男性不育精液中NO的含量与生殖细胞凋亡的关系

目的:探讨人精液中一氧化氮(nitricoxide,NO)与生殖细胞凋亡的关系。方法:采用镀铜镉还原荧光法检测NO代谢产物硝酸盐(NO-3)。用脱氧核苷酸末端转移酶(TdT)介导的缺口末端标记(TUNEL)法和透射电镜,分别检测和观察生殖细胞的凋亡及凋亡细胞的超微结构。结果:生育组精液中NO的含量为(56.83±11.65)μmol/L,生殖细胞的凋亡率(4.60±1.25)%,与不育组(128.86±23.76)μmol/L和(17.36±3.05)%相比较有非常显著性差异(P<0.01)。不育组NO的含量和生殖细胞的凋亡率呈显著的正相关(r=0.96)凋亡的生殖细胞核染色质浓缩在核周形成新月形,电子密度增高,核膜折叠,核裂解形成凋亡小体。结论:不育者精液中NO的含量与生殖细胞的凋亡率有密切关系。高浓度的NO可能是导致睾丸生殖细胞凋亡率增加而致使男性生育力下降的原因。     

一氧化氮与男性免疫性不育的相关性

男性不育症的原因非常复杂。近年已证明,免疫因素是其中的重要原因之一。精子有较强的抗原性,能刺激自身、同种生殖道局部产生特异性抗体。现已揭示,抗精子抗体（ａｎｔｉｓｐｅｒｍａｎｔｉｂｏｄｙＡＳＡｂ）是不育的重要免疫因素１．２。为进一步探讨免疫不育的机制,我们检测了１５０例ＡＳＡｂ阳性男性不育患者精浆中的一氧化氮（ｎｉｔｒｉｃｏｘｉｄｅＮＯ）含量。１材料和方法１．１研究对象男性免疫不育患者１５０例,来自１９９９年３月～２０００年６月,我院泌尿外科、中医科和妇产科门诊患者均经临床和实验室确诊。…     

男性不育与精子凋亡关系的研究

目的探讨男性不育与精子凋亡的关系。方法采用计算机辅助精液分析对正常生育组（n=20）,不育组（n=60）男性进行精液参数检测,瑞-吉染色检测精子凋亡情况。结果在不同人群中精子凋亡均有一定发生率,正常生育组为（3.52±2.11）%,不育组为（18.26±9.34）%,两者差异极显著（P〈0.01）,并且精子凋亡与精子密度、精子活力以及精子正常形态率呈显著负相关（P〈0.05）。结论精子凋亡与男性不育存在着十分密切的关系,精子凋亡增加,可能是少精症、弱精症和畸形精子症患者不育的原因之一。     

中山地区男性不育患者精液质量分析

目的对中山地区男性不育患者的精液进行常规分析,了解中山地区男性不育患者精液质量的现状。方法按照《世界卫生组织人类精液检查与处理实验室手册》（第5版）的标准,应用西班牙人类精液分析微机辅助智能系统（CASA）对2012年10月～2013年6月来中山市博爱医院生殖中心就诊的2224例不育男性患者的精液进行常规检测和采用改良巴氏染色法分析精子正常形态率。结果2224例不育男性精液中正常者1113例（31．61％）,异常者1521例（68．39％）,其中液化异常964例（43．35％）,pH值异常124例（5．58％）,精子总数异常1002例（45．05％）,精子浓度异常556例（25．0％）,精子存活率异常1031例（46．36％）,精子总活力（PR＋NP）异常1208例（54．31％）,无精子症372例（16．72％）,精子正常形态率异常1021例（45．91％）。结论精子总活力、存活率下降,精子总数减少,精子正常形态率下降和精液液化不良是引起中山地区男性患者不育的主要原因,临床上应加于重视。     

精子运动能力与一氧化氮关系的探讨

目的探讨一氧化氮(NO)与精子运动能力的关系.方法采用镀铜镉还原荧光法,测定人精液中NO代谢产物硝酸盐(NO3-).参照WHO方法,在超高倍显微镜下观察精子存活率、活动力等. 结果 80例男性不育者其中15例NO浓度明显低于正常对照组精子活动力a b级<50%(P<0.01),以运动轨迹异常和头摆动幅度下降为主, 精子存活力>75%无明显差异(P>0.05);65例NO浓度显著高于正常对照精子活动力a b 级<50%,存活率<75% (P<0.001).结论 NO与精子运动功能有着密切关系.高浓度时明显抑制精子活动力及存活率.低浓度时精子存活率及功能有着维护作用,这对男性不育的病因研究和治疗有非常重要的临床价值.     

精子动态参数在男性不育精液中的定量分析

目的评价精子动态参数在男性不育症中的作用.方法选择217例男性不育及46例正常生育对照精液标本,用精子检测系统定量测定男性不育症与对照组的精子动态参数,并进行统计学处理.结果精子动态参数中曲线速度、直线速度、平均路径速度、平均移动角度、鞭打频率、直线性、摆动性、前向性等8个参数有明显统计学差异,侧摆幅度有统计学差异.结论精子的动态参数是评估男性不育的重要指标,而这些参数是传统手工目测法不能获得的.通过两组参数的对比分析,更有利于男性不育的诊断及预后观察.     

精液白细胞与精子形态的相关性研究

目的探讨精液白细胞与精子形态之间的关系。方法按WHO精液检测方法对192例不育患者进行精子形态分析和精液白细胞检测。结果白细胞精子症组正常精子形态百分率低于非白细胞精子症组（P〈0．05）,且白细胞精子症组头部缺陷数和尾部缺陷数较非白细胞精子症组增加,差异有统计学意义。结论精液中过多的白细胞可导致精子形态异常,是男性不育重要的原因之一。     

精子形态与活率相关性的研究及其在男性不育中的应用

目的探讨精子形态与活率的相关性,以便为男性不育的诊断提供理论指导。方法分别利用改良的巴氏染色法和伊红染色法,对精子形态和精子活率进行分析。结果与精子形态正常组相比,畸形精子组中精子活率异常的比例明显上升,差异具有统计学意义(P<0.05)。结论精子活率与精子形态呈正相关。精子形态可影响精子活率,进而影响男性的生育。     

2165例男性不育患者精液分析

目的探讨精液分析各参数在男性不育的诊断与治疗中的临床应用价值.方法采用WLJY-9000伟力彩色精子质量分析系统对2165例男性不育患者与58例已生育的正常健康体检男性的精液标本进行分析.结果不育组精液量、pH值、STR、LIN、WOB、MAD略低于生育组,而ALH、BCF略高于生育组,但均无显著性差异(P＞0.05).与生育组比较,不育组30min内完全液化者率、精子密度、A级精子率、B级精子率、精子活率、VCL、VSL、VAP均呈显著性降低(P＜0.01),而畸形精子率、WBC ～ 者率、无精子症者率均呈显著性升高(P＜0.01).结论采用伟力彩色精子质量分析系统对不育患者的精液进行多项参数分析,可为男性不育的诊断和治疗提供有力的临床依据.     

乌鲁木齐地区483例男性不育患者精液分析

目的探讨彩色精子质量分析系统在男性不育的诊断与疗效评估中的应用价值。方法应用WUY-9000伟力彩色精子质量分析系统对乌鲁木齐地区483例不育男性和240例生育男性精液从精液量、精予总数、精子密度、精予活力、精子活率及各种精子动态参数进行研究和分析。结果不育组在精液量、酸碱度两项常规参数方面，与正常生育组相比较，两组间无显著性差异（P〉0．05），但在精子总数、精子密度（包括a、b、c、d级精子总数）、精子活率、A级精子率及B级精子率等方面，两组间有显著性差异（P〈0．01）。不育组STR、LIN略低于正常生育组，两组间比较有显著性差异（P〈0．05），VSL、VCL、VAP、ALH4项精子动态指标均呈显著性降低，两组之间也具有显著性差异（P〈0．01），而WOB、BCF、MAD3项指标与正常生育组比较。两组间无显著性差异（P〉0．05）。结论采用彩色精子质量分析系统对不育患者的精液进行多项常规和动态参数分析，可为临床上男性不育症的诊断和治疗提供有力的参考依据，也为生殖医学度优生学等相关科研工作提供了重要的研究工具。     

Role of caspases in male infertility

Apoptosis is characterized by a variety of changes resultingin the recognition and phagocytosis of apoptotic cells. Caspases(cysteinyl aspartate-specific proteinases) play a central rolein the regulation of apoptosis in the human seminiferous epithelium.They are expressed as inactive proenzymes and participate ina cascade triggered in response to pro-apoptotic signals. Todate, 14 caspases have been implicated in the human apoptoticpathway cascade. Among these, caspase-3 is considered to bea major executioner protease. Since apoptosis is a universalsuicide system in almost all cells, a close control via molecular,endocrine and physical factors establishes homeostasis of cellgrowth and death. The proper regulation of the caspase cascadeplays an important role in sperm differentiation and testicularmaturity. However, caspases have been implicated in the pathogenesisof multiple andrological pathologies such as impaired spermatogenesis,decreased sperm motility and increased levels of sperm DNA fragmentation,testicular torsion, varicocele and immunological infertility.Future research may provide a better understanding of the regulationof caspases, which may help us to manipulate the apoptotic machineryfor therapeutic benefits. In this review, we summarize the consequencesof caspase activation, aiming to clarify their role in the pathogenesisof male infertility.     

Increased sperm mitochondrial DNA content in male infertility

BACKGROUND: There is increasing evidence that mitochondrial DNA (mtDNA) anomalies in sperm may lead to infertility. Point mutations, deletions and the presence of a specific mtDNA haplogroup have been associated with poor sperm quality, but little attention has been paid to the role of mtDNA content. METHODS: Using density gradient separation and swim-up methods, we selected motile sperm from 32 normal and 35 abnormal sperm samples. The mtDNA/beta-globin gene ratio was determined by real-time quantitative PCR. RESULTS: The average mtDNA/beta-globin ratio of sperm collected from 100% density layers was 1.4 for normal sperm, 6.1 for sperm samples presenting at least one abnormal criterion [among the three criteria established by World Health Organization (1999), i.e. sperm count, motility and morphology], and 9.1 for sperm samples presenting two or more of these abnormal criteria. These differences are very highly significant (P < 0.0001). The mtDNA numbers were also much greater in sperm collected from the 40% density gradient layers (mean: 17.1, P < 0.001), known to contain the most abnormal sperm of the sperm samples, than in those collected from the 100% layers known to contain sperm with the best fertilizing ability. CONCLUSION: Our results showed significant mtDNA amplification in sperm collected from abnormal sperm samples.     

Genetic male infertility and mutation of CATSPER ion channels

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords ‘CATSPER'', ‘male infertility'', ‘male contraception'', ‘immunocontraception'' and ‘pharmacologic contraception'' (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.     

Human seminal plasma inhibits brain nitric oxide synthase activity

Nitric oxide is a chemical messenger which functions as a neurotransmitteror as a cytotoxic agent. Nitric oxide synthase (NOS) has beenisolated from various mammalian reproductive tissues* The presenceor absence of NOS in spermatozoa has not yet been reported.We therefore tested human and marine spermatozoa for NOS activityby measuring the conversion of argmine to citmlline. No activitywas found either in human or in murine spermatozoa. Human nativesemen and human seminal plasma exerted an inhibition on brainNOS activity, as assayed on rat brain cytosolic fractions. Thisinhibitory effect was dependent on the amount of protein presentin the human seminal plasma. No inhibitory effect was observedwhen homogenates of washed spermatozoa were tested. The humanseminal plasma did not affect the Michaelis constant (Km) ofNOS for L-arginine (endogenous NOS substrate) whereas the maximalvelocity (Vmax) was reduced, suggesting that it contains a non-competitiveinhibitor of brain NOS. This inhibitory component was virtuallyinsensitive to heat; a 10 min treatment to 95°C only slightlyreduced its ability to inhibit brain NOS. The physiologicalrelevance of our observations remains to be elucidated. Humanseminal plasma may exert an inhibition of nitric oxide synthesison cells other than spermatozoa or on cells from the male orfemale genital tract, modulating directly or indirectly (viamodulation of reactive oxygen species formation) the functionalstate of the spermatozoa.     

Clustering of male infertility in the families of couples treated with intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) is an effective treatment modality for male factor infertility, but it could promote the transgenerational transmission of genetic defects causing gametogenic failure. Cytogenetic and molecular techniques permit the diagnosis of some infertility-causing genetic aberrations, but many more probably evade detection with currently available technology. The analysis of the recurrence pattern of infertility in infertile couples' families could define the importance of heritable factors in the pathogenesis of human infertility. We have subjected 621 consecutive infertile couples treated with ICSI in a single institution to a comprehensive genetic workup including documentation of the family history, karyotyping and various DNA tests. In all, 1302 fertile couples served as controls. Of the infertile couples 6.4% were shown to have a fertility problem with a definite genetic basis. Male, but not female fertility problems displayed a distinct pattern of familial aggregation. In addition, the infertile couples had fewer siblings than the fertile controls, a finding compatible with suboptimal fertility already among the infertile couples' parents. In summary, our data indicate that male factor infertility should be considered a potentially heritable condition. The recurrence risk for infertility in the offspring of couples treated with ICSI might be substantial.     

Sub-zonal insemination for extreme male factor infertility

When in-vitro fertilization (IVF) is used for severe male infertility,the zona pellucida constitutes a major barrier to sperm —oocyte interaction, a barrier that may, in principle, be overcomeby micro-injecting one or more spermatozoa into the sub-zonalperivitelline space (‘sub-zonal insemination’ orSZI). We have defined suitable patients for SZI as having ‘extreme’male factor in that they have either shown a failure of fertilizationin previous IVF cycles or had < 50 000 motile spermatozoarecoverable after semen preparation. (This is distinct fromthose with only ‘severe’ male factor in whom sufficient(> 50 000) motile spermatozoa could be recovered from a semenpreparation.) A total of 213 SZI cycles were performed at SydneyIVF in the 4 year period September 1988 to September 1992, forextreme male factor patients with previous IVF failures or extremelylow sperm numbers for whom SZI was the first option (about two-thirdsand one-third of cases respectively). A total of 138 embryotransfers are reported, producing 20 clinical pregnancies afterperforming SZI on 1899 oocytes. One patient miscarried at 12weeks gestation and there have been nine normal deliveries (sofar) of 10 healthy infants. The first delivery was in February1990. One pregnancy was achieved in the only patient in whomspermatozoa were obtained by epididymal aspiration, and transferof three cryopreserved embryos in another patient resulted ina singleton pregnancy. Of the 492 oocytes fertilized, 282 hadtwo pronuclei (57.3%) and normal embryos were transferred in138/213 (64.8%) treatment cycles, giving an overall pregnancyrate of 14.5% per embryo transfer or 9.4% per cycle. These resultsare considerably better than those obtained in a subpopulationwho had also undergone IVF (average fertilization rate = 4.2%,no pregnancies), although lower than traditional IVF in coupleswith severe male factor. This emphasizes that selecting an appropriatepatient population for SZI is critical in establishing its trueclinical relevance. The major limitation to the technique isthe need for spermatozoa to be acrosome-reacted before injection.     

Andrology: Effects of nitric oxide on human spermatozoa: evidence that nitric oxide decreases sperm motility and induces sperm toxicity

Endogenous nitric oxide (NO) is an important functional mediatorin several physiological systems, including the reproductivesystem. However, when generated in excessive amounts for longperiods, mainly during immunological reactions, NO is cytotoxicand cytostatic for invading microbes, as well as for the cellsgenerating it and the tissues present around it. Since infertilityassociated with urogenital tract infection in males and femalesis also accompanied by reduced sperm motility and viability,it is possible that reduced fertility in these patients is dueto NO-induced sperm toxicity. We therefore evaluated the directeffects of NO, chemically derived from S-nitroso-N-acetylpenicillamine(SNAP, 0.012–0.6 mM) and sodium nitroprusside (SNP, 0.25–2.5mM), on the motility and viability of human spermatozoa. Furthermore,we tested whether inhibition of NO synthesis prevents spermmotility and viability by incubating washed total cells presentin the semen (spermatozoa, round cells) with N-nitro-L-arginine-methyl-ester(L-NAME), a NO synthesis inhibitor. Treatment of purified spermatozoawith SNAP or SNP decreased forward progressive sperm motilityand straight line velocity, and also increased the percentageof immotile spermatozoa in a concentration-dependent manner.Furthermore, the percentage of immotile spermatozoa positivelycorrelated with the percentage of dead spermatozoa. In contrastto freshly prepared SNAP, SNAP preincubated for 48 h had noeffect on the motility and viability of the spermatozoa. Furthermore,as compared to untreated controls, a significantly higher percentageof forward progressive sperm motility as well as viability (P< 0.05) was maintained in washed semen incubated with L-NAME(0.15 mM). Seminal plasma concentrations of nitrite-nitrate(stabile metabolites of NO/10⁶ spermatozoa correlated positively(P < 0.05) with the percentage of immotile spermatozoa. Ourresults suggest that NO can cause sperm toxicity as well asinhibit sperm motility. In conclusion, excessive NO synthesisin response to infection and inflammation could be an importantfactor contributing to functional change of the spermatozoa,leading to their dysfunction and to infertility.     

Diversity of antisperm antibodies bound to sperm surface in male immunological infertility

PROBLEM: The presence of antisperm antibodies (ASA) in males can reduce fecundity, however, relationship between the two is disputed. This study was performed to investigate if there is diversity of ASA bound to sperm surface using immunobead test (IBT) combined with complement dependent sperm immobilization test (SIT). METHODS: The ASA bound to sperm surface were detected using the direct IBT (D-IBT) in 275 semen samples. In some cases with ASA detected by D-IBT, sperm immobilizing antibodies bound to sperm surface were also evaluated using direct SIT (D-SIT). RESULTS: The incidence of the immunoglobulin G (IgG), IgA, and IgM classes of ASA detected by D-IBT were 2.5, 1.8, and 0.4%, respectively. Totally, nine (3.3%) infertile men had ASA on the sperm surface. D-SIT was tested positive in four (66.7%) of six cases with ASA assessed by D-IBT. CONCLUSIONS: Some of the sperm-bound antibodies are associated with complement dependent sperm immobilizing antibodies, indicating that there exists a heterogeneity of sperm-bound antibodies. This result might be one of the reasons for the controversy about the relationship between ASA and immunological infertility in men.     

Andrology: Successful treatment of severe male factor infertility in 100 consecutive cycles using intracytoplasmic sperm injection

In this report, we present the results of our first 100 consecutivecycles of intracytoplasmic sperm injection (ICSI). Overall,fertilization occurred in 98% of cycles and embryos were transferredin 94% (2.6 embryos per cycle). About 50% of patients had embryosfrozen. The overall fertilization rate was 71%, of which 4%were abnormally fertilized (three pronuclei). A total of 30clinical pregnancies were established (32% per transfer), resultingin 18 singleton, six twin and one triplet ongoing pregnancies.The implantation rate per embryo was 15%. There were no significantdifferences in the fertilization or pregnancy rates betweenpatients Who had only occasional motile spermatozoa in the ejaculate,semen that was too poor for routine in-vitro fertilization (IVF),or who had failed routine IVF and/or subzonal sperm injection(SUZI). A group of 18 patients were treated with both ICSI androutine IVF on their first cycle because of the high likelihoodof failed fertilization due to poor sperm morphology (<20%normal). In this group, ICSI oocytes had a fertilization rateof 76% compared to only 15% for the routine IVF (control) oocytes,and six patients conceived after transfer of ICSI embryos (33%),indicating that ICSI can be used successfully on 50% of theoocytes if fertilization failure is expected. Similarly, patientswho had failed to become pregnant with SUZI achieved excellentresults after ICSI. There were no significant differences betweenICSI and routine IVF in the proportions of grade 1, 2 or 3 embryoson day 3 post-oocyte recovery. In conclusion, we have achievedresults comparable to those reported from Belgium and we havefound that ICSI is universally applicable to all forms of severemale factor infertility. ICSI produces fertilization, pregnancyand freezing rates comparable to routine IVF with normozoospermicsamples and has none of the drawbacks of other assisted fertilizationtechniques.