Relative carriage rates of nuclear dehydrogenating clostridia in two populations of different colorectal cancer risk

Carriage of nuclear dehydrogenating clostridia has been associated with colon cancer and implicated in its aetiology. This study has compared the carriage of these organisms in a British population at high risk for the development of colon cancer with a low risk Nigerian population. Clostridia were found in all of the stools from both populations. Nuclear dehydrogenating clostridia were only found in the stools of the British subjects (32%). These results support the suggestion that the carriage rate of nuclear dehydrogenating clostridia in a population is related to the risk of colon cancer.     

Development of hatching blastocysts from immature human oocytes following in-vitro maturation and fertilization using a co-culture system

Recently, in-vitro maturation (IVM) of immature human oocytes recovered from non-stimulated follicles has been applied in the treatment of infertility. However, in previous reports, very few embryos cultured in conventional medium have reached the expanded blastocyst stage following in-vitro maturation and fertilization (IVM/IVF). The objective of this study was to investigate whether the developmental competence of human embryos following IVM/IVF could be enhanced by the use of a human ampullary cell co-culture system. Immature human oocytes were aspirated from small follicles at Caesarean section and then cultured in medium containing human menopausal gonadotrophin for 36 to 48 h, followed by insemination. Zygotes were randomly cultured either in conventional culture medium alone or in the co-culture system. Of 48 embryos cultured in conventional medium alone, all arrested at the 2-16- cell stage on day 3 after insemination. Of 46 embryos cultured in the co-culture system, 26 embryos (56.5%) arrested at the 2-16-cell stage. Six embryos (13%) developed to the morula stage. Fourteen embryos (30.4%) developed to expanded blastocysts and two blastocysts were hatching on day 7 after insemination. We conclude that co-culture significantly enhances the development of blastocysts in embryos resulting from IVM/IVF.      

Detection of chromosomes and estimation of aneuploidy in human spermatozoa using fluorescence in-situ hybridization

The development and application of fluorescence in-situ hybridization (FISH) has opened the way for comprehensive studies on numerical chromosome abnormalities in human spermatozoa. FISH can be rapidly applied to large numbers of spermatozoa and thus overcomes the major limitation of karyotyping spermatozoa after penetration of zona-free hamster oocytes. The simultaneous hybridization of two or more chromosome-specific probes to spermatozoa and subsequent detection of the bound probes using different fluorescent detection systems enables two or more chromosomes to be localized simultaneously in the same spermatozoon and provides a technique for undertaking reasonable estimates of aneuploidy. The most commonly used probes are those which bind to the centromeric region of specific chromosomes. Most studies to date have concentrated on estimating aneuploidy in spermatozoa from normospermic men, although reports are beginning to appear on aneuploidy in spermatozoa from subfertile and infertile men. Multi- probe FISH studies have generally reported disomy (hyperhaploidy) estimates of 0.05-0.2% per chromosome. There is preliminary evidence that some chromosomes such as X, Y and 21 are predisposed towards higher rates of non-disjunction during spermatogenesis. There are also suggestions of inter-donor variability in aneuploidy frequencies for specific chromosomes, although this requires confirmation in larger studies. While FISH is clearly a powerful technique that has many applications in reproductive medicine, it must also be realized that it does have limitations and the technology itself is still evolving and has yet to be fully validated on spermatozoa.      

Preliminary observations on polar body extrusion and pronuclear formation in human oocytes using time-lapse video cinematography

In this study, we have used time-lapse video cinematography to study fertilization in 50 human oocytes that had undergone intracytoplasmic sperm injection (ICSI). Time-lapse recording commenced shortly after ICSI and proceeded for 17-20 h. Oocytes were cultured in an environmental chamber which was maintained under standard culture conditions. Overall, 38 oocytes (76%) were fertilized normally, and the fertilization rate and embryo quality were not significantly different from 487 sibling oocytes cultured in a conventional incubator. Normal fertilization followed a defined course of events, although the timing of these events varied markedly between oocytes. In 35 of the 38 fertilized oocytes (92%), there were circular waves of granulation within the ooplasm which had a periodicity of 20-53 min. The sperm head decondensed during this granulation phase. The second polar body was then extruded, and this was followed by the central formation of the male pronucleus. The female pronucleus formed in the cytoplasm adjacent to the second polar body at the same time as, or slightly after, the male pronucleus, and was subsequently drawn towards the male pronucleus until the two abutted. Both pronuclei then increased in size, the nucleoli moved around within the pronuclei and some nucleoli coalesced. During pronuclear growth, the organelles contracted from the cortex towards the centre of the oocyte, leaving a clear cortical zone. The oocyte decreased in diameter from 112 to 106 microm (P < 0.0001) during the course of the observation period. The female pronucleus was significantly smaller in diameter than the male pronucleus (24.1 and 22.4 microm respectively, P = 0.008) and contained fewer nucleoli (4.2 and 7.0 respectively, P < 0.0001). After time-lapse recording, oocytes were cultured for 48 h prior to embryo transfer or cryopreservation. Embryo quality was related to fertilization events and periodicity of the cytoplasmic wave, and it was found that good quality embryos arose from oocytes that had more uniform timing from injection to pronuclear abuttal and tended to have a longer cytoplasmic wave. In conclusion, we have shown that time-lapse video cinematography is an excellent tool for studying fertilization and early embryo development, and have demonstrated that human fertilization comprises numerous complex dynamic events.      

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection

Aim: To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS). Methods: Ninety‐nine stools from 38 infants of median 27‐week gestation were cultured; 44 stools from 27 infants had their microbial profiles determined by 16S. Ordination analyses explored effects of patient variables on gut communities. Results: Standard microbiological culture identified a mean of two organisms (range 0–7), DGGE 12 (range 3–18) per patient. Enterococcus faecalis and coagulase negative staphylococci (CONS) were most common by culture (40% and 39% of specimens). Meconium was not sterile. No fungi were cultured. Bacterial community structures in infants with NEC and LOS differed from healthy infants. Infants who developed NEC carried more CONS (45% vs 30%) and less Enterococcus faecalis (31% vs 57%). 16S identified Enterobacter and Staphylococcus presence associated with NEC/LOS, respectively. Conclusions: Important differences were found in the gut microbiota of preterm infants who develop NEC/LOS. The relationship of these changes to current practices in neonatal intensive care requires further exploration.     

The natural history of a genetic subtype of arrhythmogenic right ventricular cardiomyopathy caused by a p.S358L mutation in TMEM43

To determine the phenotype and natural history of a founder genetic subtype of autosomal dominant arrhythmogenic right ventricular cardiomyopathy (ARVC) caused by a p.S358L mutation in TMEM43. The age of onset of cardiac symptoms, clinical events and test abnormalities were studied in 412 subjects (258 affected and 154 unaffected), all of which occurred in affected males significantly earlier and more often than unaffected males. Affected males were hospitalized four times more often than affected females (p ≤ 0.0001) and died younger (p ≤ 0.001). The temporal sequence from symptoms onset to death was prolonged in affected females by 1–2 decades. The most prevalent electrocardiogram (ECG) manifestation was poor R wave progression (PRWP), with affected males twice as likely to develop PRWP as affected females (p ≤ 0.05). Left ventricular enlargement (LVE) occurred in 43% of affected subjects, with 11% fulfilling criteria for dilated cardiomyopathy. Ventricular ectopy on Holter monitor was common and occurred early: the most diagnostically useful clinical test. No symptom or test could rule out diagnosis. This ARVC subtype is a sex‐influenced lethal arrhythmogenic cardiomyopathy, with a unique ECG finding, LV dilatation, heart failure and early death, where molecular pre‐symptomatic diagnosis has the greatest clinical utility.     

LC-MS/MS测定人血浆中对乙酰氨基酚及其人体药动学和生物等效性研究

目的 建立一种快速、灵敏的高效液相色谱-串联质谱（HPLC-MS/MS）方法以测定人血浆中对乙酰氨基酚浓度,并应用于两种对乙酰氨基酚制剂的人体药代动力学和生物等效性研究。方法 以替硝唑为内标,200μL血浆样品经5倍于其体积的乙酸乙酯液液萃取,再经Waters XBridge? C18柱等度洗脱分离后导入串联质谱,以正离子多反应监测模式进行定量分析,对乙酰氨基酚和内标的选择性反应离子对分别是m/z 152→110和248→121。方法经验证后应用于19名健康受试者单剂量空腹口服两种对乙酰氨基酚制剂500mg后药代动力学和生物等效性的研究。结果 血浆中对乙酰氨基酚在0.1～8.0 μg·mL-1范围内线性良好（r2 > 0.99）,最低检测限为 0.1 μg·mL-1,提取回收率为91.0%～98.7%,日内和日间准确度分别为98.8%～111.3% （精密度：CV ? 9.03%）和94.9%～102.6% （精密度：CV ? 10.68%）。生物等效性试验中,受试制剂与参比制剂的主要药代动力学参数Cmax、AUC0-t和AUC0-∞ 几何均值比的90%置信区间分别为83.50%～105.79%,94.25%～101.54%和93.24%～101.02%,均落在生物等效可接受标准80.00%～125.00%范围内。结论 所建立测定人血浆中对乙酰氨基酚浓度的HPLC-MS/MS法具有快速灵敏、回收率高、选择性好的特点,适用于对乙酰氨基酚片人体药代动力学和生物等效性研究。受试制剂与参比制剂在人体内吸收速度和程度相似,两种制剂生物等效。