Studies on human spermatozoal acrosome using spermac stain in fertile and infertile men including two cases of round-headed spermatozoa

Using a new acrosome stain named "Spermac stain", the relationship between the staining rate of sperm acrosome and conventional semen parameters, i.e., sp. density and sp. motility efficiency index (SMEI) was studied. The mean positive staining rate of sp. acrosome in ten fertile men was 81.5%. In 72 infertile men, a significantly higher (p less than 0.001) correlation was noted between the positive staining rate and sp. density and SMEI. 5 out of 8 male partners of unexplained infertility couples showed low staining rates (40-60%), in spite of normal sp. density and SMEI. Ejaculates from two infertile men with "round-headed" spermatozoa were examined by Spermac stain and electron microscopy. The staining rates of sp. acrosome in these two cases were 4.5% and 16.5%. The fine structures of the sperm and the spermatid were presented. Because Spermac stain is quick, reliable and easy to use, this staining method is recommended for evaluating sp. acrosomal morphology in practical use.     

精子获能前后运动轨迹变化与MTATP_8、MTCYB基因缺失

目的：为探讨精子ＭＴＡＴＰ８ 、ＴＭＣＹＢ基因缺失与精子获能障碍导致男性不育的关系。　方法：６０ 例男性不育者（其中３０ 例精子活动力正常，３０ 例精子活动力异常）和３０ 例生育男性精子获能前后运动轨迹变化，以及用ＰＣＲ技术检测其线粒体脱氧核糖核酸（ｍ ｔＤＮＡ）基因缺失情况。　结果：男性不育组获能后轨迹没有变化的共检出１６ 例，其中活动力异常组检出１０例，活动力正常组检出６ 例；生育组获能前后运动轨迹均有变化。不育组活动力异常的３０ 例标本中检出ＭＴＡＴＰ８ 基因缺失２ 例，检出ＭＴＣＹＢ基因缺失４ 例；不育组活动力正常的３０ 例标本中检出ＭＴＡＴＰ８ 基因缺失２ 例，检出ＭＴＣＹＢ基因缺失３ 例，经统计两组差异不显著（Ｐ＞ ０．０５）。生育组３０ 例标本未检出ＭＴＡＴＰ８、ＭＴＣＹＢ基因缺失，与男性不育组差异显著（Ｐ＜ ０．０５）。不育组ＭＴＡＴＰ８ 基因、ＭＴＣＹＢ基因缺失率分别为７％ 、１２％ 。　结论：ＭＴＡＴＰ８ 、ＭＴＣＹＢ基因缺失影响精子获能，可能导致男性不育。     

Significance of sperm antibodies detected by the mixed antiglobulin reaction and the tray agglutination test

Summary. The most widely used tests to detect seminal and serum sperm antibodies are the mixed antiglobulin reaction (MAR as recommended by WHO) and the tray agglutination test (TAT). It has been suggested that the prognostic significance of sperm antibody tests might be influenced by a concomitant reduction of sperm numbers and/or sperm motility. Furthermore, the relative sensitivity of these sperm antibody tests to detect sperm antibodies is not known. We therefore compared TAT, performed with serum and MAR results retrospectively for 565 infertile patients and MAR IgA and MAR IgG results for 1189 infertile patients. The association of TAT and MAR results with changes in sperm number, morphology and motility was assessed for 565 and 1185 patients, respectively. The influence of MAR and TAT results on sperm cervical mucus penetration test (SCMPT) results was investigated for 349 and 434 patients, respectively. Whereas only 23% of all MAR IgG positive patients were also MAR IgA positive, 82% of all MAR IgA positive patients were also MAR IgG positive. There was a significant ( P < 0.0001) correlation between serum TAT, and MAR results. Positive MAR and TAT results were not associated with reductions in sperm number, motility and morphology. There was a significant correlation between MAR IgG and MAR IgA results and the sperm cervical mucus penetration test (SCMPT) results. According to these results, the MAR IgG would be sufficient as an initial screening for seminal sperm antibodies. MAR IgG negative patients with strong indication for immunologic infertility should also be investigated with the MAR IgA and the serum TAT. Serum and seminal sperm antibodies do not seem to influence male fertility via reductions of sperm number, motility and morphology. However because of their significant influence on SCMPT results, MAR IgG and MAR IgA results seem to be of prognostic significance.     

计算机辅助精液分析男性不育患者精子运动参数与精子活力的相关性

目的探讨计算机辅助精液分析精子运动参数在评价男性不育患者精子活力中的价值。方法按《WHO人类精液及精子-宫颈黏液相互作用实验检验手册》标准,采用国产WLJY-9000伟力彩色精子质量检测系统对276例男性不育患者的精液进行平均直线运动速度、平均曲线运动速度、运动的前向性、运动的直线性、运动的摆动性、平均路径速度、精子活力及分级等进行检测并分析其相关性。结果276名男性不育患者的平均精子活力为（48.93±19.10）%,分级为A级（32.11±17.25）%、B级（17.03±8.91）%、C级（10.14±5.99）%。平均直线运动速度、平均曲线运动速度、运动的前向性、运动的直线性、运动的摆动性、平均路径速度与精子活力的相关系数分别为0.60（P〈0.01）、0.59（P〈0.01）、0.51（P〈0.01）、0.55（P〈0.01）、0.52（P〈0.01）、0.67（P〈0.01）。结论计算机辅助精液分析精子运动参数平均直线运动速度、平均曲线运动速度、平均路径速度是反映精子活力的有效指标,精子运动参数对男性不育的诊断和生育能力的评估具有实用意义。     

Separation of motile spermatozoa by "Separon" as measured by a videomicrography computer system

By means of a recently developed apparatus ("Separon") motile spermatozoa were concentrated in a small volume of culture medium (0,3 ml). Sperm concentration, progressive motility and mean velocity of 60 ejaculates were determined by a computerized videomicrography system. After "Separon" sperm concentration was reduced in an average of 30%, mean progressive motility was increased from 27% to 42% (asthenospermic ejaculates) and from 45% to 84% (control ejaculates) respectively. Mean sperm velocity was increased from 18,5 to 34,5 microns/sec. (control ejaculates).     

Male infertility due to asthenozoospermia and flagellar anomaly: detection in routine semen analysis

Major monomorphous teratozoospermia, due to sperm tail structural anomalies, were detected in 42 out of 4231 infertile patients during routine semen analysis. The flagella were very short or absent in 16 cases, shortened with thickness irregularity in 18 cases, and of normal length with diameter anomalies in eight cases. These syndromes were always associated with poor forward motility, while mobility and penetration into human cervical mucus were sometimes impaired only partially. With good sperm smears and an experienced observer, the diagnosis could be made without the need for electron microscopic analysis. All the affected patients were sterile and several syndromes could have been transmitted genetically.     

Seminal fibronectin in fertile and infertile males

This study aimed to assess seminal plasma fibronectin in fertile and infertile males. Ninety infertile males were investigated; asthenozoospermia (n = 27), asthenoteratozoospermia (n = 30), oligoasthenoteratozoospermia (n = 33) compared with 20 healthy fertile controls. They were subjected to semen analysis, seminal plasma fibronectin estimation by radial immune diffusion, serum testosterone (T) and follicle stimulating hormone (FSH) estimation by ELISA. There was significant increase of seminal plasma fibronectin among different infertile groups compared with the controls. Significant negative correlation was elicited between seminal fibronectin and sperm count, sperm motility grades A, B, A + B, sperm velocity, linear velocity, linearity index, sperm normal forms and serum T. Seminal fibronectin showed significant positive correlation with grade D sperm motility and serum FSH. ROC curve analysis discriminating controls and other infertile groups demonstrated criteria value of < 674 mg l(-1) (sensitivity 100% and specificity 96.4%). It is concluded that increased seminal fibronectin is associated with decreased sperm count and sperm motility.     

精索静脉曲张患者精液质量及其不育患者手术前后精液变化的观察

目的观察精索静脉曲张（VC）患者的精液质量和精子形态学改变，以及VC不育患者手术前后精液的变化。方法121例VC患者精液按WHO标准常规分析并对精子形态学进行评价，23例健康男性精液检查结果作为对照。并对21例VC不育患者术前及术后的精液进行检测分析。结果121例VC患者的精子密度、（A＋B）级活动力精子（％）、活率、有效精子数、活动精子数、活力指数以及正常形态精子比例较对照组明显降低（P〈0．01）；畸形精子中小头、锥形头和无定形头精子数较对照组增多（P〈0．01）。21例VC不育患者手术后精子质量和精子形态学较术前明显改善。结论VC可以引起精液质量下降导致不育，精子形态学分析是判定VC患者精子受损的一个敏感指标，手术能有效地改善精液质量。     

Influence of fibronectin on the motility of human spermatozoa

The objective of the present study was to scrutinize the concentration of seminal fibronectin and the potential effects of exogenous fibronectin on human sperm motility. In addition, variability in the localization of fibronectin on human spermatozoa from andrological patients was studied, at both the light and electron microscopic levels. A total of 58 freshly ejaculated semen samples from patients attending for infertility treatment were submitted to sperm motility analysis and ELISA quantification of seminal plasma and cell-bound fibronectin. Immunofluorescence and immunoelectron microscopy revealed a relatively broad distribution pattern of fibronectin immunoreactivity on sperm heads and testicular spermatids. Addition of a fibronectin antiserum to vital spermatozoa in vitro at a moderate dilution (1:50) resulted in a significant increase in sperm motility. Purified plasma fibronectin, added at various concentrations to a preparation of live spermatozoa, was found to inhibit sperm motility in a dose-dependent manner. At concentrations from 0.18 to 0.5 mg fibronectin per ml ejaculate, no motile spermatozoa were recorded. Seminal plasma fibronectin ranged between 0.8 and 1000 μg/ml in infertility patients. There was a significant inverse correlation between sperm motility and seminal fibronectin in patients with oligo-astheno-teratozoospermia. In a preliminary study in patients with varicocele or hypogonadism, no such correlation was found.     

Pentoxifylline increases the level of nitric oxide produced by human spermatozoa

Pentoxifylline (PF) is a xanthine derivative drug primarily used to treat peripheral vascular disorders. It is currently used in assisted reproductive technologies to enhance human sperm motility. However, the mechanism by which this enhancement occurs is not fully understood. Given that nitric oxide has been identified as a trigger to sperm motion, we asked whether nitric oxide modulates the stimulatory effect of PF on sperm motility. A total of 41 semen samples from infertile males were studied. Nitric oxide production in the presence of 5 mm PF was tested using different bio‐analytical methods (spectrophotometry, fluorometry and fluorescence microscopy). The spectrophotometric determination showed higher levels of nitrite, an indirect measure for nitric oxide, in sperm samples supplemented with PF compared to controls. The fluorometric experiment showed higher 4, 5‐diaminofluorescein triazole, a product from the reaction between nitric oxide and 4, 5‐diaminofluorescein diacetate, after adding PF to spermatozoa. The fluorescence microscopy images of the spermatozoa supplemented with PF showed higher green fluorescence, indicating higher 4, 5‐diaminofluorescein triazole levels, compared to controls. It is concluded that PF enhances nitric oxide production in human spermatozoa, which explains, at least in part, the mechanism by which PF stimulates human sperm motility.     

司机职业对男性不育患者精子活力和动力学参数的影响及相关性研究

目的 探讨乌鲁木齐地区司机职业对男性不育患者精子活力和动力学参数的影响及相关性研究.方法 应用WLJY-9000型精子质量检测系统对157例不育男性(司机组72例、非司机组85例)和125例正常生育男性精子的直线运动速度(VSL)、曲线速度(VCL)、平均路径速度(VAP)、平均移动角度(MAD)、侧摆幅度(ALH)、鞭打频率(BCF)、前向性(STR)、直线性(LIN)、摆动性(WOB)精子活力及其分级进行检测并分析其相关性.结果 不育组与正常生育组相比,精子活力、VSL、VCL、VAP、LIN和STR显著降低(P<0.05或P<0.01);司机组与非司机组相比,精子活力、VSL、VCL、VAP和MAD显著降低(P<0.05或P<0.01),而BCF显著升高(P<0.01);驾龄10年～组与0年～和5年～两组相比,精子活力、VSL、VCL、VAP、MAD、LIN、WOB和STR显著降低(P<0.05或P<0.01),而BCF显著升高(P<0.01);司机职业男性不育患者精子活力与VSL、VCL、VAP有显著正相关性,而与BCF有显著负相关性.结论 (1)司机职业可引起男性精子活力和动力学参数异常,驾车车龄对男性精子质量的影响存在时效关系,长时间驾车可能是引起男性不育的重要原因之一;(2)VSL、VCL、VAP和BCF是反映司机职业不育男性精子活力的有效指标.     

Hormonal and Seminal Parameters in Infertile Men

500 infertile patients (250 with and 250 without left side varicocele) and 33 fertile men were evaluated as far as seminal parameters and the hormonal status were concerned. Sperm motility was constantly lower in infertile patients also when infertile group was compared to fertile one with the same sperm density. Serum testosterone levels were lower in infertile groups when compared to fertile men, and this confirms the existence of an androgenic deficit as a common finding in infertility associated or not to varicocele. FSH and LH increased (p less than 0.001) when sperm density dropped to less than 5 X 10(6) spermatozoa/ml. A negative correlation was found between both gonadotropins and sperm count (p less than 0.001), also after exclusion of azoo- and oligozoospermic (less than 5 X 10(6) spermatozoa/ml) patients (p less than 0.01). Gonadotropins were moreover tightly correlated between each other (p less than 0.001). Our data suggest that both gonadotropins are tightly tuned with sperm output and thus with the spermatogenic potential.     

Correlation Between the Sperm Motility and the Adenylate Cyclase Activity in Infertile Men/Über die Beziehungen zwischen der Motilität der Spermatozoon und der Adenylcyclase-Aktivität bei unfruchtbaren Männern

The relationship between the sperm motility and the adenylate cyclase activity in spermatozoa from patients with male infertility was investigated. Cyclic AMP contents were measured from 34 semen specimens and adenylate cyclase activity were assayed from 50 specimens. All semen specimens were collected from infertile men at our clinic by masturbation after 5 days sexual abstinence. Spermatozoa were washed and concentrated according to the method described by Harrison. The cAMP contents and the adenylate cyclase activity of spermatozoa were determined by radioimmunoassay. Positive correlations were found among the cAMP contents, the adenylate cyclase activity in human spermatozoa and the sperm motility. This result suggests that the sperm motility is regulated by the adenylate cyclase activity via cAMP and that poor sperm motility observed in infertile men is partially caused by the impairment of adenylate cyclase system.     

捐精者精子冷冻复苏率影响因素分析

目的:探讨季节、血型及精液参数等对捐精者精子冷冻复苏率的影响。方法:回顾性分析陕西省人类精子库捐精者4 088份精液标本,研究季节、血型、禁欲时间、精液量、精子形态、冷冻前精子活力及浓度对精子冷冻复苏率的影响。结果:捐精者精子冷冻复苏率随着精子浓度增高而增加,相关性分析提示精子浓度与冷冻复苏率呈正相关(r=0.247,P0.01)。而精子冷冻前活力和精子冷冻复苏率呈负相关(r=-0.262,P0.01)。禁欲第6天组的精子冷冻复苏率[(70.2±5.4)%]明显高于其他禁欲时间组(P0.01)。精子正常形态率20%组的精子冷冻复苏率[(71.4±5.1)%]要高于其他各组(P0.01)。A型血精子冷冻复苏率明显高于B型血[(69.1±4.8)%vs(69.8±4.7)%,P0.01];季节、精液量与精子冷冻复苏率之间无明显相关性(P0.05)。结论:捐精者的精子浓度、活力、形态及禁欲时间对于预测精子冷冻复苏率有一定的价值,而季节、血型、精液量与捐精者精子冷冻复苏率无明显相关性。     

速冻和缓慢冷冻法对精子运动特征的影响

目的了解冷冻方法对人精子运动特征的影响。方法精液标本进行速冻和缓慢冷冻保存,应用计算机精液分析仪进行精子运动特征分析。结果冷冻复温后精子运动能力与冷冻前精子运动能力比较明显下降(P<0.001,P<0.05);速冻与缓慢冷冻方法保存的精子运动参数相比较差异均无显著性(P>0.05)。结论冷冻保存易导致精子运动能力下降,速冻与缓慢冷冻方法对精子运动参数影响无明显差异。     

Glycerophosphocholine in seminal plasma of fertile and infertile men

Glycerophosphocholine (GPC) was measured in seminal plasma from 65 fertile men, 276 infertile men and 10 men before and after vasectomy, using a new enzymatic method. Extra-epididymal excretion of GPC accounted for 30% of the total seminal levels of GPC. From a diagnostic point of view, GPC determination did not appear to be a specific tool which could discriminate between secretory and excretory azoospermia. Although the seminal content of GPC was related positively to the total sperm count in both fertile and infertile men, there was an inverse relationship between the level of GPC and sperm motility when considering classes displaying the same total sperm count. This was observed in all classes from infertile men as well as in fertile men with a total sperm count lower than 200 x 10(6) sperm/ejaculate. These results suggest a possible role of GPC in the regulation of human sperm motility, which warrants further investigation.     

Development of the NBT assay as a marker of sperm oxidative stress

Oxidative stress is a well-established cause of male infertility, with reactive oxygen species (ROS) causing infertility principally by impairing sperm motility and DNA integrity. Currently, most clinics do not test their infertile patients for the presence of oxidative stress because the available tests are expensive or difficult to perform. As antioxidant therapy may improve sperm DNA integrity and pregnancy outcomes, it has become apparent that there is an unmet clinical need for an inexpensive and easy-to-perform assay to identify sperm oxidative stress. The aim of this study was to develop a standardized protocol for performing a photometric nitro blue tetrazolium (NBT) assay for the measurement of seminal ROS production via production of coloured formazan, whilst correlating these results with impaired sperm function (motility and DNA integrity). Semen samples from 21 fertile and 36 male aetiology infertile men were assessed for ROS production (NBT assay), sperm DNA integrity (TUNEL), apoptosis (Annexin V) and sperm motility. Infertile men's semen contained on average fourfold higher levels of ROS than fertile men. The production of ROS by sperm was positively correlated with sperm DNA fragmentation and apoptosis, whilst being negatively correlated with sperm motility. Receiver-operating characteristic plot analysis established a cut-off point of 24 μg formazan/10⁷ sperm having a sensitivity of 91.7% and a specificity of 81% for determining the fertility status of an individual. This study has been successful in establishing a standardized protocol for performing a photometric seminal NBT assay that has significant clinical utility in identifying men with impaired fertility because of oxidative stress.     

Clinical significance of reactive oxygen species in semen of infertile Indian men

Reactive oxygen species (ROS) levels in semen are believed to play both physiological and pathological roles in male fertility. The study was aimed to find the clinical significance of ROS levels in infertile Indian men. This pilot study included 33 idiopathic infertile men and 18 proven fertile controls. ROS levels in the washed sperm were measured using chemiluminescence assay and expressed as 10⁶ cpm per 20 million spermatozoa. Sperm count, percent sperm motility, and percent normal sperm morphology were found to be significantly ( P  < 0.0001) reduced in infertile men compared with the controls. Median (minimum, maximum range) ROS levels of the infertile group [24.90 (6.89, 44.71)] were found to be significantly ( P  < 0.0001) elevated compared with the fertile controls [0.167(0.15, 2.78)]. No significant correlation was seen between ROS levels and semen parameters. Elevated ROS levels in the idiopathic Indian infertile men may be one of the underlying reasons for impaired fertility. Therefore measurement of seminal ROS levels may be used in Indian infertile men for better understanding of the aetiology and selection of antioxidant regimen in the treatment of male infertility. However, large studies may be urgently warranted to find out the role of antioxidants in ROS elevated Indian infertile men through randomised, controlled clinical study.     

Investigations on the Motility of Human Spermatozoa in a Defined Medium in the Presence of Metabolic Inhibitors and of Carnitine

When washed human sperm were incubated in a modified Krebs-Ringer buffer solution in the absence of exogenous metabolizable substrates at 30 degrees C they maintained progressive motility for at least six hours. Under these conditions the spermatozoa apparently utilize endogenous substrates but addition of exogenous substrates (glucose, fructose, acetate, short-chain fatty acids, branched chain amino acids) did not affect the % progressive motility or % total motility of the cells. The phospholipase inhibitors, quinacrine and Upjohn No. 1002, inhibited progressive motility when added to sperm utilizing endogenous substrate, and subsequent addition of oxidative or glycolytic substrates did not reverse the inhibition. In contrast, the inhibition by KCN of progressive motility based upon utilization of endogenous substrate was reversed upon addition of glycolyzable compounds (glucose or fructose). The addition of carnitine or its acetyl-, propionyl-, isobutyryl-, valeryl- or isovaleryl esters did not consistently affect progressive or total motility of sperm samples. The inhibitor, octylsulfobetaine, inhibited sperm motility at a concentration higher than that required for inhibition of carnitine acetyltransferase or translocase activity. On the basis of these results it does not appear that exogenous carnitine has an effect on the motility of human sperm incubated under the conditions described here.     

Estimate of oxygen consumption and intracellular zinc concentration of human spermatozoa in relation to motility

Aim: To investigate the human sperm oxygen/energy consumption and zinc content in relation to motility. Methods: In washed spermatozoa from 67 ejaculates, the oxygen consumption was determined. Following calculation of the total oxygen consumed by the Ideal Gas Law, the energy consumption of spermatozoa was calculated. In addition, the zinc content of the sperm was determined using an atomic absorption spectrometer. The resulting data were correlated to the vitality and motility. Results: The oxygen consumption averaged 0.24μmol/106 sperm×24 h, 0.28μmol/106 live sperm×24 h and 0.85μmol/106 live & motile sperm×24 h. Further calculations revealed that sperm motility was the most energy consuming process (164.31 mJ/106 motile spermatozoa×24 h), while the oxygen consumption of the total spermatozoa was 46.06 mJ/106 spermatozoa×24 h. The correlation of the oxygen/ energy consumption and zinc content with motility showed significant negative correlations (r= -0.759; P<0.0001 and r=-0.441; P<0.0001, res