【作者】 蔡文娟；

【导师】 谷翊群； 于和鸣； 梁小薇； 卢文红；

【作者基本信息】 中国协和医科大学 ， 外科学， 2010， 硕士

【摘要】 目前,临床上还没有鉴别OA和NOA敏感性和特异性都非常高的血清学标志物。研究血清INHB的鉴别效果是当务之急。INHB自被发现迄今已有七十余年。在已经完成的INHB试验研究中,发现其预测精子发生状态效果较好,是一个非常有临床应用前景的激素。INHB研究的目的是预测睾丸内精子发生的状况,从而为男性不育症的临床诊治提供依据。然而,抑制素B在国内男性的正常参考值范围以及在鉴别OA和NOA的应用价值这两个问题一直没有得到解决。由于血清INHB在不同种族差异具有显著性,因此国外的正常参考范围以及鉴别OA和NOA的切点不能在国内得以应用。尽管国内一些临床研究给出了参考范围,但是均由于标本量不够以及实验方法落后等原因而没有被普遍采用。FSH、精浆中性α-Glu在OA和NOA患者之间存在差异。近年来,国外越来越多的证据显示,INHB比FSH更能够预测精子的发生。通过研究比较血清INHB和FSH、精浆中性α-Glu在预测OA或者NOA的敏感性和特异性,可以阐明INHB在鉴别OA和NOA的应用价值,从而为更科学的应用INHB提供理论指导与实验依据。目的：本研究通过测定血清抑制素B (INHB)并与卵泡刺激素(FSH)和精浆中性α-葡糖苷酶(α-Glu)等经典指标比较,评价INHB在鉴别诊断梗阻性(OA)和非梗阻性无精子症(NOA)中的应用价值,并对睾丸精子发生障碍作出预判。方法健康生育男性组(n=60),以睾丸活检为金标准确定OA组(n=39)和NOA组(n=77),留取血液和精液标本,进行精液常规分析,检测血清INHB、FSH和精浆中性α-Glu的水平,采用受试者工作特征(ROC)曲线法,通过计算ROC曲线下面积,确定切点值并分析评价检测指标的敏感性和特异性。结果本实验室健康育龄男性血清INHB的95%参考值范围为：20.37-206.21pg/ml。血清INHB. FSH.精浆中性α-Glu、血清INHB/FSH比值以及INHB+FSH联合在OA组与NOA组之间均差别显著,具有统计学意义(P<0.01)。其中血清INHB的曲线下面积最大,为0.985,诊断价值最高,敏感性为97.4%,特异性为92.2%,切点值为49.89pg/ml。结论血清INHB比血清FSH、精浆中性α-Glu、血清INHB/FSH比值、或INHB+FSH联合指标在鉴别OA与NOA方面具有最佳的敏感性与特异性。Y染色体上的无精子症因子(azoospermiafactor, AZF)基因位点的微缺失会导致非梗阻性无精子症(non-obstructive azoospermia, NO A)的发生。因此,NOA患者有必要进行Y染色体微缺失检测以明确病因。Y染色体微缺失可以通过PCR技术进行检测。尽管一些研究通过单重或者多重PCR技术研究了不同男性人群Y染色体从2个到22个位点的微缺失,然而,检测技术以及检测位点一直是大家争论的议题。已有研究发现NOA患者人群会发生Y染色体微缺失,缺失率从1%到55%各不相同。检测结果差异产生的可能原因与以下因素有关：(1)研究对象选择标准不同；(2)检测所用STSs标记位点密度和位置不同；(3)检测方法不同；(4)群体遗传背景和环境不同。目的：评价多重PCR和单重PCR检测Y染色体微缺失的优劣,检测6个位点和15个位点的利弊,分析NOA患者Y染色体微缺失的患病率。通过研究患者是否有Y染色体微缺失的状况,可以明确病因,从而为医生临床诊疗提供指导和实验依据。方法：将15个经典微缺失位点分成6组,摸索各组进行多重PCR的最佳反应条件；摸索15个引物进行单重PCR的反应条件；继而分别进行NOA患者DNA的检测,确定每位患者Y染色体微缺失的情况。再运用统计学方法进行多重PCR和单重PCR的比较；以及比较经典的15个微缺失位点和传统的6个微缺失位点的检出率。结果：获得各组进行多重PCR的最佳反应条件。73名患者经传统的6个微缺失位点的检测和15个微缺失位点的检测发现：两者在微缺失人数和位点缺失数目上的比较均具有统计学差异(χ2=5.06,P<0.05和χ2=15.06,P<0.05)。单重PCR和多重PCR的检测率进行比较后发现,微缺失人数没有统计学差异(χ3=2.25,P>0.05),位点数目的差异在统计学上具有显著性(χ2=22.04,P＜0.05)。在12名NOA且存在Y染色体微缺失的患者中,有1、2、3、6名患者分别缺失4、3、2、1个位点。结论：通过检测15个微缺失位点发现NOA患者中Y染色体微缺失的发生率为16.44%。15个位点的Y染色体微缺失检测比6个位点的检测更有临床意义和价值。Y染色体微缺失的多重PCR检测会导致假阴性结果,需要单重PCR进行确认。研究还发现AZFb+c的缺失对生精功能损伤可能较为严重、Y染色体微缺失和睾丸体积无相关性以及不同PCR仪上的反应条件不一定能够通用。更多还原

【Abstract】 Currently in the world, the hormone distinguishing OA and NO A with very high sensitivity and specificity does not exit in clinical usage. The need for researching serum INHB has been greater. It has been seventy years when INHB was found. Most trials demonstrated that INHB had good effects in predicting spermatogenesis status and was very promising in clinical applications.The study of INHB is to predict the status of spermatogenesis within the testis. Thus provide the basis for clinical diagnosis and treatment. However, the normal reference range for chinese men as well as the cut point in identifying OA and NOA has not been obtained till now. Because of significant racial differences in serum INHB, the normal reference range as well as the cutoff point in identifying OA and NOA with INHB aborad can not be applied internally. Although some references are given by chinese clinical researcheres, they are not widely used because of small subject number or inferior experimental methods.FSH, seminal plasma neutral a-Glu have differences in patients with OA and NOA. Within recent years an increasing body of evidence has shown that INHB is better than FSH and seminal plasma neutral a-Glu in predicting spermatogenesis.A study can clarify the application values of INHB in distinguishing OA and NOA to provide guidance in theory and experimental evidence for INHB to doctors. The study compared the serum INHB and FSH, seminal plasma neutral a-Glu in forecasting OA and NOA.Objective:This research aims to evaluate the applicable values of serum inhibin B(INHB) in distinguishing obstructive and non-obstructive azoospermia and comparing it with classical parameters including follicle-stimulating hormone(FSH) and seminal plasma neutralα-glucosidase. Meanwhile, to predict the spermatogenetic malfunction in testes. Methods:Semen and blood samples were collected from healthy fertile males(n=60), non-obstructive azoospermia(NOA, n=77) and obstructive azoospermia(OA, n=39) that was defined by testicular biopsy as golden standard for diagnosis. Semen parameters were analyzed. Levels of the serum INHB、FSH and seminal plasma neutralα-glucosidase were determined and their area under curve (AUC), cut-off points, sensitivities and specificities were calculated through receiver operating characteristics(ROC) curve.Results:The 95% confidence interval of serum inhibin B in healthy fertile males was between 20.37 pg/ml and 206.21 pg/ml in our laboratory. There were significant differences in levels of serum inhibin B, FSH, seminal plasma neutralα-glucosidase, INHB/FSH ratio and combination of INHB plus FSH between the OA and NOA(P<0.01), among which, serum INHB alone had the largest AUC of ROC with diagnostic value of 0.985 while the sensitivity and specificity were 97.4% and 92.2% respectively.Conclusion:Serum inhibin B has better sensitivity and specificity in distinguishing OA and NOA than FSH, seminal plasma neutralα-glucosidase, INHB/FSH ratio and combination of INHB plus FSH. Y chromosome microdeletions in loci of azoospermia factor (AZF) will lead to non-obstructive azoospermia(NOA). Therefore, patients with NOA are necessary to be tested. Microdeletion loci can be tested through the PCR technique. However, there have been arguments on the methods and loci in testing Y chromosome microdeletions. Although some researches showed two to twenty-two microdeletion loci situations through single or multi-PCR technique in different males groups, the way on how to measure it have become what we debate.Some researchers have found that patients with NOA had incidence of Y chromosome microdeletions from 1% to 55%. The possible causes of different incidences are given as follows:(1) different selection criteria for subjects; (2) different STSs marker density and locations; (3) different detection methods; (4) different genetic background and environment.Objective:This study compared multi-PCR and single PCR detecting Y chromosome microdeletions, evaluated using 6 or 15 microdeletions loci in testing patients with NOA, so that causes of azoospermia can be verified for clinical diagnosis and treatment in order to provide guidance for clinical practices using this technique.Methods:The 15 classical microdeletion loci were divided into six groups and the optimal multi-PCR reaction conditions in each group and single PCR reaction conditions were found. Then each patient was detected with their own DNA. The multi-PCR and single PCR detection rates,15 and 6 loci microdeletion rates were compared using statistical methods.Results:The six groups’optimal multi-PCR reaction conditions were obtained. Each single PCR reaction condition was explored. In the same 73 patients, statistical significances in either microdeletion patient number and microdeletion locus number (x2=5.06, P＜0.05; x2=15.06, P＜0.05) were found with both traditional 6 loci test and classical 15 loci test. However, no statistically significant (χ2= 2.25, P> 0.05) was found in patient number of microdeletion by comparing single PCR and multi-PCR, while the locus number was statistically significant (χ2=22.04, P<0.05). Among the 12 patients, microdeletion loci at frequency of 4、3、2、1 were detected in 1、2、3、6 patients, respectively.Conclusion:The incidence of 15 microdeletion loci detected by multi-PCR in patients with NOA is 16.44%. Testing 15 Y-chromosome microdeletion loci have much more clinical values than testing 6 microdeletion loci only. Multi-PCR reaction sometimes may lead to false negative results and needs to be verified by single PCR method.The research also found that microdeletion of AZFb+c can lead to very serious damage to spermatogenic function.Y chromosome microdeletion has no relationship to testicular volume.Different PCR machines cannot use the same reaction condition.更多还原