This was confirmed by direct analysis by gene-specific reverse transcription-PCR on a limited number of lysed embryos (one gene analysed per embryo). Thus, the amplified cDNA may be used to rapidly identify those imprinted genes expressed in preimplantation development and, hence, those genes amenable to investigation of the epigenetic mechanisms regulating mono-allelic expression. Confirmation of preimplantation expression also identifies those imprinted diseases amenable to preimplantation diagnosis, and the imprinted genes which may be used in assessment of possible perturbations of imprinting following new procedures in assisted reproduction. Our series of single embryo amplified cDNA are established as a valuable resource for comparative studies of gene expression within one embryo and between embryos throughout early human development. The amplified cDNA thus circumvent the need for a continuous supply of human embryos for studies on embryonic gene expression.

Detection of human novel developmental genes in cDNA derived from replicate individual preimplantation embryos.

We have constructed amplified cDNA preparations from replicate samples of human oocytes and individual preimplantation embryos. Differential display of the cDNA preparations shows disparate patterns of gene expression in the individual embryos at all stages of preimplantation development. The variation in patterns of genes expressed is in part due to the low starting cell number undergoing the reverse transcription-polymerase chain reaction (RT-PCR) step in the preparation of the amplified cDNAs. Despite this variability, the use of replicate embryo samples makes it possible to identify and isolate human genes specifically expressed at the different stages of human preimplantation development from the unfertilized oocyte to the blastocyst stage.

Developmental expression of specific genes detected in high-quality cDNA libraries from single human preimplantation embryos.

We describe an improved highly sensitive method for generating cDNA libraries containing a high proportion of cDNAs enriched with 5'-coding sequences from single human preimplantation embryos and a 10 week old whole foetus. The embryonic mRNA was isolated using oligo-(dT) linked to magnetic beads. First-strand cDNA synthesis was carried out directly on the bound mRNA, followed by PCR designed to amplify the cDNA molecules synthesized in their entirety. The complexities of the libraries are between 10(5) and 10(6) independent clones. The average cDNA size is 1.0 kb, and the size range is 0.5-3.0 kb. PCR analysis of the embryonic libraries for specific genes has revealed transcripts for genes known to be transcribed in preimplantation stages, such as the imprinted gene SNRPN, developmental genes WNT11, HOX, OCT-1 and the embryonic OCT-4, cytoskeletal genes keratin-18 and beta-actin, the cell cycle gene C-MOS, and housekeeping genes GAPDH and HPRT. Sequencing of random clones showed the presence of a variety of sequences, such as human chorionic gonadotrophin, ubiquitin, TFIIA, guanine nucleotide-binding protein (beta-subunit), annexin I, a gene encoding a kinesin-like protein, and TWIST, which encodes a basic helix-loop-helix (bHLH) transcription factor implicated in Saethre-Chotzen syndrome (characterized by craniofacial and limb anomalies). Approximately 40% of these randomly analysed clones were full length. In addition to cDNAs matching known ESTs (Expressed Sequence Tags) in the GenBank and dbEST databases, novel sequences were detected at a frequency of 16% of randomly picked clones. The libraries are a valuable resource, providing longer cDNAs representing genes expressed during human preimplantation development.

A prospective comparison of 'in house' and commercially prepared Earle's balanced salt solution in human in-vitro fertilization.

A prospective, randomized study was undertaken to compare the use of Earle's balanced salt solution (EBSS) prepared 'in house' with that produced commercially, in 448 cycles of therapeutic in-vitro fertilization. Outcome was assessed in terms of fertilization and cleavage rates, embryo morphology, and implantation rates following embryo transfer. The only differences that were found between the two media in any of the outcome parameters were in the number of cycles with failed fertilization (1/218 in 'in house' medium compared with 10/230 in commercially prepared medium; P = 0.0186), and in the rate at which embryos cleaved. Thus, while the median number of blastomeres per embryo was no different in the two groups at 46-49 h post insemination (three in embryos cultured in 'in-house' medium, compared with four in those cultured in commercially prepared medium; P > 0.1), the number of embryos per cycle that had cleaved to the 4-cell stage by 46-49 h post insemination was significantly greater in the Medi-Cult than in the EBSS medium (P < 0.001).

A prospective randomized study comparing the outcome of in-vitro fertilization and embryo transfer following culture of human embryos individually or in groups before embryo transfer on day 2.

A prospective randomized trial of in-vitro fertilization and embryo transfer was undertaken to investigate the reported beneficial effects of culturing preimplantation human embryos in groups, rather than individually. A total of 159 treatment cycles, in which the women were matched for age, basal gonadotrophin concentrations and number of previous attempts, were included in the study. Of these, 78 cycles were randomized to the 'individual culture' group, and 81 cycles were randomized to the 'group culture' group. The groups did not differ in terms of the median number of oocytes or embryos obtained per cycle. There was no statistically significant difference between the two groups in terms of treatment outcome, as assessed by pregnancies or clinical pregnancies.

Synthetic profiles of polypeptides of human oocytes and normal and abnormal preimplantation embryos.

There is considerable variation in the rate of development in vitro of individual preimplantation human embryos. The relationship between the rate of development and patterns of polypeptide synthesis in individual embryos was examined using SDS-PAGE and autoradiography. After incubation in [35S]methionine, 19 polypeptide bands were identified that change between fertilization and the morula stage. Although changes in two of the bands occurred in embryos that were developing normally and in ageing oocytes, and are thus independent of fertilization, the changes identified in the remaining 17 bands occurred only after fertilization. In embryos that were developing abnormally, as assessed by delayed cleavage, cleavage arrest or extensive fragmentation, the alteration in polypeptide synthetic profiles increased with increasing abnormality.

Distribution of alpha3, alpha5 and alpha(v) integrin subunits in mature and immature human oocytes.

The distribution of three integrin subunits, alpha3, alpha5 and alpha(v), in immature and mature human oocytes has been examined using immunofluorescence and confocal microscopy. The results demonstrate that both alpha5 and alpha(v) are present at the germinal vesicle stage, while alpha3 was only detected in oocytes after germinal vesicle breakdown, in metaphase I and II stage oocytes. The cortical concentration of integrin subunits alpha3 and alpha5 is consistent with their localization in the oolemma. In contrast, the homogeneous distribution of alpha(v) throughout the oocyte suggests the existence of cytoplasmic reservoirs of this protein in the oocyte.

Imprinted expression of SNRPN in human preimplantation embryos.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two clinically distinct neurogenetic disorders arising from a loss of expression of imprinted genes within the human chromosome region 15q11-q13. Recent evidence suggests that the SNRPN gene, which is defective in PWS, plays a central role in the imprinting-center regulation of the PWS/AS region. To increase our understanding of the regulation of expression of this imprinted gene, we have developed single-cell-sensitive procedures for the analysis of expression of the SNRPN gene during early human development. Transcripts of SNRPN were detected in human oocytes and at all stages of preimplantation development analyzed. Using embryos heterozygous for a polymorphism within the SNRPN gene, we showed that monoallelic expression from the paternal allele occurs by the 4-cell stage. Thus, the imprinting epigenetic information inherited in the gametes is recognized already in the preimplantation embryo. The demonstration of monoallelic expression in embryos means that efficient preimplantation diagnosis of PWS may be made by analysis for the presence or absence of SNRPN mRNA.

Detection of a novel splice variant of the hypoxanthine-guanine phosphoribosyl transferase gene in human oocytes and preimplantation embryos: implications for a RT-PCR-based preimplantation diagnosis of Lesch-Nyhan syndrome.

We have detected a novel splice variant of the hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene in two human oocytes and four preimplantation embryos from the 4-cell to the 8-cell stage of development. The novel HPRT transcript lacks exons 4, 5 and 6 of the normal HPRT gene. The same parental origin for the two oocytes and two of the preimplantation embryos, in which the alternatively spliced transcript was detected, might suggest that the alternative splicing is influenced by genetic background. Mutations in the HPRT gene which cause alternative mRNA splicing are implicated in Lesch-Nyhan syndrome. However, the relatively high frequency of detection of this novel HPRT transcript described here (6/109 oocytes and preimplantation embryos) suggests that it is not involved in Lesch-Nyhan syndrome. It is probable that the alternative HPRT transcript is derived from the aberrant splicing of a small percentage of the total mRNA produced from normal HPRT alleles. The presence of this alternative transcript in human preimplantation embryos may complicate an reverse transcription-polymerase chain reaction-based preimplantation diagnosis of Lesch-Nyhan syndrome.

Cytogenetic analysis of human preimplantation embryos following developmental arrest in vitro.

The relationship between chromosomal abnormalities in the human preimplantation embryo and developmental arrest in vitro was investigated. Cytogenetic analysis of 171 embryos that had arrested between the pronucleate and the 8-cell stages demonstrated that the overall incidence of chromosomal abnormality among these embryos was 63.4%. Of the embryos that arrested at the pronucleate stage (n = 48), 47.9% were chromosomally abnormal, compared with 59.5% of those that arrested between the 2- and 4-cell stages (n = 50), and 82.8% of those arrested between the 5- and 8-cell stage (n = 73). The rate of abnormality in embryos with poor morphology (irregular shaped blastomeres and considerable extracellular fragmentation) was significantly higher (86.8%; n = 33) than those with good morphology (60%; n = 51; P<0.005). These results suggest that there is an association between chromosomal abnormality, developmental arrest in vitro, and poor morphology.

Transcription of tissue-specific genes in human preimplantation embryos.

We have recently detected de-novo transcripts of the predominantly muscle-specific myotonin protein kinase gene in human preimplantation embryos from the 1-cell to the 4-cell stages. Others have shown de-novo transcripts of the Y-linked genes, ZFY and SRY, in the 1-cell zygote. In order to assess the significance of early transcription of these predominantly tissue-specific genes in preimplantation development, we have analysed individual human oocytes and preimplantation embryos for the presence of transcripts of two further tissue-specific genes, alpha-globin and beta-globin, and two house-keeping genes, HPRT and APRT. Reverse transcriptase polymerase chain reaction assays were developed to the required single cell sensitivity, using human red blood cells and fibroblasts, prior to their application to human oocytes and embryos. As expected, transcripts of the house-keeping genes, HPRT and APRT, were detected at all stages of preimplantation development. Transcripts of 'tissue-specific' alpha-globin were readily detected in preimplantation embryos from the 1-cell stage. However, transcripts of beta-globin were detected only rarely (in only one of the 11 embryos analysed). This difference may be due to the fact that alpha-globin contains a CpG island. A survey of the data on gene expression in early human development suggests that CpG-island-containing genes may be expressed in preimplantation embryos. Expression of these genes in gametes and early embryos may be involved in the survival of CpG islands in evolution.

cDNA libraries from single human preimplantation embryos.

In this paper, the construction, evaluation, and application of cDNA libraries from eight unfertilized oocytes and single four-cell-, seven-cell-, and blastocyst-stage embryos are described. Rapid, reproducible, and efficient procedures for the construction of PCR-based cDNA libraries from fewer than 10 cells were first developed in small populations of fibroblast cells. The human embryo libraries display complexities sufficient (between 10(5) and 10(6) clones) to represent the entire active gene population at these early stages of human development. The ubiquitous cytoskeletal elements, beta-actin, keratin-18, and alpha-tubulin, were detected at the expected frequency. Sequencing of consecutively picked random clones, without selection, showed the presence of a variety of sequences, such as the human transposable element, LINE-1 and Alu repeat sequences, housekeeping genes, and tissue-specific genes, such as alpha-globin and FMR-1. In addition to cDNAs corresponding to known ESTs (expressed sequence tags) in the GenBank and dbEST databases, a high proportion of novel sequences were detected. Applications of the libraries to several areas of interest, such as expression of CpG-island-containing "tissue-specific" genes, developmental genes expressed in a stage-specific manner, and a search for monoallelic expression of imprinted genes, are described. The libraries are a valuable resource for the study of gene expression during human preimplantation development and obviate the need for research on the human embryos themselves.