严重少精子症患者血清相关激素测定与精子超微形态及Y染色体微缺失检测研究
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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。
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.
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。
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.
引文
1．马乐，潘柏年，陈宝英，主编．男性不育与辅助生殖技术．人民卫生出版社．北京．2002．1-8
2．李江源．生殖功能的内分泌调节．男子不育诊疗规范化研讨班讲义．上海．2006．26
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18. Ramaswamy S, Marshall GR, Mcneilly AS, et al. Dynamics of the follicle-stimulating hormone (FSH)-inhibin B feedback loop and its role in regulating spermatogenesis in the adult male rhesus monkey (Macaca mulatta) as revealed by unilateral orchidectomy[J]. Endocrinology,2000,141(1):18-27.
19. Anderson RA, Irvine DS, Balfour C, et al. Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis[J]. Hum Reprod,1998,13(4):920-926.
20. Anderson RA, Wallau EM, Groome NP, et al. Physiological relationships between inhibin B, follicle stimulating hormone secretion and spematogenesis in normal man and response to gonadotrophin suppression by exogenous testosterone[J].Hum Reprod, 1997,12(4):746—751.
21. Eckardstein SV, Simoni M, Bergmmann M, et al. Serum inhibin B in combination with serum follicle stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men ,but cannot predict the presence of sperm in testicular tissue samples [J]. J Clin Endo Metab,1999,84(7):2496-2501.
22. Pierik FH, Vreeburg JTM, Stijnen T, et al. Serum inhibin B as a marker of spermatogenesis[J]. J Clin Endo Metab,1998,83(9):3110—3114.
23. Andersson AM, Toppari J, Haavisto AM, et al. Longitudinal reproductive hormone profiles in infants: Peak of inhibin B levels in infant boys exceeds levels in adult men[J].J Clin Endo Metab,1998,83(2):675-681.
24. Ballesca JL, Balasch J, Calafell JM, et al. Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia[J]. Hum Repord, 2000,15(8):1734-1738.
25. Pierik FH, Vreeburg JTM, Stijnen T, et al. Serum inhibin B as a marker of spermatogenesis [ J]. J Clin Endo Metab, 1998, 83(9) :3110-3114
26. Meachem SJ, Nieschlag E, Simoni M. Inhibin B in male reproduction: pathophysiology and clinical relevance[J]. Eur J Endocrino, 2001, 145(5):561-571
27. Anderson RA. Clinical studies: inhibin in the adult male[J].Mol Cell Endocrino 1,2001,180 (1-2): 109-116.
28. Hayes FJ, Pitteloud N, DeCruz S, et al. Importance of inhibin B in the regulation of FSH secretion in the human male[ J]. J Clin Endo Metab, 2001, 86(11):5541-5546.
29. Carlsen E, Olsson C, Petersen JH, et al. Diurnal rhythm in serum levels of inhibin B in normal men: relation to testicular steroids and gonadotropins[J]. J Clin Endo Metab, 1999, 84(5) : 1664-1669.
30. Jose LB,Juan B, Josep MC,et al.Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospernia[J].Hum Reprod,2000,15(8),1734-1738
31. Keel BA, Quinn P, Schmidt CF Jr, et al. Results of the American Association of Bioanalysts National Proficiency Testing Programme in andrology[J]. Hum Reprod, 2000,15(3): 680-686
32．黄宇烽，Philip S．Li，精液分析标准化刻不容缓，中华男科学杂志．2005，11(2)：83-84
33．孙桂勤，印洪林，周晓军，人类精液标本的电镜制备与染色技术，中华男科学．2002，8(12)：461
34. Ralf RH, Wolf-Bernhard S, Sperm Preparation for ART[J], Reprod Biol and Endocrin, 2003,14(11): 108
35．张景强，朴英杰，蔡福筹等编著，生物电子显微技术[M]．(第二版)．广州．中山大学出版社，1993．34-62
36．M．ARABI，B．SHAREGHI，尼古丁的抗生育效应，中华男科学杂志．2005，5(11)：323-330
37．Cheng D，Zheng XM，Li S W，et al．表皮生长因子对精索静脉曲张大鼠精子密度及活动能力的影响[J]．Asian Journal of Andrology，2006，8(6)：713-717
38．El-Hanggar S，El-Ashmawy,A Attia，et al．不育男性血清和精浆中的Beta-内啡肽[J]．Asian Journal of Andrology，2006，8(6)：703-712
39. Simoni M, Bakker E, Krarsz C. EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions.State of the art 2004[J]. International Jourmal of andrology,2004,27:240-249
40. Tiepolo L, Zuffardi O, Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm[J].Hum Genet, 1976,5(7):933-943
41. Reijo R, Lee TY, Salo P, et al. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding ppotein gene[J].Nat Genet, 1995,10(4):383-393
42. KN Jha, AM Salicioni, E Arcelay, O Chertihin, et al. Evidence for the involvement of proline-directed serine/threonine phosphorylation in sperm cpacitation[J].Mol Hum Reprod 2006,12(12):781-789
43. M.Lynch, D.S.Cram, A.Reilly, et al. The Y chromosome gr/gr subdeletion is associated with male infertility[J]. Mol Hum Reprod, 2005,ll(7):507-512
44. Ali Hellani, Saad Al-Hassan, Adel Al-Duraihim, et al. Y chromasome microdeletions: are they implicated in teratozoospermia?[J]. Hum Reprod, 2005,20(12): 3505-3509
45. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of th human Y chromosome long arm[J]. Hum Genet, 1976,34 : 119-124.
46. Skaletsky H,Kuroda KT,Minx PJ, et al.The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.Nature 2003,432,825-837
47. Yen P. The fragility of fertility[J].Nature Genetics,2001,29,243-244
48. Repping S,Skaletsky ,Lange J.et al. Recombination between palindromes P5and P1 on the human Y chromosome carses massive deletions and spermatogenic failure. American J of Human Genetics,2002,71,906-922
49. Repping, S,Skaletsky H, Brown L,et al.Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploed selection[J].Natrue Genetices,2003,35,247-251
50. Vogt PH, Edelmann A, Kirsch S, et al. Human Y chromosome azoospermia factors(AZF)mapped to different subregions in Yqll[J].Hum Mol Genet, 1996,5(7):933-943
51. Saxena R, de Vries, Repping S,et al, Four DAZ genes in two clusters fornd in the AZFc region of the human Y chromosome[J].Genomics , 2000,67,256-267
52. Oates RD, Silber S,Brown LG,et al. Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome ,and of 18 children conceived via ICSI[J].Hum Reprod,2002,17(11):2813-2824
53. Cram DS,Ma K, Bhasin S,Arias M,et al,Y chromosome analysis of infertile men and their sons conceived through intracytoplasmic sperm injection :vertical transmission of deletions and rarity of de novo deletions[J].Fertility and Sterility,2000,74:909-915
54. Krausz C, Rajpert DM, Larsen LF, et al. Double blind screening for microdeletions of Y chromosome genes in infertile and fertile/normospermic Danish men. [J]Joumal of Clinical Endocrinology and Metabolism,2001,86,263 8-2642
55. Femandes S, Huellen K, Goncalves J, et al.High frequency of DAZ1/DAZ2 gene deletions in patients with severe oligozoospermia[J].Mol Hum Reprod, 2002,8(3):286-289
56. Dohle GR,Halley KJ, Van Hemel JO, et al. Genetic risk factors in infertile men with severe oligozoospermia and azoospermia[J].Hum Reprod, 2002,17(1):13-16
57. Foresta C, Ferlin A, Gianaroli L, et al.Guidelines for the appropriate use of genetic tests in inferile couples[J].Eur J Hum Gent, 2002,10(5):303-312
58. Krausz C , Quintana ML, McElreavey K, et al. Prognostic value of Y deletion analysis:what is the clinical prognostic value of Y chromosome microdeletion analysis? [J].Hum Reprod,2000,15,1431-1434
59. Krausz C, Forti G, McElreavey K. The Y chromosome and male fertility and infertility[J].International Journal of Andrology, 2003,26,70-75
60. Skaletsky H, Kuroda-KT, Minx PJ, et al. The male-specific region of the human Y Chromosome is a mosaic of discrete sequence classes[J].Nature,2003,423,825-837
1. Matthew RN, Nicholas LC. Structural Features of Sterols Required to Inhibit Human Sperm Capacitation[J]. Biology of Reproduction, 2003 ;68:1308-1317.
2. M.lynch,DS Cram, A Reilly, et al. The Y chromosome gr/gr subdeletion is associated with male infertility[J].Mol.Hum.Reprod.2005,11 (7): 507-512.
3．王国洪，许瑞吉，张中书等，少精症患者血清和精浆性激素水平的测定及其意义．Labeled Immunoassays & Clin Med，2005，12(2)：82-84．
4．周桂香，刘雨生，骆丽华等，严重生精功能障碍患者助孕前遗传学检查的意义[J]．Chinese Journal of Andrology．2005，19(5)：26-28．
5．谷翊群，陈振文，于和鸣等，译．人类精液及精子-宫颈粘液相互作用实验室检验手册(第四版)．
6．李仁良综述，抑制素B与男性生殖的研究进展[J]．中华男科学杂志2005，11(4)：299-302
7. Chada M, Prusa R, Bronsky J, et al. Inhibin B,follicle stimulating hormone ,luteinizing hormone and testosterone during childhood and puberty in males:changes in serum concentrations in relation to age stage of puberty [J]. Physiol Res, 2003, 52(1):45-51.
8. Andersson AM, Carlsen E, Petersen JH, et al. Variation in levels of serum inhibin B, testosterone, estradiol, luteinizing hormone, follicle-stimulating hormone, and sex hormone-binding globulin in monthly samples from healthy men during a 17-month period: possible effects of seasons [J].J Clin Endocrinol Metab, 2003, 88(2):932-937.
9. F Morel, V Amice, C Roux, et al. Analysis by FISH of the meiotic segregation in spermatozoa from two pericentric inversion carriers. Abstracts of the 21st Annual Meeting of the ESHRE, Copenhagen, Denmark, 19-22 June 2005,186.
10. P.Navarro C, C.Proenca, P Marques-V, et al. The high polymorphism rates of the DAZ3+DAZ4 cluster suggest a functional clustering of DAZ genes. Abstracts of the 21st Annual Meeting of the ESHRE,Copenhagen, Denmark,19-22 June 2005,187.
11. S Femandes, P Costa, C Ferras, et al. DAZ1/DAZ2 deletion analysis in hypospermatogenesis. Abstracts of the 21st Annual Meeting of the ESHRE, Copenhagen, Denmark,19-22 June 2005,187
12．朱平主编 临床分子遗传学．第一版．北京．北京医科大学出版社，2002年 146-158.
13. Kamp C, Hirschmann P, Voss H, et al.Tvo Long homologous retroviral sequence blocks in proximal Yq11 cause AZFa microdeletions as a result of intrachromosomal recombination wvents[J].Hum Molecular Genetics,2000,9,2563-2572
14. Kamp C,Huellen K, Fermandes S, Sousa M, et al.High deletion frequency of the complete AZFa sequence in men with Sertoli-cell-only syndrome[J].Molecular Human Reproduction,2001,7,987-994
15. Kent-first MG, Kol S, Muallem A, et al.The incidence and possible relevance of Y-linked microdeletions in babies born after imtracytoplsmic sperm injection and their infertile fathers[J].Molecular Human Reproduction, 1996,2,943-950
16. Krausz C, Quintana-M, L Mcelreavey K, et al. Prognostic value of Y deletion analysis; what is the clinical prognostic value of Y deletion analysis?[J]Hum Reprod.2000,15,1431-1434
17. Krausz C, Rorti G, McElreavey K, et al. The Y chromosome and male fertility and infertility[J].Intermational Jounal of Andrology,2003,26,70-75
18. Luetjens CM, Gromoll J, Engelhardr M, et al.Manifestation of Y-chromosomal deletions in the human testis:a morphometrical and immunohistochemical evaluation[J]. Hum Reprod, 2002,17,2258-2266
19. Tates RK, Silber S, Brown LG, et al.Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome and of 18 children conceived via ICSI[J].Hum Reprod,2002,17,2813-2824
2．李江源．生殖功能的内分泌调节．男子不育诊疗规范化研讨班讲义．上海．2006．26
3. Keel BA.Quality control quality assurance and proficiency testing in the andrology laboratory[J].Arch Androl,2002,48 (6):417-431.
4. Keel BA,Quinn P, Schmidt CF Jr,et al. Results of the American association of bioanalysis national proficiency testing programme in andrology[J].Hum Reprod,2002,15(3):680-686.
5. Keel BA.How reliable are results for the semen analysis?[J].Fertil Steril,2004,82(1):41-44
6. Wernaeve W, Tournaye H, Schiettecatte J, et al. Serurn inhibin B cannot predict Testicular sperm retrieval in patients with non-obstructive azoospermia[J].Fertil Steril, 2002,78(6):1195-1198
7．谷翊群，陈振文，于和鸣等，译．人类精液及精子．宫颈粘液相互作用实验室检验手册(第四版)．
8．丛玉隆， 秦小玲， 邓新立，主编．现代医学实验室管理与实践．人民军医出版社．北京．2005．2～3
9．周俊宜，主编．分子医学技能．科学出版社．北京．2006．218-220．
10．李仁良综述，抑制素B与男性生殖的研究进展[J]．中华男科学杂志2005，11(4)：299-302．
11. Kinniburgh D, Anderson RA. Differential patterns of inhibin secretion in response to gonadotrophin stimulation in normal men[J].Int J Androl,2001,24(2):95-101
12. Hipler UC, Hochheim B, Knoll B, et al. Serum inhibin B as amarker for spermatogenesis[J].Arch Androl, 2001,46(3) :217-222.
13. Chada M, Prusa R, Bronsky J, et al. Inhibin B, follicle Stimulating hormone, luteinizing hormone and testosterone during childhood and puberty in males : changes in serum concentrations in relation to age and stage of puberty [J].Physiol Res, 2003,52(1):45-51
14. Bohring C, Krause W. Serum levels of inhibin B in men of different age groups[J].Ahing Male, 2003,6(2):73-78
15. Andersson AM, Carlsen E, Petersen JH, et al. Variation in levels of serum inhibin B, testosterone, estradiol, luteinizing hormone,follicle-stimulationg hormone, and sex hormone-binding globulin in monthly samples from healthy men during a 17-month period:possible effects of seasons[Jj.J Clin Endocrinol Metab, 2003,88(2):932-937
16. Yalti S, Gurbuz B, Ficicioglu C. Serum levels of inhibin B in men and their relationship with gonadal hormones, teticular volume, testicular biopsy results and sperm parameters[J].J obstet Gynaecol, 2002,22(6):649-654
17. Bohring V, Schroeder PI, Weidner W, et al. Serum lewels of inhibin B and follicle-stimulating hormone may predict successful sperm retrieval in men with azoospermia who are undergoing testicular sperm extraction[J].Fertil Steril, 2002,78(6): 1195-1198
18. Ramaswamy S, Marshall GR, Mcneilly AS, et al. Dynamics of the follicle-stimulating hormone (FSH)-inhibin B feedback loop and its role in regulating spermatogenesis in the adult male rhesus monkey (Macaca mulatta) as revealed by unilateral orchidectomy[J]. Endocrinology,2000,141(1):18-27.
19. Anderson RA, Irvine DS, Balfour C, et al. Inhibin B in seminal plasma: testicular origin and relationship to spermatogenesis[J]. Hum Reprod,1998,13(4):920-926.
20. Anderson RA, Wallau EM, Groome NP, et al. Physiological relationships between inhibin B, follicle stimulating hormone secretion and spematogenesis in normal man and response to gonadotrophin suppression by exogenous testosterone[J].Hum Reprod, 1997,12(4):746—751.
21. Eckardstein SV, Simoni M, Bergmmann M, et al. Serum inhibin B in combination with serum follicle stimulating hormone (FSH) is a more sensitive marker than serum FSH alone for impaired spermatogenesis in men ,but cannot predict the presence of sperm in testicular tissue samples [J]. J Clin Endo Metab,1999,84(7):2496-2501.
22. Pierik FH, Vreeburg JTM, Stijnen T, et al. Serum inhibin B as a marker of spermatogenesis[J]. J Clin Endo Metab,1998,83(9):3110—3114.
23. Andersson AM, Toppari J, Haavisto AM, et al. Longitudinal reproductive hormone profiles in infants: Peak of inhibin B levels in infant boys exceeds levels in adult men[J].J Clin Endo Metab,1998,83(2):675-681.
24. Ballesca JL, Balasch J, Calafell JM, et al. Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospermia[J]. Hum Repord, 2000,15(8):1734-1738.
25. Pierik FH, Vreeburg JTM, Stijnen T, et al. Serum inhibin B as a marker of spermatogenesis [ J]. J Clin Endo Metab, 1998, 83(9) :3110-3114
26. Meachem SJ, Nieschlag E, Simoni M. Inhibin B in male reproduction: pathophysiology and clinical relevance[J]. Eur J Endocrino, 2001, 145(5):561-571
27. Anderson RA. Clinical studies: inhibin in the adult male[J].Mol Cell Endocrino 1,2001,180 (1-2): 109-116.
28. Hayes FJ, Pitteloud N, DeCruz S, et al. Importance of inhibin B in the regulation of FSH secretion in the human male[ J]. J Clin Endo Metab, 2001, 86(11):5541-5546.
29. Carlsen E, Olsson C, Petersen JH, et al. Diurnal rhythm in serum levels of inhibin B in normal men: relation to testicular steroids and gonadotropins[J]. J Clin Endo Metab, 1999, 84(5) : 1664-1669.
30. Jose LB,Juan B, Josep MC,et al.Serum inhibin B determination is predictive of successful testicular sperm extraction in men with non-obstructive azoospernia[J].Hum Reprod,2000,15(8),1734-1738
31. Keel BA, Quinn P, Schmidt CF Jr, et al. Results of the American Association of Bioanalysts National Proficiency Testing Programme in andrology[J]. Hum Reprod, 2000,15(3): 680-686
32．黄宇烽，Philip S．Li，精液分析标准化刻不容缓，中华男科学杂志．2005，11(2)：83-84
33．孙桂勤，印洪林，周晓军，人类精液标本的电镜制备与染色技术，中华男科学．2002，8(12)：461
34. Ralf RH, Wolf-Bernhard S, Sperm Preparation for ART[J], Reprod Biol and Endocrin, 2003,14(11): 108
35．张景强，朴英杰，蔡福筹等编著，生物电子显微技术[M]．(第二版)．广州．中山大学出版社，1993．34-62
36．M．ARABI，B．SHAREGHI，尼古丁的抗生育效应，中华男科学杂志．2005，5(11)：323-330
37．Cheng D，Zheng XM，Li S W，et al．表皮生长因子对精索静脉曲张大鼠精子密度及活动能力的影响[J]．Asian Journal of Andrology，2006，8(6)：713-717
38．El-Hanggar S，El-Ashmawy,A Attia，et al．不育男性血清和精浆中的Beta-内啡肽[J]．Asian Journal of Andrology，2006，8(6)：703-712
39. Simoni M, Bakker E, Krarsz C. EAA/EMQN best practice guidelines for molecular diagnosis of y-chromosomal microdeletions.State of the art 2004[J]. International Jourmal of andrology,2004,27:240-249
40. Tiepolo L, Zuffardi O, Localization of factors controlling spermatogenesis in the nonfluorescent portion of the human Y chromosome long arm[J].Hum Genet, 1976,5(7):933-943
41. Reijo R, Lee TY, Salo P, et al. Diverse spermatogenic defects in humans caused by Y chromosome deletions encompassing a novel RNA-binding ppotein gene[J].Nat Genet, 1995,10(4):383-393
42. KN Jha, AM Salicioni, E Arcelay, O Chertihin, et al. Evidence for the involvement of proline-directed serine/threonine phosphorylation in sperm cpacitation[J].Mol Hum Reprod 2006,12(12):781-789
43. M.Lynch, D.S.Cram, A.Reilly, et al. The Y chromosome gr/gr subdeletion is associated with male infertility[J]. Mol Hum Reprod, 2005,ll(7):507-512
44. Ali Hellani, Saad Al-Hassan, Adel Al-Duraihim, et al. Y chromasome microdeletions: are they implicated in teratozoospermia?[J]. Hum Reprod, 2005,20(12): 3505-3509
45. Tiepolo L, Zuffardi O. Localization of factors controlling spermatogenesis in the nonfluorescent portion of th human Y chromosome long arm[J]. Hum Genet, 1976,34 : 119-124.
46. Skaletsky H,Kuroda KT,Minx PJ, et al.The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.Nature 2003,432,825-837
47. Yen P. The fragility of fertility[J].Nature Genetics,2001,29,243-244
48. Repping S,Skaletsky ,Lange J.et al. Recombination between palindromes P5and P1 on the human Y chromosome carses massive deletions and spermatogenic failure. American J of Human Genetics,2002,71,906-922
49. Repping, S,Skaletsky H, Brown L,et al.Polymorphism for a 1.6-Mb deletion of the human Y chromosome persists through balance between recurrent mutation and haploed selection[J].Natrue Genetices,2003,35,247-251
50. Vogt PH, Edelmann A, Kirsch S, et al. Human Y chromosome azoospermia factors(AZF)mapped to different subregions in Yqll[J].Hum Mol Genet, 1996,5(7):933-943
51. Saxena R, de Vries, Repping S,et al, Four DAZ genes in two clusters fornd in the AZFc region of the human Y chromosome[J].Genomics , 2000,67,256-267
52. Oates RD, Silber S,Brown LG,et al. Clinical characterization of 42 oligospermic or azoospermic men with microdeletion of the AZFc region of the Y chromosome ,and of 18 children conceived via ICSI[J].Hum Reprod,2002,17(11):2813-2824
53. Cram DS,Ma K, Bhasin S,Arias M,et al,Y chromosome analysis of infertile men and their sons conceived through intracytoplasmic sperm injection :vertical transmission of deletions and rarity of de novo deletions[J].Fertility and Sterility,2000,74:909-915
54. Krausz C, Rajpert DM, Larsen LF, et al. Double blind screening for microdeletions of Y chromosome genes in infertile and fertile/normospermic Danish men. [J]Joumal of Clinical Endocrinology and Metabolism,2001,86,263 8-2642
55. Femandes S, Huellen K, Goncalves J, et al.High frequency of DAZ1/DAZ2 gene deletions in patients with severe oligozoospermia[J].Mol Hum Reprod, 2002,8(3):286-289
56. Dohle GR,Halley KJ, Van Hemel JO, et al. Genetic risk factors in infertile men with severe oligozoospermia and azoospermia[J].Hum Reprod, 2002,17(1):13-16
57. Foresta C, Ferlin A, Gianaroli L, et al.Guidelines for the appropriate use of genetic tests in inferile couples[J].Eur J Hum Gent, 2002,10(5):303-312
58. Krausz C , Quintana ML, McElreavey K, et al. Prognostic value of Y deletion analysis:what is the clinical prognostic value of Y chromosome microdeletion analysis? [J].Hum Reprod,2000,15,1431-1434
59. Krausz C, Forti G, McElreavey K. The Y chromosome and male fertility and infertility[J].International Journal of Andrology, 2003,26,70-75
60. Skaletsky H, Kuroda-KT, Minx PJ, et al. The male-specific region of the human Y Chromosome is a mosaic of discrete sequence classes[J].Nature,2003,423,825-837
1. Matthew RN, Nicholas LC. Structural Features of Sterols Required to Inhibit Human Sperm Capacitation[J]. Biology of Reproduction, 2003 ;68:1308-1317.
2. M.lynch,DS Cram, A Reilly, et al. The Y chromosome gr/gr subdeletion is associated with male infertility[J].Mol.Hum.Reprod.2005,11 (7): 507-512.
3．王国洪，许瑞吉，张中书等，少精症患者血清和精浆性激素水平的测定及其意义．Labeled Immunoassays & Clin Med，2005，12(2)：82-84．
4．周桂香，刘雨生，骆丽华等，严重生精功能障碍患者助孕前遗传学检查的意义[J]．Chinese Journal of Andrology．2005，19(5)：26-28．
5．谷翊群，陈振文，于和鸣等，译．人类精液及精子-宫颈粘液相互作用实验室检验手册(第四版)．
6．李仁良综述，抑制素B与男性生殖的研究进展[J]．中华男科学杂志2005，11(4)：299-302
7. Chada M, Prusa R, Bronsky J, et al. Inhibin B,follicle stimulating hormone ,luteinizing hormone and testosterone during childhood and puberty in males:changes in serum concentrations in relation to age stage of puberty [J]. Physiol Res, 2003, 52(1):45-51.
8. Andersson AM, Carlsen E, Petersen JH, et al. Variation in levels of serum inhibin B, testosterone, estradiol, luteinizing hormone, follicle-stimulating hormone, and sex hormone-binding globulin in monthly samples from healthy men during a 17-month period: possible effects of seasons [J].J Clin Endocrinol Metab, 2003, 88(2):932-937.
9. F Morel, V Amice, C Roux, et al. Analysis by FISH of the meiotic segregation in spermatozoa from two pericentric inversion carriers. Abstracts of the 21st Annual Meeting of the ESHRE, Copenhagen, Denmark, 19-22 June 2005,186.
10. P.Navarro C, C.Proenca, P Marques-V, et al. The high polymorphism rates of the DAZ3+DAZ4 cluster suggest a functional clustering of DAZ genes. Abstracts of the 21st Annual Meeting of the ESHRE,Copenhagen, Denmark,19-22 June 2005,187.
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