Factors involved in the biochemical etiology of human seminal plasma hyperviscosity

Semen rheology was studied to elucidate the biochemical basis of seminal plasma hyperviscosity. Semen proved to fit in with a power law model, by presenting a pseudoplastic behavior. Apparent viscosity at 230 s(-1) and 25 degrees C (eta(a)) was 4.3 /- 0.2 cp and 5.4 +/- 0.4 cp in normal and high-consistency semen, respectively. The effect of enzymes and mucolytic agents on human seminal plasma viscosity were evaluated by incubating normal and hyperviscous semen pool aliquots with trypsin, dithiothreitol, EDTA, alpha-amylase and deoxyribonuclease I. After incubation, trypsin treatment reduced eta(a) by 36% in normal semen and by 44% in hyperviscous semen. There was a decrease in eta(a) following incubation of hyperviscous samples with dithiothreitol (33%) and alpha-amylase (44%) that was not observed in the normal consistency samples. No decrease was observed in eta(a) after EDTA or DNAse treatment of both groups. Comparison of normal and hyperviscous seminal plasmas revealed no difference in the concentration of total proteins, DNA, or in the percentage of water content. These findings indicate that the primary substances responsible for basic normal semen rheologic behavior are proteins. A comparison of rheological properties between normal and hyperviscous semen samples indicates the existence of a highly organized network in the latter group, in which disulfide bonds and oligosaccharide chains complexed to the peptide core may play a key role.     

Adenosine 5''-Triphosphate (ATP) Activity in Normal and Pathological Human Semen. Relationship with Round Cells of the Ejaculate

The aim of the present paper is deleted to determine ATP activity in the human seminal cells by bioluminescence and to investigate the relationship between these values and the number of round cells either peroxidase positive or peroxidase negative. We studied 244 untreated men, divided into six groups. Our mean value of ATP activity in normal semen was 20.02 +/- 0.65 n moles per 10(8) spermatozoa. In asthenozoospermic or oligoasthenozoospermic patients with less than 1,000,000 round cells per ml, the concentration of ATP was significantly lower than normal (P less than 0.001) or (P less than 0.05). Semen with normal characteristics or samples from asthenozoospermic or oligoasthenozoospermic patients but with more than 1,000,000 round cells, per ml and with predominance of peroxidase negative cells, had an ATP concentration higher than normal subjects (P less than 0.001). We believe that the knowledge of the quantity and quality of round cells of the ejaculate is important to interpret the seminal values of ATP.     

弱精子症、少弱精子症患者血清、精浆和精子锌含量分析

目的:检测弱精子症和少弱精子症患者血清、精浆和精子锌的含量,分析锌含量的变化与精子密度和精子运动之间的关系。方法:按照WHO《人类精液及精子-宫颈粘液相互作用实验室检验手册》第四版的标准进行精液质量分析,随机筛选出90例弱精子症、60例少弱精子症患者以及20例精液质量正常的生育者作为研究对象,利用原子吸收光谱法检测其血清、精浆、精子的锌含量并进行统计学分析。结果:3组间血清锌含量没有显著差异;弱精子症、少弱精子症患者精浆锌含量均显著低于正常生育者(P<0.05);少弱精子症患者精子锌含量显著高于弱精子症患者和正常生育者(P<0.01)。结论:弱精子症、少弱精子症患者精子的发生及运动功能下降可能与精浆锌含量的低下呈正相关;但过高的精子锌含量与精子的发生和运动功能的关系尚不十分明了,有待进一步研究。     

Severe antioxidase deficiency in human semen samples with pathological spermiogram parameters

Summary.  Spermatozoa of 103 ejaculates from infertile patients and fertile healthy individuals were separated from seminal plasma and purified on Percoll gradient to determine the activities of superoxide dismutase (SOD) and catalase (CAT) in seminal plasma as well as in spermatozoal supernatants after hypotonic disintegration of the sperm plasma membrane. Out of collected specimens, a subgroup of ejaculates from 40 individuals was examined whose female partners had developed malignant processes in the cervix uteri (oncological subgroup). All sperm samples were classified into normal and pathological semen samples according to WHO criteria. While no significant differences of SOD levels were detected in seminal plasma of patients with primary infertility, a catalase deficiency seemed to be associated with combined sperm pathology—oligoastheno-teratozoospermia (OAT). Liberated concentrations of both SOD and catalase were diminished by 10–70% in the oncological subgroup compared to normozoospermia. In four OAT samples obtained from infertile males of the oncological subgroup, total depletion from both antioxidases was observed. A lack of sufficient antioxidase protection in cases of severe sperm pathology (OAT) may also lead to cervical dysplasia.     

生育与不育男性精浆总抗氧化能力分析

目的:分析生育与不育男性精浆中总抗氧化能力(TAC)及其在男性生育中意义。方法:225例男性不育患者分为6组,分别为:梗阻性无精子症组(n=10),非梗阻性无精子症组(n=42),少精子症组(n=20),弱精子症组(n=78),少弱精子症组(n=57),以及正常精子症组(n=18)。28例正常生育男性作为对照(生育组)。分别采用计算机辅助精液分析(CASA)系统进行精液参数分析,采用比色法检测精浆TAC水平。结果:生育组男性精浆TAC为(19.82±6.33)U,梗阻性无精子症组(1.71±1.33)U,非梗阻性无精子症组(12.73±9.44)U,少精子症组(10.85±6.64)U,弱精子症组(13.88±8.24)U,少弱精子症组(11.20±7.02)U,正常精子症组(18.07±8.73)U;与生育组精浆TAC[(19.82±6.33)U]相比,在各不育症组中,除正常精子症组精浆TAC与生育组差异无显著性外,其余各组均显著低于生育组(P<0.01)。精浆TAC与精子密度(r=0.182,P<0.05)和a级精子(r=0.150,P<0.05)呈显著正相关。结论:精浆中TAC水平与男性不育密切相关,精浆中过低的TAC水平可能是引起男性不育的病因之一。     

Lack of correlation of selenium level in human semen with sperm count/motility

Considering the importance of selenium (Se) in male fertility, its concentration was measured in 211 semen samples from 211 normozoospermic, oligozoospermic, asthenozoospermic, and azoospermic men using the hydride generation atomic absorption spectrophotometry. No significant correlation of any kind existed between Se level in the seminal plasma and sperm count or motility. In view of the known poor correlation of these two frequently used semen parameters with the incidence of pregnancy, the assessment of the fertilizing potential of normozoospermic ejaculates with low Se levels is warranted.     

Analysis of lipid peroxidative levels in seminal plasma of infertile men by high-performance liquid chromatography

For the purpose to evaluate the significance of lipid peroxidative products on male infertility, the levels of malondialdehyde (MDA), which is one of the final products of lipid peroxidation in seminal plasma, were determined. Ninety-three male infertile patients were divided into obstructive azoospermic group (12 cases), non-obstructive azoospermic group (15 cases), oligozoospermic group (21 cases), asthenozoospermic group (19 cases), oligoasthenozoospermic group (16 cases) and oligoasthenoteratozoospermic group (10 cases). Eighteen fertile males were included in the control group. MDA concentrations of seminal plasma in the fertile and infertile men were detected by high-performance liquid chromatography (HPLC). The results showed that the concentration of MDA in seminal plasma differed significantly between the control group and all the infertile groups (P < 0.01) except the obstructive azoospermic group, between the oligoasthenozoospermic group and the oligozoospermic and asthenozoospermic groups (P < 0.01), and between the oligoasthenoteratozoospermic group and the oligozoospermic and asthenozoospermic groups (P < 0.01). MDA concentration of seminal plasma in the oligoasthenoteratozoospermic group differed significantly from that in the oligoasthenozoospermic group (P <0.05). The results suggested that detection of MDA concentrations in seminal plasma by HPLC has an indicative value on the diagnosis of male infertility induced by overproduction of reactive oxygen species in male reproductive system.     

Investigation of, the cause of low sperm motility in asthenozoospermic patients by multiple quantitative tests

Multiple tests were done on the ejaculates of 10 asthenozoospermic patients and nine healthy normozoospermic volunteers in an attempt to identify individually the cause of low sperm motility in these patients. Possible defects in the sperm plasma membrane and the motility apparatus of sperm, and in epididymal function affecting the development of motility, were investigated. The presence of seminal sperm antibodies or any motility-inhibiting factors in the seminal plasma that could be removed by washing were also tested. Each test was positive in only one or two patients but axonemal dysfunction was identified in nine patients. Removal of seminal plasma from asthenozoospermic samples did not improve sperm motility to any greater extent than with donor ejaculates, and the motile sperm of these patients exhibited characteristics mostly similar to those of donors under various incubation conditions. Selection procedures are, therefore, required to obtain samples of good quality sperm from such asthenozoospermic ejaculates.     

Differences in osmolality, pH, buffering capacity, superoxide dismutase and maintenance of sperm motility in human ejaculates according to the degree of coagulation

The purpose of this study was to compare various seminal plasma parameters in fresh human ejaculates exhibiting different amounts of coagulum. Poorly coagulating samples demonstrated significantly lower osmolality and buffering capacity but had a higher pH than did samples with good coagulation. However, no correlation was obtained between the activity of superoxide dismutase and the amount of coagulum. Eight hours after ejaculation, sperm motility had decreased by 15 and 80% in samples with good and poor coagulation, respectively. It is suggested that subfertility may be associated with poor coagulation of ejaculates.     

Reactive oxygen species in semen of infertile patients: levels of superoxide dismutase- and catalase-like activities in seminal plasma and spermatozoa

Reactive oxygen species (ROS) can be detected in the semen of 40% of infertile men, whereas none is detected in semen from normal men. The ROS detected in semen are a reflection of the imbalance between ROS production and degradation. The aim of the present study was to determine whether a lowered scavenging capacity or an increased production of ROS was responsible for the ROS detected in semen samples from infertile men. Two activities were investigated: (1) catalase-like activity, which is responsible for the degradation of H₂O₂, and (2) superoxide dismutase-like (SOD-like) activity which is responsible for the degradation of O₂-_-. Catalase-like and SOD-like activities were found in whole seminal plasma, in dialyzed seminal plasma (> 12 kD), in an ultrafiltrate of seminal plasma (< 5 kD) and in spermatozoa. There was no significant difference in the SOD-like activities measured in spermatozoa, or in seminal plasma (whole or fractionated) from samples that did or did not produce ROS. SOD-like activity originated mostly from the high molecular weight components of seminal plasma. However, the catalase-like activity of whole seminal plasma and of spermatozoa was significantly greater ( P = 0.01) in those samples that produced ROS as compared to those that did not. The catalase-like activity in dialyzed seminal plasma, and an ultrafiltrate of seminal plasma from semen samples that did or did not produce ROS were not statistically different. The catalase-like activity of the seminal plasma originated equally from high and low molecular weight components. In conclusion, the data suggest that the ROS detected in the semen of infertile patients are likely due to increased ROS production rather than to decreased ROS scavenging capacity.     

Relationship between Zinc Concentrations in Seminal Plasma and Various Sperm Parameters

The zinc concentration in seminal plasma from 98 infertile male patients and 8 fertile males was measured. The zinc concentration of the seminal plasma in azoospermic and oligoasthenozoospermic patients was significantly lower than that in the other groups (each, p<0.05). The seminal plasma zinc concentration in asthenozoospermic males was significantly higher than that in any other group (p<0.05). There was a positive correlation of zinc concentration with sperm concentration (r=0.33, p<0.05) and with sperm motility (r=0.22, p<0.05), while there was no correlation with sperm morphology. A correlation between zinc concentration and plasma testosterone concentration was observed (r=0.24, p<0.05). It is concluded that excessively high zinc concentration is apparently related to defective motility in asthenozoospermic patients, even though adequate seminal plasma content of the element is required for normal sperm function.     

Fresh versus frozen seminal plasma for enhancing sperm motility in asthenozoospermic males

Previous data demonstrated improvement in severe asthenozoospermia in men with retrograde ejaculates by adding donor seminal plasma (DSP); interestingly, no such improvement was demonstrated following suspension in Ham's F-10 medium. A study was performed to examine whether DSP would also improve asthenozoospermic ejaculates from men with antegrade semen. In addition, the study further explored whether the ameliorative effect of DSP could be maintained after freeze-thawing. The mean increase in motility following fresh DSP in 37 specimens was 102% and was -1.3% for DSP frozen at -20 degrees C and 16.7% for DSP frozen at -196 degrees C. The only statistical difference (using Student's t test) was seen in the comparison of fresh DSP to DSP frozen at -20 degrees C (p = .001). Nine of twenty-four men exhibited doubled baseline motility rates following addition of fresh DSP, compared to only 1 of 20 and 1 of 5, respectively, following addition of semen treated with DSP frozen at -20 degrees C and -196 degrees C, respectively. The data suggest that poor motility may improve when DSP is added to the specimen. However, proper quarantine may not be possible because efficacy is lost after freezing.     

Caffeine increased progressive motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples and enhanced activity of seminal creatine kinase

Even though the effect of caffeine on humans' health has been revealed in various research studies, its effect on semen quality has yet to be well explained. Here, we measured the effect of caffeine at 1, 5, 10 and 20 mM on motility of human spermatozoa in normozoospermic and asthenozoospermic semen samples, level of seminal nitric oxide, chelation of seminal calcium ions and activity of seminal creatine kinase. Fifty-one normozoospermic and ten asthenozoospermic semen samples were recruited in this study. Sperm motility was evaluated by Makler counter, and seminal nitric oxide, seminal-free calcium and activity of seminal creatine kinase were measured spectrophotometrically. Caffeine at 10 mM significantly (p < .05) increased progressive motility of spermatozoa in both tested groups. Also, caffeine significantly increased (p < .05) activity of creatine kinase and insignificantly (p > .05) altered nitric oxide and free calcium levels in seminal plasma. In conclusion, progressive motility of human spermatozoa was found to be higher in the presence of caffeine at 10 mM in normozoospermic and asthenozoospermic semen samples; this increase, albeit partially, could be due to increased activity of seminal creatine kinase, but not to increased production of nitric oxide or chelation of free calcium ions.     

Ejaculate fractions of asthenozoospermic and teratozoospermic patients have differences in the sperm DNA integrity

The initial fraction of the human ejaculate mainly contains prostatic secretions and the subsequent fraction holds majority of the spermatozoa suspended in the secretions from the seminal vesicle. Apart from large series of proteins, human ejaculate also contains antioxidants and reactive oxygen species that are specific to certain accessory sexual glands; however, the influence of these components on the sperm DNA integrity has not been elucidated till date. The present investigation was conducted using split (first and second) ejaculate fractions of forty-one subjects having various semen abnormalities. Sperm DNA integrity was assessed in the individual fractions by comet assay and quantified. The amount of sperm DNA damage between the split fractions is not significantly different in normozoospermic semen samples. In contrast, split fraction-2 had significantly elevated level of DNA-damaged spermatozoa in asthenozoospermic (P < 0.01) and teratozoospermic groups (P < 0.001) when compared to whole ejaculate. The split fraction analysis using various types of ejaculates demonstrated the difference in sperm DNA integrity, which has not been reported till date. Hence, in a clinical point of view, the use of initial ejaculate fraction may be considered superior to whole ejaculate in assisted conception if the DNA integrity is a concern especially in asthenozoospermic and teratozoospermic samples.     

Quantitative Determination of High Molecular Weight Serum Proteinase Inhibitors in Human Semen

Immunochemical determinations of serum proteinase inhibitors in human semen showed the presence of alpha1-antitrypsin and alpha1,x-antichymotrypsin, whereas inter-alpha-trypsin inhibitor, antithrombin III, alpha2-neuramino-glycoprotein and alpha2-macroglobulin could not be detected. Both serum proteinase inhibitors were determined in the seminal vesicle secretions of two patients with prostatic cancer. Employing the Ouchterlony double immunodiffusion technique pattern of identity was found between alpha1-antitrypsin resp. alpha1,x-antichymotrypsin in seminal plasma, seminal vesicle secretions and serum.Mean alpha1-antitrypsin concentration in seminal plasma of 129 andrological patients was 97.7 mug/ml and that of alpha1,x-antichymotrypsin 32.8 mug/ml. There were no differences in the mean alpha1-antitrypsin concentrations of normozoospermic and oligozoospermic ejaculates and those with seminal plasma fructose deficiency. Azoospermic ejaculates, however, showed a significant decrease of the mean alpha1-antitrypsin concentration (p less than 0.05). Alpha1,x-antichymotrypsin concentrations of normozoospermic ejaculates were significantly higher compared to those of oligozoospermia and azoospermia (p less than 0.05). Alpha1,x-antichymotrypsin levels in semen samples were fructose deficiency were not different from those of the total ejaculate population. The cause and significance of the observed differences in the inhibitor concentrations within the different ejaculate types is not known. However, there are no indications for the involvement of both proteinase inhibitors in male reproductive processes.     

精浆和血清生殖激素与精液质量的关系

目的为了评估精液质量不同的男性精浆和血清生殖激素的浓度与精子浓度及活动力的关系,探索精浆与血清生殖激素的关系。方法对301名男性进行精液检查,按照精液的质量参数将受试对象分成4组:精液正常组(n=176),弱精子症组(n=66),少精子症组(n=40)和非梗阻性无精子症组(n=19)。采用电化学发光免疫法测定各组受试对象血清卵泡刺激素(FSH)、黄体生成素(LH)、泌乳素(PRL)、孕酮(P)、睾酮(T)和雌二醇(E2)六项生殖激素和精浆PRL、T、P和E2四项生殖激素的浓度,比较组间差异并进行相关性分析。结果精液正常组和弱精子症组血清FSH和E2的浓度显著低于少精子症组和非梗阻性无精子症组(P0.05),精液正常组血清LH和P的浓度显著低于弱精子症、少精子症和非梗阻性无精子症的人群(P0.05);而精液正常、弱精子症和少精子症三组精浆PRL的浓度则高于非梗阻性无精子症组(P0.05)。除了非梗阻性无精子症组,受试者血清FSH的浓度与其精子浓度呈负相关(r分别为-0.350、-0.273和-0.448,P0.05)。精液正常组精浆PRL的浓度和精子的浓度之间呈正相关(r=0.269,P0.05);在少精子症组中,亦有相同趋势的相关性(r=0.432,P0.05)。结论精浆PRL及血清FSH的浓度能够反映精子浓度或活动力,在男性不育的病因分析中具有一定的指导价值。     

Catalase-like and superoxide dismutase-like activities in human seminal plasma

Human spermatozoa are highly susceptible to oxidative injury but are naturally protected from such injury by the antioxidant properties of seminal plasma. We measured catalase-like and superoxide dismutase (SOD)-like activities in the seminal plasma of fertile and vasectomized men in order to gain insight into the potential source(s) and function(s) of these antioxidants in semen. Semen samples were obtained from fertile men ( n=11) and men post-vasectomy ( n=16). Catalase-like activity was measured by the decrease in hydrogen peroxide concentration after incubation with seminal plasma. SOD-like activity was measured as the inhibition of nitroblue tetrazolium reduction due to superoxide anion generation by xanthine plus xanthine oxidase. Mean seminal catalase-like activity (+/-1SD) in the fertile group was not significantly different from that of the post-vasectomy group (389+/-163 and 325+/-119 U/ml, respectively). Similarly, mean seminal SOD-like activity in the fertile group was not significantly different from that of the post-vasectomy group (37+/-10 and 36+/-10 U/ml, respectively). Our data suggest that the testis and epididymis are not an important source of catalase-like and SOD-like activities in semen. These findings indicate that antioxidants in semen are primarily of post-testicular origin and probably serve to protect ejaculated spermatozoa from oxidative stress such as that which occurs in the female reproductive tract.     

A new CentriSwim procedure to increase the recovery of motile spermatozoa

A prospectively controlled in vitro study was performed to compare sperm concentration, sperm motility and progressive sperm motility recovered following the standard swim-up procedure and a new CentriSwim procedure. The CentriSwim procedure involves creating a centrifugal force to counteract the force of gravity during sperm swim-up procedure. Two aliquots of semen from 12 normozoospermic ejaculates and 12 laboratory-induced oligoasthenozoospermic specimens were diluted, centrifuged, and 1.0 ml of media layered over the sperm pellet. One aliquant was processed by standard swim-up technique. The other aliquant was processed by CentriSwim procedure involving centrifugation at 200 rpm on a 2-cm radius upward-directing arm, at an angle of 60 degrees for 10 min, creating roughly 0.8 g centrifugal force at room temperature (22-24 degrees C) to counteract the force of gravity. The numbers of spermatozoa recovered from the upper 0.5 ml of the medium following CentriSwim from the normozoospermic ejaculates and laboratory-induced oligoasthenozoospermic specimens were significantly higher than following standard swim-up procedure. No statistical differences in the recovery of percentage sperm motility and progressive sperm motility between the two techniques were observed. In conclusion, the CentriSwim procedure yields higher numbers of motile spermatozoa than the standard swim-up technique.     

Evidence for the prostatic origin of immunoreactive inhibin-like material in human seminal plasma

Inhibin is defined as a gonadal peptide exerting an inhibitory effect on the secretion of follicle-stimulating hormone (FSH) by the pituitary. Using a radioimmunoassay (RIA) procedure developed for a homogeneous inhibin-like peptide with a molecular weight of 14 000 daltons isolated from human seminal plasma, immunoreactive inhibin-like matrial (ILM) was quantitated in serum, urine and semen of men in order to investigate its origin. Vasectomy did not result in a significant reduction in seminal plasma ILM. Determination of ILM immunoreactivity in ejaculates form normal men and semen samples characterized by prostate-rich and prostate-deficient secretions, indicated high levels of ILM in the prostatic secretions. Immunoreactive ILM levels estimated in different fractions of split ejaculates from normal men paralleled those of zinc and acid phosphatase activity and were significantly higher in fractions representing prostatic secretions compared to those representing the secretions of seminal vesicles. Estimation of ILM in semen, serum and urine from bilaterally gonadectomized men showed that immunoreactive ILM levels remained high after gonadectomy. It is concluded that the bulk of the immunoreactive ILM present in the semen, blood and urine of men is not secreted by the testes. The principal site of origin of this material, at least in semen, appears to be the prostate.     

Comparison of glycosidase levels in bovine seminal plasma

Seven glycosidases (beta-N-acetylglucosaminidase, alpha-fucosidase, beta-galactosidase, acid alpha-glucosidase, beta-glucuronidase, acid and neutral alpha-mannosidase) were analysed in seminal plasma from the first and second successive ejaculates in normal Ayrshire bulls. In comparison to our previous data the results indicate that beta-N-acetylglucosaminidase, beta-galactosidase and beta-glucuronidase are derived mainly from epididymal secretions, while alpha-fucosidase and particularly neutral alpha-mannosidase originate additionally from the spermatozoan cytoplasmic droplets. The seminal vesicles appear to contribute particularly to the seminal plasma acid alpha-glucosidase and acid alpha-mannosidase activities. The seminal plasma enzymes derived from the epididymis and cytoplasmic droplets were suppressed in semen samples with low sperm density or with high numbers of abnormal spermatozoa. The epididymal and seminal vesicle enzymes could be utilized in assessment of the secretory/functional capacity of these glands.