Effect of timing of oocyte denudation and micro-injection on survival, fertilization and embryo quality after intracytoplasmic sperm injection

In human in-vitro fertilization (IVF), the oocytes are surrounded by cumulus and corona cells at the time of insemination so that their maturity cannot easily be evaluated. The best IVF results are obtained if the oocytes are inseminated 2-6 h after retrieval. In the intracytoplasmic sperm injection (ICSI) procedure, the oocytes are denuded by enzymatic and mechanical treatment in order to be able to perform the injection. As a consequence, the nuclear maturity of the oocytes can be evaluated and only those that have extruded the first polar body are injected. However, metaphase-II oocytes that have not yet reached cytoplasmic maturity cannot be recognized. The purpose of this study was to investigate the effect of different timing of cumulus- corona cell removal and injection on the outcome of ICSI. For this we allowed the oocytes to complete in-vitro cytoplasmic maturation in two different culture conditions: (i) surrounded by their cumulus and corona cells or (ii) totally denuded. We performed three different studies on sibling oocytes obtained after a standardized buserelin/human menopausal gonadotrophin (HMG) protocol. We investigated the effect of early (1-2 h after retrieval) and late (5-6 h after retrieval) oocyte denudation and injection on the survival and fertilization of the injected oocytes and on embryo cleavage after fertilization. We found no statistically significant differences between early and late injection, indicating that after a standardized buserelin/HMG protocol the metaphase-II oocytes do not need time for further cytoplasmic maturation. Furthermore, a different timing of cumulus-corona cell removal has no effect on the outcome of ICSI, suggesting that the surrounding cells are not necessary for survival, fertilization and cleavage after ICSI.      

广东省2005-2007年麻疹病原学监测

目的 开展有效的麻疹病原学监测,分离麻疹病毒,了解广东省2005-2007年流行的麻疹野病毒分离株基因特征,为控制、消除麻疹提供科学依据.方法 用Veto/Slam细胞从暴发和散发麻疹疑似病例的咽拭子和尿液标本中分离麻疹病毒,并对所有分离到的麻疹病毒通过逆转录-聚合酶链反应(RT-PCR)方法,扩增出核蛋白(N)基因碳末端450个核苷酸片段,对其产物测定基因序列以定型.结果 2005-2007年共收到380份标本,包括咽拭子或尿液标本.共分离到82株麻疹野病毒.2005年病毒分离率为23.58%,2006年病毒分离率为17.11%,2007年病毒分离率为39.13%.病毒分离成功率与病例出疹天数和标本的采集质量有密切关系.结论 我省已经掌握了麻疹病毒的分离和分子生物学鉴定技术,分离率较高;我省多年来流行的麻疹毒株均为H1基因型,与国内流行的优势基因型一致.     

HIV-1 B''亚型R5或R5/X4嗜性毒株在GHOST细胞的感染性分析

目的分析我国HIV-1B′亚型R5或R5／X4嗜性毒株在GHOST细胞的感染性。方法采用传统的共培养方法从HIV-1感染者PBMC中分离并培养病毒，用表达CD4和趋化因子受体CCR5或CXCR4的GHOST细胞系，测定毒株的辅助受体利用情况，使用相同病毒量即2mg的HIV-1 p24分别感染表达不同受体的GHOST细胞系，通过流式细胞仪检测分析绿色荧光蛋白（GFP）的表达反映病毒感染细胞的能力。结果35例B′亚型毒株利用CCR5受体，占22例（62．85％），双嗜性即CCR5／CXCR4均阳性占13例（37．15％）。GHOST-R5／X4细胞的感染性分析结果显示，R5／X4双嗜性毒株的感染性明显高于CD4〉200／μl的R5毒株的感染性（P〈0．05）；R5／X4毒株与CD4≤200／μl的R5毒株感染性比较差异无统计学意义（P〉0．05）；CD4〉200／μl的R5毒株与CD4≤200／μl的R5毒株感染性比较差异有统计学意义（P〈0．05）；GHOST-CCR5细胞感染性分析结果显示：R5／X4双嗜性毒株的感染性明显下降，与CD4〉200／μl的R5毒株的感染性比较差异无统计学意义（P〉0．05）。利用相同剂量的双嗜性毒株分别感染R5、X4或R5／X4的GHOST细胞系，显示双嗜性毒株可同时利用CCR5和CXCR4辅助受体，但69．23％的R5／X4毒株以CCR5受体为主，30．77％的R5／X4毒株以CXCR4受体为主。结论HIV-1B′亚型R5／X4病毒不仅有更广泛的宿主细胞嗜性，而且在GHOST-R5／X4细胞中感染性明显提高。持续使用CCR5受体的毒株在疾病进展的过程中虽然辅助受体的利用是一样的，但病毒感染细胞的能力增加。     

Interpretation of gram-stained sputa containing Moraxella (Branhamella) catarrhalis.

Sputum specimens culture positive for Moraxella (Branhamella) catarrhalis were Gram stained with three decolorizer solutions (slow, 95% ethyl alcohol; intermediate, 1:1 ratio of 95% ethyl alcohol and acetone; and fast, acetone alone) for 5, 10, 20, and 30 s. Optimal results were obtained with acetone alone after 10 s or with a 1:1 mixture of acetone and ethanol after 20 s. Inadequate decolorization of M. catarrhalis in sputa is likely if the decolorization solution and exposure time are not optimal and may contribute to underreporting of this organism.     

大鼠肠系膜淋巴结高内皮微静脉与淋巴迷路之间的网状纤维支架的研究

目的 研究大鼠肠系膜淋巴结内高内皮微静脉与淋巴迷路之间淋巴细胞归巢的通路.方法 用镀银染色光镜观察法和冻裂割断扫描电镜观察法观察健康、成熟Wistar大鼠肠系膜淋巴结的基质网状结构.结果 位于高内皮微静脉和淋巴迷路周围有网状纤维支架,在二者相临近部位有密集交织的网状纤维网.结论 淋巴结内高内皮微静脉和淋巴迷路之间密集交织的网状纤维网,为细胞的居留和迁移提供结构支持和适宜的微环境,可能是淋巴细胞归巢的重要通路.     

The occurrence of Chlamydia trachomatis in the semen of men participating in an IVF programme

Chlamydia trachomatis is one of the most common causative agentsof sexually transmitted diseases. The authors studied the occurrenceof C.trachomatis in the semen of 184 asymptomatic men participatingin the IVF programme. Twenty-six (14.1%) of the 184 tested werepositive for C.trachomatis, these patients and their wives receivingdoxycycline capsules twice, 100 mg on the first day and 100mg/day for the following 13 days. This treatment was effectivein 88.5% of the cases and in the rest, treatment continued witherithromycin 250 mg four times/day for 2 weeks. The authorscompared the semen parameters (cell count, motility, morphology,bovine mucus penetration and hypo-osmotic swelling test) inthe infected and non-infected groups and observed no significantdifference between these two groups     

Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.     

Percoll gradient centrifugation can be omitted in sperm preparation for intracytoplasmic sperm injection

Prior to intracytoplasmic sperm injection (ICSI), seminal fluid is currently washed out from the ejaculated semen and further sperm selection is carried out by a discontinuous Percoll gradient. Possible deleterious effects from the sperm-separating substance Percoll on sperm function or embryo cleavage after in-vitro fertilization (IVF) have, to our knowledge, not yet been reported and the use of Percoll has been widely accepted in IVF. In this study, we examined whether the omission of the Percoll step in the sperm preparation has any influence on the outcome of the ICSI procedure. Two methods of sperm preparation for ICSI were compared in a controlled study on sibling oocytes: washing the semen sample once, followed by a Percoll gradient centrifugation versus washing the sperm sample twice without a Percoll gradient centrifugation. The mean fertilization rates were similar for the two sperm preparation methods: 78.2 +/- 21.4 and 75.0 +/- 24.1% respectively of the intact oocytes displaying two pronuclei. Cleavage rates did not differ statistically between the two groups. Whereas in both groups similar percentages of excellent, good and poor quality embryos were obtained, the percentage of fair quality embryos was significantly higher in the group without Percoll (16.3 +/- 20.1 versus 9.1 +/- 15.7%). However, no statistical differences were observed in either the percentage of transferable embryos or in the percentage of embryos actually transferred or frozen in the two groups. In conclusion, spermatozoa from ejaculates that are washed out from the seminal fluid without any further selection can be used for ICSI without any adverse effect on fertilization and embryo cleavage.