【作者】 马艳华；

【导师】 糜若然； 白晓红；

【作者基本信息】 天津医科大学 ， 妇产科学， 2007， 博士

【摘要】 研究背景：据世界卫生组织的研究显示，人类的生殖能力在近半个世纪的时间内呈持续下降趋势，特别是男性的生殖能力，据统计精子的数量和质量平均值从五十年前的每毫升一亿一千万到现在的二千六百万。在我国统计学分析发现不孕不育患者中有一半是男性因素造成的。单精子胞浆内注射技术(intracytoplasmic sperm injection，ICSI)的成功使男性少精子症、无精子症患者看到了希望，通过治疗能够成为生物学父亲。那么造成男性不育的原因如何，外周血的激素水平哪项更能够准确反应睾丸的生精功能，精子在形态学方面有何改变以及男性不育的遗传学问题等等已成为当今辅助生殖领域的热点问题。目的：1．研究严重少精子症患者外周血清相关激素：睾酮(testosterone，T)、游离睾酮(free testosterone，FT)、性激素结合球蛋白(Sex hormone-binding globulin，SHBG)、血清抑制素B(inhibin B，IHN-B)水平与睾丸生精功能间的关系。2．运用扫描电子显微镜(scanning electron microscope，SEM)观察测定严重少精子症患者精子表面结构特点，检测精子畸形指数(sperm deformity index，SDI)，与精子常规染色(Diff-Quik法)光学显微镜观察进行对比研究。3．进行精子DNA提取，Y染色体微缺失(Y cheomosome microdeletion analysis)研究，比较体细胞Y染色体微缺失与配子Y染色体微缺失发生率。方法：1．严格按照WHO标准筛选分组，严重少精子症患者为实验组(重复精子常规计算机分析检洲精子密度低于5×10~6／ml，经2次以上检查并离心沉淀未见精子者除外)。对照组为已生育男性。于晨起间隔2小时抽取外周血两次，离心分离血清，将两次血清混匀后取适量混合，运用ELSA法测定外周血T、FT、SHBG、IHN-B进行检测。2．采取精液后运用梯度离心分离精子，运用锡箔纸作为精子SEM载体，按SEM制样程序制样，观察精子表面超微形态特点及缺陷；运用Diff-Quik法涂片常规染色精子，在光镜下进行精子头部缺陷、颈部缺陷、尾部缺陷记数观察；比较两种精子形态学检测方法。3．提取精子DNA，运用聚合酶链反应(polymerase chainreaction，PCR)特异地扩增Y染色体上的目标区域，进行精子Y染色体微缺失检测和外周血Y染色体微缺失率检测。4．结果进行统计学分析。结果：1．严重少精子症患者外周血血清IHN-B较对照组明显降，差异有显著性。血清T、FT、SHBG与对照组相比差异无显著性。2．严重少精子症患者的精子存在超微形态异常，SEM观察发现严重少精子症患者SDI与对照组比较明显增高(1.64±0.55和0.86±0.23，P＜0.05)。3．严重少精子患者精子Y染色体微缺失检测发现存在无精子症因子(azoospermia factor，AZF)C区(AZFc)缺失，其发生率为15％，较外周血AZFc检测发生率6.7％明显增高，差异具有显著性差异。未发现AZFa、AZFb等其它缺失。结论：1．严重少精子症患者的血清抑制素B检测较T、FT、SHBG等检测指标能准确预测睾丸的生精功能。2．严重少精子症之精子存在表面超微结构异常，其SDI明显高于对照组，SEM观察精子较光学显微镜普通染色技术更能够发现精子的头颈部缺陷。3．严重少精子症忠者精子Y染色体微缺失检测发现AZFc缺失阳性率明显高于外周血检测结果。讨论：1．男性不育呈不断上升趋势，使得我们不得不正视这个问题，多年以来学术界一直寻求更好、更准确的研究指标来反应睾丸的生精功能，本研究选取了目前较为关注的几个指标进行检测比较，发现IHN-B水平的高低能够更为准确的反应精子的生成情况。2．在精子扫描电子显微镜标本制样观测，具有独特的优点，能够更为直观发现精子的缺陷。通过对于一些反复进行助孕治疗失败病例的诊治将有所帮助。3．精子DNA提取有无可比拟的先进性、精确性，研究结果发现严重少精子症患者的AZFc缺失率较外周血明显增加，使过去一部分难以解释的严重少精子症的原因得到解释。4．ICSI技术使男性不育症患者看到了希望，有可能得到生物学予代，其子代的遗传性状如何，是我们将来需要深入研究的问题。更多还原

【Abstract】 Introduction: According to WHO report, Human reproduction capability is poor thanbefore. However, sperm density and motility were being decreased from 120×10~6/mlto 26×10~6/ml during 50 years. Recent work by National study indicates the reason thatabout 50％couples with infertility is male reason. Intracytoplasmic sperm injection(ICSI) is a recent major advance in the treatment of such couples. ICSI offersidiopathic oligospermia couples the possibility of fathering own genetic children.However, what is a useful marker of spennatogenesis in serum7 How is spermmorphology construction by scanning electron microscope (SEM) and by spermmorphology Quick Staining kit? The reason of gene idiopathic oligozoospermia is theaim of present study.Objective:To investigate the correlation of serum hormone and sperm morphology construction by scanning electron microscope (SEM) and the Y-chromosome microdeletions with idio-pathic oligozoospermia patient.Materials and methods:A total of 60 consecutive men who presented with clinical and laboratory dataindicating idiopathic oligospermia. Mean male age was 32.1±2.8(rang 29～36)(group1), The control group included 60 men having normal seminal parameters was31.0±2.2(rang 28～35)(groug 2).1. Patients and seminal analyses: Semen samples were produced by masturbationafter 2～7 days of sexual abstinence and collected into sterile containers. Thesperm density was detected by computer-aided sperm analysis(CASA) at least two seminal analyses which were carried out as described in the World HealthOrganization Manual(WHO, 1999). Each patient underwent physicalexamination. The sperm density in semen was detected by routine semen analysis.Group 1 sperm density was less than 5×10~6/ml and more than 0. Group 2 spermdensity was higher than 20×10~6/ml.2. Hormone Analyses: Hormones were measured using commercially available kits.testosterone (T), free testosterone (FT), sex hormone-binding globulin (SHBG)and inhibin B(IHN-B). Blood and semen samples were collected from two groups.Serum was collected two times at 8:00and 10:00 am, and mixed serum amply.Serum concentration was measured by using specific an enzyme linkedimmunosorbent assay (ELISA) with two monoclonal antibodies in serum ofpatients with oligospermia.3. Semen processing: Semen samples were obtained by masturbation after 2～7 daysof sexual abstinence. After liquefaction at incubator, semen parameters wereassessed as outlined by the WHO criteria. Sample was examed by spermmorphology Quick Staining (Diff-Quik) Kit. Semen analyses were performed toseparate motile spermatozoa from non-sperm cells, The ejaculate was layeredover the gradient and centrifuged at 2000r/min for 20min. After centrifugation ,thesupernatant was removed, and sperm resuspended in 2ml freshmedium(Sperm-washing medium, COOK). Sperm washing according to assistedReproduction Technology (ART). The semen was divided into two parts equally.One part of semen was fixed in 2.5％(w/v) glutaraldehyde solution in a sodiumacetone series, dried in a critical point drier using carbon dioxide, mounted on thespecimen holder, coated with gold, and examined under a scanning electronmicroscope (SEM), detecting the sperm deformity index (SDI).The another part ofsemen was performed and checked by PCR amplification of selected regions ofthe Y-chromosome. The male-specific region of the Y-chromosome (MSY) STSprimers amplify both anonymous sequences of the chromosome or genes. At the same time lymphocytes Y- chromosomal microdeletions of blood was detected.The PCR amplification genomic DNA for clinical diagnosis requires strictcompliance with excellently laboratory practice and basic principles of qualitycontrol.Results:1. Inhibin B significantly lower in group 1 compared with group 2. Serum inhibin Bconcentrations were significantly higher with group 2(mean values25.07±19.49pg/ml and 189.06±47.06 pg/ml, P＜0.01). In contrast, no differenceswere detected between these two groups with respect to serum T(mean values437.27±109.1 ng/dl and 436.66±97.52 ng/dl)、FT(mean values 17.03±5.94 pg/mland 18.63±4.32 pg/ml)、SHBG(mean values 186.21±68.74 pg/ml and184.24±67.68 pg/ml) or testicular size respectively(P＞0.05).2. The study of sperm morphology construction by SEM detecting SDI, there wassignificant differences between two groups(1.644±0.55 vs 0.864±0.23, P＜0.05). SDIof sperm of Idiopathic oligozoospermia had more deformity than that of normalgroup. The study of sperm morphology by SEM is better way than other method.3. Multi-PCR and PCR-RFLR were used to analyze the sperm deletion of DAZ genecopies in the AZFc region of Y-chromosome. Chromosoma(quantity andconstruction were detected by G-band in the two groups). In the severeoligozoospermic patients and fertile males, the rates of AZFc deletion was 15％, noother type Y-chromosomal microdeletions was found, but no deletion was detectedin the normal control group. There is significant differences in the semenY-chromosomal microdeletions between two groups. The rates of oligospermiaAZFc microdeletion (15％)is higher than lymphocytes.Conclusion:1. Measuremcnt of serum IHN-B concentration could reflect the function of tesis,which is useful for early diagosis and treatment of oligospermia. Inhibin Bmeasurement is a useful non-invasive predictor of spermatogenesis, and thus, all infertile males should have serum inhibin B concentrations determined in additionto other measurement.2. SDI of oligospennia is significant higher than fertile group by SEM. SEM may bea better method to exam sperm of infertile males.3. There is a high frequency of sperm Y-chromosomal microdeletions in patientswith oligosperia than that of serum lymphocytes Y-chromosomal microdeletions,which suggests that Y-cbormosomal microdeletions might be important genoticcauses sperm density abnormal and rnale spermatogensesis failure.更多还原