精子膜的完整性与男性不育的相关性研究

目的探讨精子膜完整性与精子功能的相关性。方法收集140例精液标本，其中生育组50例，不育组90例，采用伊红-Y水试验，低渗肿胀试验，检测精子形态和精子膜低渗肿胀率。结果生育组和不育组比较，精子头部未着色率、低渗肿胀率有显著性差异（P〈0．01）；同时，精子膜低渗肿胀率与精子活率密切相关，精子低渗肿胀率越高，其活率也越高（P〈0．01）。结论精子膜功能可能与精液参数密切相关，精子膜功能是评价男性生育力的重要指标之一。     

冷冻保存对人类精子膜完整性的影响

本文建立“低渗肿胀－伊红”（ＨＯＳ－ＥＹ）结合试验探讨冷冻保存人类精子头部和毛部膜完整性的影响，ＨＯＳ－ＥＹ试验按照精子尾部肿胀与否和头部着色与否区分为４种膜类型。冷冻保存后，头膜－尾膜均完整的Ⅳ型精了率和头膜－尾膜的均损伤的Ｉ型精子率分别显著减少和增多（ｎ＝５０，Ｐ〈０．００１），表明冷冻－解冻过程对精子膜完整性有严重影响，头膜损伤－尾膜完整的Ⅲ型精子率在冷冻组显著增多，提示精子头部或尾部的冷东     

不育男性精子膜表面抗精子抗体水平与精液主要参数及精子功能的相关性

目的探讨男性不育症患者精子膜表面抗精子抗体与精液主要参数的相关性。方法收集男性不育症患者350例,均采用免疫珠法测定精子膜表面抗精子抗体(AsAb,IgA或IgG型),根据结果分为AsAb阳性组(43例)和阴性组(307例)。结果在这350例男性不育症患者中精子膜表面抗精子抗体检出率为14.00%。AsAb阳性组患者的精液主要参数(精液密度、活动率、活动力、精液量)均低于AsAb阴性组患者,两组间比较差异具有显著性(P<0.05)。结论精子膜表面抗精子抗体与精液主要参数及精子功能下降有明显相关性,是导致男性免疫性不育的重要因素之一。     

解脲支原体感染对男性不育患者精子DNA完整性和精液常规参数的影响

目的研究解脲支原体（UU）感染对男性不育患者精子DNA完整性和精液常规参数的影响。方法216例男性不育患者分为UU阳性组（120例）和UU阴性组（96例）,另外选取60例健康体检者作为对照组。分别进行DNA完整性、精子密度、精子活动率和精子形态等指标的检测,其中DNA完整性以DNA断裂指数（DFI）表示。结果与UU阳性组相比,UU阴性组和对照组的精液精子密度和精子活动率均明显升高（P〈0．05）,而精子畸形率和DFI却明显降低（P〈0．05）;与UU阴性组相比,对照组精子密度和精子活率均明显增高（P〈0．05）,而精子畸形率和DFI没有明显差异。结论解脲支原体感染可能会损伤男性精子DNA及影响精子数量活力及形态,是引起男性不育的原因之一。     

精液常规参数与人工授精临床妊娠率关系的研究

目的探讨精液常规参数对宫腔内人工授精（IUI）临床妊娠率的影响。方法回顾分析125例患者的178个IUI治疗周期,检测记录每个周期患者精液常规参数,并根据妊娠结果分成两组：妊娠组与非妊娠组。对主要精液常规参数精子密度、精子形态及精子总数与IUI临床妊娠率的关系进行分析。结果 125例患者IUI治疗178个周期,临床妊娠34个周期,周期妊娠率为19.10%（34/178）,IUI治疗累计临床妊娠率为27.20%（34/125）。妊娠组与非妊娠组精液常规参数无明显差别;精子总数〈100×106或精子密度〈20×106/ml患者临床妊娠率明显降低（P〈0.05）。精子形态对IUI临床妊娠率无影响。结论不孕夫妇进行IUI治疗,当精子总数≥100×106或精子密度≥20×106/ml时才有可能获得较为理想的临床妊娠率。     

正常液化精液标本不同时间检测对精液参数的影响

目的探讨正常液化后的精液标本不同时间检测对精液参数的影响。方法参照世界卫生组织操作标准及最新版的全国临床检验操作规程,将15 min内完全液化的400例精液标本,分别于液化后15、30、45、60、90、120 min,采用计算机辅助精液分析系统(CASA)进行分析,测定精子活力、精子活动率、精子浓度及圆细胞浓度。利用统计软件分析检测时间对精液参数的影响。结果通过Friedman检验,各时间点精子浓度与圆细胞浓度差异均无统计学意义(P0.05),而精子活力及精子活动率差异均有统计学意义(P0.05)。通过Wilcoxon检验,两两比较可得液化后精子活力15min与30 min差异无统计学意义(P0.05),其余各组差异均有统计学意义(P0.05)。液化后精子活动率15 min、30 min与45 min差异无统计学意义(P0.05),其余各组差异均有统计学意义(P0.05)。通过Pearson相关性分析,检测时间与精子活力及精子活动率均存在负相关(P0.05)。结论液化后精子活力及活动率均随时间的延长而下降,为避免时间延长对精子活力及活动率的影响,精液常规检测应在完全液化后的30 min内尽快完成。     

一种人精子膜蛋白基因的克隆与鉴定

应用本实验室制备的抗人精子膜蛋白的单克隆抗体(YWK-Ⅱ),自大鼠睾丸λgtll表达型cDNA库中表位筛选了一个阳性克隆,其插入cDNA长度为1.8kb,命名为RSD-2。RSD-2片段的部分酶谱分析确定该片段具有PstⅠ及BglⅡ的单一酶切位点和PvuⅡ等酶的多个位点。此外,采用RNA-DNA杂交技术还研究了RSD-2片段的种属特异性,表明人及大鼠睾丸间有较高的同源性。     

精液白细胞对精液主要参数及精子功能的影响分析

目的探讨精液白细胞含量与精液主要参数和指标的关系。方法按照WHO人类精液实验室手册要求检测精液中的白细胞、主要参数、精子形态分析、精子顶体酶活性、精浆抗体（AsAb）、解脲支原体等，分析精液白细胞与男性不育相关因素的关系。结果238例男性不育患者中有75例（31．5％）精液中白细胞〉1×10^6个／ml，设为白细胞精子组；163例（68．5％）患者精液中白细胞≤1×10^6个／ml，设为非白细胞精子组。白细胞精子组的精子密度、精子活动率、a＋b级活力精子率、精子顶体酶阳性率均低于非白细胞精子组（P〈0．05）；而精子畸形率、精浆抗体（AsAb）、解脲支原体阳性率均高于非白细胞精子组（P〈0．05）。两组的精液量、pH值和液化时间差异无显著性。结论精液中白细胞含量与精液质量有密切的关系，是导致男性不育的重要原因。     

改良中性α－糖苷酶与其它精液参数的关系

采用ＷＨＯ１９９２中性α－糖苷酶测定法检测了１８４例男性不育症者精浆，用以探讨中性α－糖苷酶和其它精液参数之间的联系。在精子计数和α－糖苷酶活性（ｍｕ／ｍｌ）（ｒ＝０．４５，Ｐ＜０．０１）、总活性（ｍｕ／Ｔ）（ｒ＝０．３８，Ｐ＜０．０１）之间，精液量和α－糖苷酶活性、总活性之间均有线性正相关；快速前向精子活力和α－糖苷酶之间不相关；ｐＨ和α－糖苷酶之间有抛物线关系。结果显示，在阻塞性无精子症的诊断中，精液参数和α－糖苷酶活性反映附睾阻塞及附睾小管损害的变异程度方面，改良中性α－糖苷酶检测法优于普通总α－糖苷酶检测法。     

精液白细胞含量与精液质量主要参数的关系

目的探讨精液白细胞含量与精液质量主要参数的关系。方法依据世界卫生组织（WHO）的相关标准,精液中白细胞〉1×10^6/ml可诊断为白细胞精子症,据此分为白细胞精子组和精液白细胞≤1×10^6/ml的正常对照组。应用伟力彩色精子质量分析系统检测男性精液量、液化时间、精子总数、精子密度、精子活力、精子活率等参数进行分析。结果当精液中白细胞〉1×10^6/ml时,精液的主要参数液化时间、精子总数、精子密度、a级精子百分率、a＋b级精子百分率、精子活率与正常对照组比较差异具有显著性（P〈0.05）。结论精液中白细胞含量与精液质量有密切的关系,是导致男性不育的重要因素。     

Andrology: Effects of sperm activity on zinc and fructose concentrations in seminal plasma

Attempts to correlate zinc and fructose concentrations in seminalplasma with andrological parameters have produced inconsistentresults. To assess further this relationship, a prospectivestudy was performed measuring zinc and fructose concentrationsin seminal plasma in 1178 patients referred for fertility treatmentSeminal analysis was performed with biochemical measurementsof seminal zinc and fructose. The main outcome measures werethe correlation between motile sperm concentration and seminalzinc and fructose concentrations. Zinc concentrations were notinfluenced by the motile sperm concentration (r = 0.039). Fructoseconcentrations were found to be negatively correlated with motilesperm concentration (r = 0.062). We conclude that seminal plasmazinc is an unreliable marker of spermatogenic activity. Whilethere does appear to be a negative correlation between seminalplasma fructose concentrations and motile sperm concentrationthis relationship is far from linear. Due to the biochemicalcomplexity of seminal fluid attempts to perform such simplecorrelations between seminal plasma components and andrologicalparameters are likely to produce inconsistent results and theirrole in the assessment of sperm function must therefore be calledinto question.     

Studies on the membrane integrity of human sperm treated with a new injectable male contraceptive

BACKGROUND: The aim of this study was to evaluate the integrityof sperm surface characteristics in the presence of a new malecontraceptive, RISUG [1 mg styrene maleic anhydride (SMA)/100µl dimethylsulphoxide (DMSO) in 1 ml sperm solution].METHODS: Progressively motile human sperm were treated in vitrowith RISUG. The cells were analysed for the release of 5'-nucleotidase(5'-NT) (a plasma membrane marker) using 3 mmol/l 5'-AMP and3 mmol/l -glycerophosphate as substrates. Hyaluronidase (anacrosomal membrane marker) was analysed using hyaluronic acidas a substrate. The contents of free and total acrosin, and% proacrosin (all acrosome markers) were assayed using 0.5 mmol/l-N-benzoyl-L-arginine ethylester (BAEE). RESULTS: RISUG causedalmost complete disintegration of the plasma membrane leadingto significant (P<0.0001) release of 5'-NT into the surroundingmedia. Complete dissolution of the acrosome with concomitantvesiculation of the membrane system, as judged from the lossof hyaluronidase, was observed. Total acrosin content in thesperm was also reduced to almost 10%, and proacrosin droppedto 13.2% in the presence of RISUG in comparison to 90.2% incontrol (P<0.0001), indicating dispersion of acrosomal contents.CONCLUSION: Under in vitro conditions, RISUG, at a concentrationof 1 mg SMA dissolved in 100 µl of DMSO, caused significantdamage to the acrosome and its contents, indicating loss offunctional ability of sperm.     

Antisperm antibodies modify plasma membrane functional integrity and inhibit osmosensitive calcium influx in human sperm

BACKGROUND: The hypo-osmotic swelling (HOS) test evaluates the ability of the functional sperm plasma membrane to stretch following cell swelling when exposed to hypo-osmotic solutions. Sperm samples with low HOS scores show low fertilization and pregnancy rates during assisted reproductive techniques, though data are controversial. The aim of this study was to compare the results of the HOS test in a group of normozoospermic men with those in a group of subjects affected by autoimmune infertility due to the presence of antisperm antibodies (ASA) bound to the sperm surface. METHODS: Sperm from normozoospermic and from infertile subjects affected by autoimmune infertility were exposed to hypo-osmolar conditions to verify the effects on intracellular calcium concentrations and acrosome reaction. RESULTS: Sperm samples from infertile men with ASA showed HOS test scores that were significantly lower than those of normozoospermic subjects despite similar sperm viability percentages. Sperm with ASA bound to their plasma membrane showed a reduced rise in intracellular calcium concentrations and acrosome reaction after hypo-osmotic challenge with respect to sperm from normozoospermic subjects without ASA. CONCLUSIONS: Infertile subjects with ASA have a reduced sperm plasma membrane functional integrity that could explain, at least in part, the low fertilization and pregnancy rates observed in these subjects during assisted reproductive procedures. Evaluation for the presence of ASA in all sperm samples showing low HOS test scores in the presence of normal sperm viability percentages is suggested.     

厦门地区不同职业对男性不育患者精子DNA完整性和精子参数的影响

目的探讨厦门地区不同职业对精子DNA完整性和精子参数的影响。方法将就诊的1509例男性不育患者按照职业分为司机组、计算机工作者组、工人组。将600例正常生育组做为健康对照组。采用SQA—V全自动精子分析仪进行精液常规分析,精子DNA完整性检测采用精子染色质扩散试验（SCD）,以DNA断裂指数（DFI）表示。结果司机组、计算机工作者,工人组的精子密度、活动率、前向运动率和DFI与对照组比较差异有统计学意义（P〈0．05）;司机组和计算机工作者组的精子活动率、前向运动率、精子密度及和DFI与工人组比较差异有统计学意义（P〈0．05）;4组的精液量、精液pH值差异无统计学意义（P〉0．05）。结论司机、计算机工作者和工人可以影响精液质量,从而导致男性不育。     

Protamine 2 precursors, protamine 1/protamine 2 ratio, DNA integrity and other sperm parameters in infertile patients

BACKGROUND: The protamine 1-to-protamine 2 ratio (P1/P2) is altered in the sperm cells of some infertile patients. Also, evidence for increased protamine 2 precursors (pre-P2) in a few patients has been reported. But so far, there have been no studies measuring simultaneously these two variables in a large number of patients. METHODS: We measured the P1/P2 ratio and the presence of pre-P2 using, for the first time, an antibody specific to the precursor pre-P2, together with other sperm parameters in 224 infertile patients. Additionally, the DNA integrity was assessed by terminal transferase dUTP nick-end labelling (TUNEL) in a subset of the samples. RESULTS: Pre-P2 levels show a significant positive correlation with the P1/P2 ratio, with the presence of other proteins and, at low pre-P2 levels, with TUNEL-positive sperm. An inverse correlation with sperm count, normal morphology and motility was detected. CONCLUSIONS: The levels of pre-P2 may provide clues into the pathogenic mechanisms of infertility. The increased proportion of pre-P2 in some patients with increased P1/P2 ratio suggests an involvement of pre-P2 processing. The positive correlation between TUNEL-positive sperm and pre-P2 at low pre-P2/P2 ratios also suggests a link between deficient protamine processing and decreased DNA integrity.     

Andrology: The presence of Mullerian inhibiting substance in human seminal plasma

Müllerian inhibiting substance (MIS), produced by testicu-IarSertoli cells, is present in adult male serum. The first aimof this study was to determine if MIS is present in seminalplasma. Using an enzyme-linked immunosorbent assay (ELISA),we measured MIS concentrations in seminal plasma from 23 donorsexhibiting normal (WHO criteria) sperm qualities, and 169 patientswith subnormal sperm parameters. The second aim of this studywas to examine a potential relationship between MIS and spermmotility. MIS concentrations in seminal plasma ranged from 0.5to 3.6 ng/ml in donors and from 0.5 to 17.8 ng/ml in patients.Motility index (MI, mean ± SEM) for all patient sampleswas lower compared with donors (113.3 ± 3.2 and 1983± 13.5, P < 0.00001), while mean MIS concentration(± SEM) was higher (4.2 ± 03 and 1.4 ±0.2, P < 0.0003). When the patients were stratified intoGroups I (motility < 50%, n = 42) and D (motility >50%,n = 127), the MI (mean ± SEM) values were 623 ±3.8 and 130.2 ± 2.7 respectively (P < 0.0001 for bothcompared with donors) and mean MIS concentrations (±SEM) were 5.4 ± 0.6 and 3.9 ± 03, respectively(P < 0.0001 and P < 0.001 compared with donors). The inverserelationship between MIS concentration in seminal plasma andmotility index suggests that MIS may have a function in modulatingmotility.     

Transferrin in seminal plasma and in serum of men: its correlation with sperm quality and hormonal status

Transferrin concentrations in the seminal plasma and in serum were measured and correlated with sperm quality (concentration, motility and morphology) and hormonal status (FSH, LH and testosterone) of 75 men aged from 21 to 46 years. A significant positive correlation was found between the seminal plasma concentration of transferrin and the sperm concentration (r = 0.69, P less than 0.0001) and motility (r = 0.39, P less than 0.0001). No other correlations were found. These data confirm that seminal plasma levels of transferrin can be used as a reliable index of sperm quality, and possibly as a Sertoli cell marker.     

Andrology: Effectiveness of various sperm processing methods in removing seminal plasma from insemination media

The effectiveness of the wash and swim-up (1 and 2 wash cycle),two layer Percoll gradient, SpermPrep and underlay sperm preparationmethods in removing seminal plasma from insemination suspensionswas investigated. The number of wash cycles needed to rid spermsuspensions of seminal plasma (n = 15) was also determined.All sperm preparation methods were compared to the control washand swim-up method, performed using Earle's buffered salt solution(EBSS), without serum supplementation so as not to mask seminalplasma concentrations. Control processing comprised 1 ml semensubjected to two wash cycles in EBSS followed by a swim-up periodof 60 min under 5% CO₂ in air. The Percoll method comprised1 ml semen layered on a discontinuous 36 and 81% Percoll gradient(n = 14). In the SpermPrep method, 1 ml semen was run througha SpermPrep II column (n = 10), and underlay samples (n = 12)were processed by two wash cycles, after which sperm pelletswere resuspended in the remaining medium, layered under 1.2ml EBSS and allowed to swim up under 5% CO₂ in air for 1 h.Seminal plasma and supernatant fractions obtained after eachprocessing phase were stored at -20°C and protein concentrationswere determined by spectrophotometry. The wash and swim-up methodwas the most effective in removing seminal plasma from spermsuspensions, followed by the two-layer Percoll gradient, underlayand finally the SpermPrep II processing methods.