79例卵母细胞浆内单精子注射术(ICSI)治疗男性不育临床及安全性研究

目的对比分析应用卵母细胞浆内单精子注射(ICSI)技术和常规的体外受精与胚胎移植(IVF-ET)技术治疗不育的临床资料,为卵母细胞浆内单精子注射技术的临床应用提供安全性依据。方法回顾性分析79例接受技术治疗不育的资料,并与同期288例接受常规的体外受精与胚胎移植(IVF-ET)技术的病例对比分析。结果在受精率及流产率方面,ICSI组均低于IVF-ET组,卵裂率、妊娠率及胚胎形态方面,围生期结果两组比较无显著性差异(P>0.05)。结论ICSI治疗男性严重少弱精子症的效果是肯定的,出生后代安全性与IVF-ET相似。但ICSI出生后代安全性的确定仍需大样本研究及长期随访。     

严重生精功能障碍患者细胞和分子遗传学检查

目的检测严重生精功能障碍患者外周血染色体及Y染色体微缺失,探讨Y染色体微缺失的遗传机制,为拟行ICSI(intracytoplasmic sperm injection,ICSI)技术助孕的患者提供遗传咨询。方法按常规方法分析严重生精功能障碍患者的外周血淋巴细胞核型,对核型正常的患者,用多重PCR技术对Y染色体AZF(azoospermua fctor,AZF)所在区域的 15个序列标签位点(sequence tag site,STS)进行扩增,应用琼脂糖凝胶电泳分离扩增产物,对有AZF缺失的患者,随访其父亲的生育及AZF缺失情况。结果 71例无精子症患者中有25例染色体核型异常,异常率35．1％,1例核型为46,XY (小Y);75例严重少精子症患者中有4例46,XY(小Y);45例染色体核型正常的严重生精功能障碍患者,14例发生 AZF缺失(31．11％)。严重生精功能障碍患者总遗传缺陷发生率26．7％。所有AZF缺失患者父亲平均生育4．0个子女,其中有5例AZF缺失患者的父亲接受AZF检测,均无缺失。结论 1．染色体异常和AZF的缺失是引起无精子和严重少精子并造成男性不育的重要原因之一。AZF缺失可能并非有父亲遗传而来,其遗传机制尚有待进一步探讨。2．ICSI助孕前,夫妇双方须行遗传学检查以避免遗传缺陷后代的出生。     

改良卵胞浆内单精子显微注射技术在男性不育中的应用

目的 探讨改良卵胞浆内单精子显微注射技术(intracytoplasmic sperm injection,ICSI)及其对男性不育的治疗效果。方法 对ICSI进行改良及应用此技术对97例不孕不育患者、女方常规超促排卵、男方采用手淫(91例)、睾九穿刺取精(TESA6例)进行治疗。结果 改良ICSI在正常受精率(97.5％)、卵裂率(99.05％)、囊胚获得率(28.9％)、临床妊娠率(40.2％)等方面具有显著优势;精子参数与来源不同在ICSI治疗结果上无显著性差异。结论 ICSI是治疗男性不育的有效方法,依据生殖医学理论改良ICSI技术可提高治疗效果,非技术因素也是影响ICSI的重要因素,精子参数与来源不影响ICSI治疗结果。     

胞质内单精子注射与遗传学检测

胞质内单精子注射 (ICSI)为治疗男性不育发挥了重要的作用 ,但是由于男性的遗传因素 ,如染色体异常、CF基因突变、Y染色体微缺失等所引起的男性不育将会给所生后代带来遗传学缺陷。本文综述了 ICSI技术为治疗上述男性不育所要进行的遗传学检测及其存在问题与展望     

特发性男性不育的分子遗传学诊断与生育指导

特发性男性不育是指男性不育症患者除有精子异常外,其余情况均正常,而患者出现精子异常的原因不明.研究证明,Yq11中无精子因子(azoospermia factor,AZF)的缺失,可引起不同程度的生精障碍[1],但随着辅助生殖技术的发展,AZF缺失患者依然可以生育后代,并且不同缺失类型辅助治疗对策各异.因此,有必要对特发性男性不育患者进行Y染色体AZF区域微缺失研究,从而对该类患者的诊断和生育指导提供依据.     

Y染色体异常的临床表现与遗传学相关分析

采用外周血淋巴细胞培养技术对720例检查者实施染色体分析,其中有34例存在Y染色体异常,主要表现为不良孕产史、性别畸形、无精子症等.说明人类Y染色体异常通常会导致男性性发育异常或生殖异常等遗传效应.     

辅助生殖技术出生儿童的生长发育情况研究

目的随着辅助生殖技术(ART)在世界范围内的开展,通过体外受精-胚胎移植(IVF-ET)、胞浆内单精子注射(ICSI)、冻融胚胎移植(FET)等技术出生的儿童日益增加,这些通过ART出生的儿童的生长发育情况如何,成为全世界关注的问题。方法本文通过回顾近10年有关ART出生儿童生长发育情况的文献,总结通过ART出生的不同年龄段儿童的结局及生长发育情况。结论资料表明:ART出生儿童的生长发育同自然受孕儿比较基本上无明显差异。     

无精子症患者染色体核型和性激素水平分析

目的探讨无精子症患者的血清生殖激素和染色体核型的临床诊断意义。方法检测并分析无精子症患者的染色体核型以及血清生殖激素水平。结果染色体异常总数64例,异常发生率达57.14%。性染色体异常56例,占异常总数的87.50%,常染色体异常8例,占异常总数12.50%。无精子症组卵泡刺激素(FSH)、黄体生成素(LH)、垂体催乳素(PRL)均显著高于正常精液组(P<0.01),睾酮(T)呈显著性降低(P<0.01),雌二醇(E2)无显著性变化(P>0.05)。结论对无精子症患者进行染色体核型和血清生殖激素测定对其病因分析、诊断及治疗具有重要的临床意义。     

15例47,XYY患者病例分析并文献复习

目的探讨47,XYY患者染色体异常的原因、辅助生殖结局及其子代遗传情况。方法总结从9725个辅助生殖周期中筛查的15例47,XYY患者的一般临床特征、精液参数、妊娠结局、子代情况、子代核型及配偶情况。结果 15例患者中有5例是单纯女方因素所致不育,表现为输卵管梗阻或卵巢功能不全。男方因素中,有4例无精子症,其中2例睾丸穿刺未见精子,其他2例附睾穿刺可见精子,行ICSI处理。1例隐匿性精子症,1例不射精症,1例极重度少、弱精症,1例畸形精子症,1例轻度少、弱精子症,2例轻度弱精子症,治疗后行ICSI处理。4例精液正常患者中1例考虑性取向有问题,行IUI处理,其余3例行IVF-ET和ICSI处理。15例夫妇共分娩活婴7人,4男3女,1例失访外,其余核型均正常。结论非整倍体精子在受精或者胚胎形成之前选择性地除去了额外的Y染色体,其后代出现异常的风险极低,IVF-ET和ICSI是不育患者接受辅助生殖时首选的技术方式,而PGD不推荐使用。     

浙江省台州地区167例男性不育的染色体畸变及生殖激素水平研究

目的研究男性不育与染色体畸变及生殖激素水平关系。方法对167例男性不育患者进行染色体核型分析和精液常规检测,用电化学发光法测定生殖激素水平。结果不育男性患者染色体异常发生率为10.8%(18/167),其中性染色体异常占7.2%(12/167),常染色体异常占3.6%(6/167)。染色体核型异常患者FSH、LH均不同程度升高,而T水平偏低;染色体核型正常无精子症组FSH、LH显著高于少弱精子症与对照组,少弱精子组也显著均高于对照组,另四项T、PRL、E2、P三组间不存在差异。结论染色体异常是造成男性不育重要因素,而生殖激素水平能反映不同程度的生精障碍,对男性不育者有必要进行细胞遗传学检查和生殖激素检测。     

Unmasking antigens for immunohistochemistry

Preservation of tissue by fixation in a formalin solution, followed by dehydration and paraffin-wax embedding remains the predominant method of preparation for microscopic analysis of morphology. Whilst this may be optimal for morphological assessment, this technique has major disadvantages for subsequent immunohistochemical study as a result of the structural alteration of antigens that occurs during the processing procedure. However, the introduction of a range of antigen unmasking procedures has revolutionised immunohistochemistry, establishing the technique as a powerful tool in diagnostic pathology. Here, the development of enzymatic, non-enzymatic and heat-based antigen retrieval techniques is reviewed, and the methods in use currently are appraised.     

Human mixed lymphocyte cultures. Evaluation of microculture technique utilizing the multiple automated sample harvester (MASH)

Use of lymphocyte cultures for in vitro studies such as pretransplant histocompatibility testing has established the need for standardization of this technique. A microculture technique has been developed that has facilitated the culturing of lymphocytes and increased the quantity of cultures feasible, while lowering the variation between replicate samples. Cultures were prepared for determination of tritiated thymidine incorporation using a Multiple Automated Sample Harvester (MASH). Using this system, the parameters that influence the in vitro responsiveness of human lymphocytes to allogeneic lymphocytes have been investigated.     

Computer-assisted quantitative videoscanning technique for measuring cross-sectional areas and dimensions of tissue components

A computer-assisted image quantiser has been used to provide a means of measuring cross-sectional areas of placental peripheral villi. A standardising technique was developed to adapt the instrument for this purpose. Application of this technique has demonstrated that the average reduction of peripheral villous mass in placentas from a malnourished Central American population, as demonstrated by other techniques, results from a reduction in the number of villi rather than in the dimensions of the existing villi.     

Use of near-infrared spectroscopy in the investigation of brain activation during cognitive aging: A systematic review of an emerging area of research

The cognitive neuroscience of aging is a growing and stimulating research area. The development of neuroimaging techniques in the past two decades has considerably increased our understanding of the brain mechanisms that might underlie cognitive performance and resulting changes due to normal aging. Beside traditional metabolic neuroimaging techniques, such as Positron Emission Tomography and functional Magnetic Resonance Imaging, near infrared spectroscopy (NIRS), an optical imaging technique allowing to monitor real-time cerebral blood oxygenation, has gained recent interest in this field. The aim of the present review paper, after briefly presenting the NIRS technique, is to review and to summarize the recent results of neuroimaging studies using this technique in the field of cognitive aging. The reviewed literature shows that, despite low spatial resolution and cerebral depth penetration, this technique provides consistent findings on the reduced hemodynamic activity as a function of chronological age, mainly in the prefrontal cortex. Important moderators of brain hemodynamics, such as cognitive load, subjects’ characteristics and experimental conditions, for which the NIRS technique is sensitive, are discussed. Strengths and weaknesses of functional NIRS in the field of cognitive aging are presented and finally, novel perspectives of research are proposed.     

HotSHOT Plus ThermalSHOCK, a new and efficient technique for preparation of PCR-quality mite genomic DNA

The present study adapted the HotSHOT method, a technique which has been successfully applied on different kinds of tissues, to studies of Sarcoptes. Some modifications of this technique were made which allowed the quick preparation of PCR-quality Sarcoptes genomic DNA (gDNA), namely applying sodium hydroxide as a substrate for three cycles of thermal shock, followed by a short incubation and pH adjustment with a Tris solution (HotSHOT Plus ThermalSHOCK). The performance of this technique was tested by amplifying a ~450-bp rDNA fragment of the second internal transcribed spacer (ITS-2) and by multi-locus genotyping using ten microsatellites on 520 individual Sarcoptes samples. No difference in performance was observed between gDNA samples prepared using the HotSHOT Plus ThermalSHOCK technique and those prepared using a commercial kit utilizing proteinase K digestion. The results demonstrated that the HotSHOT Plus ThermalSHOCK technique is time-saving, economic, and easily automatable for the preparation of PCR-quality mite gDNA, which has implications for studying the molecular biology of mites with human and animal health significance. Although tested in the present study using Sarcoptes mites as a model, this technique may find broad applicability in extraction of gDNA from other parasites with small sizes and hard bodies.     

Quantitative sputum culture as a means of excluding false positive reports in the routine microbiology laboratory

A relatively simple technique for sputum homogenization and dilution is described. Results show that this technique is reliable for isolation and quantitative culture of organisms from cases of chest infection. The technique has resulted in significant reduction in false positive reports, particularly when a system of interpretative reporting is utilized based on 10(7) organisms per millilitre of sputum being accepted as evidence of significant infection.     

Confocal fluorescent intravital microscopy of the murine spleen

Intravital microscopy has provided many insights into cellular interactions in various secondary lymphoid tissues. Because this technique allows for the visualization of cellular movement in real-time, it has been very powerful. However, until now, it has been difficult to apply this technique to the spleen. We report a technique that utilizes the Nikon RCM-8000 scanning laser, confocal microscope that allows for visualization of cellular movement in real-time in the rodent spleen. Using fluorescently labeled high molecular weight dextran or monoclonal antibodies, we are able to visualize fluorescently labeled cells rolling, tethering, and adhering in the spleen. In addition, we show that the majority of blood flow to the spleen remains within the white pulp nodules, as do most transferred erythrocytes at early time points. This is the first report of intravital microscopy of the spleen using a method that allows for easy identification of transferred cells.     

Noise considerations in dual energy CT scanning.

Dual energy CT scanning (tomochemistry) has been proposed as a method for determining various parameters relating to the elemental composition of the tissues. In this paper, our aim is to study the relative noise inherent in two proposed techniques for dual energy scanning; a "two crystal" technique and a "two kV" technique. In the two crystal technique, a split crystal detector is used to simultaneously obtain the high and low energy data during one scan at high kV. The two kV technique requires two scans taken with widely different kV settings. We first review three commonly used approaches for utilizing the scan data to compute the relevant parameters. A theoretical formalism is constructed which aids in understanding these methods. Then this formalism is used to study the influence of CT image noise on measurement precision in the case where the unknown parameters are densities. It is shown that, (1) the unavoidable overlap in the spectral data obtained by the two crystal technique results in a much lower signal-to-noise ratio than can be obtained by using the two kV technique, (2) the necessity for hard filtration of the high energy beam in the two kV technique has not heretofore been appreciated, and (3) the dose for a given x-ray tube heat load is also lower with the two kV technique.     

Determination of HL-A2 Gene Dosage Effect on Lymphocytes by Release of Radioactive Chromium

A reproducible, well-defined technique is described for measurement of lytic rate of lymphocytes in the presence of HL–A antisera. Using this technique, it has been possible to determine HL–A2 zygosity in related and unrelated people using both fresh and stored cells. Homozygous HL–A2 individuals show lysis times approximately half those of heterozygotes, and this is interpreted as indicating possession of twice the amount of antigen. The application of the technique to the determination of HL–A zygosity in donors and recipients is discussed.     

Modification of the silver impregnation technique of Bielschowsky for use in glycol methacrylate-embedded brain tissue

The modified Bielschowsky stain has become the standard silver impregnation technique for use in paraffin-embedded tissue sections for identifying abnormalities of neuritic processes in a variety of neurodegenerative disorders such as Alzheimer's disease. Recently, glycol methacrylate embedding has been used increasingly as a substitute for paraffin embedding of tissue, because it results in less tissue distortion during processing and provides greater clarity and resolution of cellular architecture and structural details. We have been able to modify the Bielschowsky stain for use in glycol methacrylate-embedded brain sections. The use of this technique will permit more accurate quantitation of the histopathologic changes in neuritic processes in neurodegenerative diseases.