Relationship of hamster ovum sperm penetration assay to seminal fluid analyses in the evaluation of infertile couples

The males of 279 infertile couples were evaluated with hamster ovum sperm penetration assay (SPA) and seminal fluid analysis. The mean SPA score for the total population was 23.0% penetration with a range of 0-97%. Twenty five percent of the patients demonstrated scores within the abnormal range (0-10%), and 15% were in the "equivocal" range (11-14%). Comparing each individual with the total population using linear regression analysis, it was noted that sperm concentration, percent motility, and percent oval forms varied directly with the SPA, and the slopes of the relationships are positive and statistically significant (p less than 0.0001, 0.002, and 0.0001, respectively). The relationship between SPA and volume is not statistically significant (p greater than or equal to 0.354). To determine whether the SPA could be utilized to establish appropriate normal parameters for various components of SFA, these were analyzed in 169 men who had SPAs of greater than or equal to 15%. Although most SFA values fell within the normal range for this group, there were several exceptions, particularly with respect to percent motility and the presence of leukocytes in the semen. Comparing the percentage of males with abnormal SPA in groups of couples with or without a demonstrable abnormality affecting fertility in the wife, no statistically significant differences could be found. The value of the SPA and SFA in investigating males of infertile couples is discussed.     

Physiological integrity of human sperm in the presence of Ureaplasma urealyticum

Several species of Mycoplasma have been isolated from the human genital tract, the most common being M. hominis and Ureaplasma urealyticum. A causal relationship between such infections and sperm dysfunction and infertility has yet to be established. It was the purpose of this study to examine the effects of U. urealyticum infection on the function of sperm as assessed by seminal fluid analysis (SFA), in vitro penetration of bovine cervical mucus (BCMP), and the hamster sperm penetration assay (SPA). No significant differences were noted in the SFA of infected and uninfected samples, either fresh or frozen, fertile or infertile. In addition, no differences were noted in the BCMP or SPA. In sperm from U. urealyticum-infected individuals the basic physiological mechanisms underlying mucus penetration and ovum fertilization seem intact.     

男性生育力的研究：三种精子功能测定方法的比较

应用精液常规分析(SFA),精子尾部低渗肿胀试验(HOS)和去透明带地鼠卵穿透试验(HOP)对15名能宵男性和15名不育患者的精液样本进行了综合检测和相关分析。能育组SFA参数异常者占20％,不育组SFA参数正常者占27％;精子尾部低渗肿胀率在能育组与不育组间无显著性差异(P>0.05);精子穿透率和受精指数在两组间差异非常显著(P<0.005,P<0.001)这表明HOP试验用于男性生育力的评价是一种较为准确,可靠的手段。本文还对各实验参数间的相关关系进行了分析和讨论。     

Problems in evaluating male fertility: valuable factors in evaluating male fertility and normal values of seminal parameters

To determine the valuable factor for evaluating male fertility, a comparative study was done as to various seminal parameters between fertile and infertile groups. The fertile group consists of 57 proven fertile males and the infertile group consists of randomly chosen 67 infertile patients. Seminal parameters assessed were sperm concentration, motility, mean velocity, total sperm output, total motile sperm output, sperm morphology, acrosin activity and sperm penetration rate on zona-free hamster egg penetration assay (SPA). The infertile group was significantly different from the fertile group in every parameter except acrosin activity. However, the range of each parameter in the two groups overlapped each other. The diagnostic rate of each parameter, which is the percentage of an infertile male correctly diagnosed as infertile, was calculated by using 95% specificity threshold value of fertile males. The 95% specificity threshold values of sperm concentration, motility and % normal shaped sperm were 24.9 x 10(6)/ml, 34.9% and 55%, respectively, and they could be acceptable for the normal limit of seminal parameters. The diagnostic rate was highest in penetration rate (72.4%). In other words, penetration rate is the most valuable factor in various parameters for making a distinction between fertile and infertile males. Sperm motility and mean velocity showed the next highest diagnostic rate. On the other hand, sperm concentration showed a poor diagnostic rate (36.8%). In addition, there was no significant correlation between penetration rate and any other seminal parameters. These results suggest that the SPA will be an essential test for evaluating male fertility and penetration rate may be a marker of male fertility in the treatment of male infertility.     

Flow cytometric measurement of sperm nuclear DNA fragmentation in infertile men with normal standard sperm parameters

BackgroundMale-factor infertility plays a role in approximately 50% of infertile couples. In at least 30% of cases, repeated standard semen analyses of the male partner of an infertile couple reveal normal results. When diagnostic work-up of the female partner is also normal, they are classified as idiopathic. The objective of this study was to evaluate the levels of sperm nuclear DNA fragmentation in a population of infertile men with normal standard semen parameters and to compare their results with those from men who had abnormal semen parameters, as well as with a control group of fertile men.MethodsSemen samples were obtained from 202 infertile men and 30 fertile donors. Standard semen analysis was performed according to the World Health Organization guidelines. Flow cytometry has been extensively used to study sperm DNA fragmentation and the results are expressed as the percentage of sperm DNA fragmentation index (DFI).ResultsOf the 202 patients, 48 (23.8%) had normal standard sperm parameters, while 154 (76.2%) had an abnormality in one or more of these parameters. DFI in infertile men with normal sperm parameters was significantly higher than in fertile donors (p = 0.03), but not significantly different from infertile men with abnormal sperm parameters (p = 0.10). There were statistically significant negative correlations between DFI and the percentage of motile sperm from infertile men with abnormal and normal semen parameters, but not in fertile donors (r = ?0.26, p = 0.001 and r = ?0.48, p = 0.0001, respectively).ConclusionSperm from infertile men with normal standard sperm parameters may have significant levels of DNA fragmentation that are comparable to levels in infertile men with abnormal sperm parameters. Sperm DNA fragmentation analysis is an independent test of sperm quality and has an important diagnostic value in the evaluation of male infertility.     

Study of sperm fertilizing ability in male infertility using the zona free hamster egg penetration assay

Zona free hamster egg-sperm penetration assay (SPA) was performed on 50 normal fertile men and 58 infertile men. The median penetrating rate was 68% (13%-100%) for the fertile men and 11% (0-100%) for the infertile men. These existed no clear relationship between SPA and any other sperm parameters. SPA showed the best diagnostic rate (72.4%) for male infertility, which was evaluated by the 95% specificity threshold values of normal males. Therefore, SPA is best correlated with male impregnating ability. On the contrary, the infertile diagnostic rate of sperm density was relatively low (36.8%). SPA was also carried out on male partners of unexplained infertility couples, and 8 out of 36 (22%) showed decreased sperm penetration rate (less than 10%). In these couples, the deterioration of fertilizing ability was thought to be one of the causative factors of infertility.     

Relationship between sperm telomere length and sperm quality in infertile men

Telomeres, noncoding and repetitive DNA sequences play a significant function in chromatin integrity. Telomere length is age-dependent in somatic cells, while it increases in sperm cell with age. Therefore, we aimed to assess sperm chromatin, leucocyte and sperm telomere length (LTL, STL) in spermatozoon of 38 infertile and 19 fertile men aged between 20 and 50 years. Protamine deficiency (chromomycin A3 test), DNA fragmentation (TUNEL assay), lipid peroxidation (Bodipy probe) and telomere length (quantitative real-time PCR) were assessed. A significant decrease in mean of sperm concentration and motility and a significant increase in means of sperm abnormal morphology, DNA fragmentation, lipid peroxidation and protamine deficiency were observed in infertile compared with fertile men. In addition, the mean of LTL and STL were significantly shorter in infertile men compared with fertile individuals. We observed significant associations between telomere length with sperm concentration, DNA fragmentation and lipid peroxidation. We hypothesised that increased oxidative stress in spermatozoa of infertile men can result in abnormal packaging of chromatin, damage of DNA and shorter sperm telomere length. Together, these anomalies may account for fertility failure in these individuals.     

Time-related decline in sperm motility patterns in men with cytotoxic antibodies

Sperm motility patterns in semen from 10 fertile nonautoimmune men (fertile control group) and 33 infertile men with various titers of cytotoxic sperm antibodies in their seminal plasma (group 1: titers less than or equal to 16, n = 6; group 2: titers 64 to 512, n = 12; group 3: titers greater than or equal to 1024, n = 15) were evaluated every 2 hours for 12 hours and finally at 24 hours. A significant decline in sperm swimming speed and linearity was observed at 6 hours in semen from 27 infertile men with sperm antibodies. Beginning at 8 hours, semen from sperm antibody-positive men in group 2 showed a significant decline in percentage motile sperm (p less than 0.001) compared to the fertile controls. The percentage motility in semen of donors in groups 1 and 3 was significantly lower than that in semen of fertile donors at 10 hours (p less than 0.05), 12 hours (p less than 0.01), and 24 hours (p less than 0.001). The mean velocity in groups 2 and 3 was significantly less than that in fertile controls at 10 and 12 hours (p less than 0.05). The linearity of sperm motility started to decline 4 hours after semen samples were obtained from sperm antibody-positive groups 2 and 3 in contrast to sperm antibody-negative fertile or infertile groups (p less than 0.05). It is concluded that the presence of cytotoxic sperm antibodies in the seminal plasma adversely affects sperm linearity as early as 4 hours after semen collection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Are conventional sperm morphology and motility assessments of predictive value in subfertile men?

Sperm morphology and motility are believed to be important prognostic factors for fertility. Results of a group of 67 men investigated for primary infertility who had mean sperm concentrations greater than 5 million per ml and who later produced pregnancies, were compared with those of 67 matched controls who remained infertile. All female partners were potentially fertile. The groups were matched for other known prognostic factors for fertility, namely, wife's age, the duration of infertility, sperm concentration and varicocele size. There were no significant differences between the two groups overall in the (mean +/- SEM)% of sperm with normal morphology (58.3 +/- 2.1; 58.5 +/- 2.2), or motility (40.6 +/- 1.8; 37.0 +/- 2.0). However, among oligospermic men from the two groups, sperm motility was significantly higher (P less than 0.05) in the subsequently fertile group (43.1 +/- 2.6%) than in the persistently infertile group (33.3 +/- 3.7). These results indicate that sperm morphology as currently assessed may not be important in predicting fertility in subfertile men with a mean sperm concentration over 5 million/ml and the % sperm motility may only be a relevant predictor in oligospermic men.     

Relationship between the human sperm hypo-osmotic swelling test and sperm penetration assay

The hypo-osmotic swelling (HOS) test has been proposed as a useful assay in the diagnosis of the infertile male. A good correlation between the HOS test and the sperm penetration assay (SPA) in fertile and normal semen samples was initially found, but subsequently, no significant correlation was demonstrated with fertile and infertile patients. To validate the potential clinical usefulness of the HOS test, we evaluated 92 ejaculates using the HOS test, SPA, and traditional semen parameters. The methodology originally described by Jeyendran et al (1984) was used for the HOS test. The SPA was performed by the original procedure using an 18-hour preincubation period, and for 28 ejaculates, a modified procedure using TEST-yolk buffer was performed. Values of 60% or more for the HOS and 1% or more for the SPA were considered positive, and less than 60% for HOS and 0% for SPA were considered negative when the standard SPA was performed. For the TEST-yolk buffered SPA, values of 20% or more were considered positive. The sensitivity of the HOS test was 87%, but the specificity was 36%. The association of the two tests over and above that expected by chance (Kappa) was only 0.23. Using logistic regression, both sperm count (P less than 0.001) and morphology (P less than 0.025) were significant predictors of the SPA classification, but the HOS test did not improve the predictive results (P greater than 0.50).(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of zinc concentrations in blood and seminal plasma and the various sperm parameters between fertile and infertile men

The aim of the study was to examine the relationships between concentrations of zinc in blood and seminal plasma and sperm quality among infertile and fertile men. One hundred seven male (infertile group) partners of couples who were undergoing investigation for infertility with no known cause for the infertility and 103 men (fertile group) whose wives were pregnant at the time of the study were recruited. The subjects' blood and seminal plasma concentration of zinc were determined by atomic absorption spectroscopy. Except for semen volume, all the other semen parameters for the infertile men were significantly lower than those for the fertile group. The geometric means of the seminal plasma zinc concentration were significantly lower in the infertile group compared with those in the fertile group; 183.6 mg/L (range, 63-499) versus 274.6 mg/L (range, 55-420). There were no significant differences in the geometric means of the blood zinc concentration between the 2 groups. Seminal plasma zinc concentration was significantly correlated with sperm density (r = 0.341, P < .0001), motility (r = 0.253, P < .0001), and viability (r = 0.286, P < .0001). On the basis of the findings of this study and those of other reports, zinc may contribute to fertility through its positive effect on spermatogenesis.     

人类精子低渗试验指数分析

目的：探讨精子低渗试验指数（ＳＨＯＴＩ）对评估男性生育力的临床价值。方法：采用两种不同渗量的低渗液结合活体染色技术，对１２９例男性精液进行精子低渗试验。结果：生育组与不育组比较精子卷尾率、ｇ型肿胀精子率和精子低渗试验指数差异均显著（Ｐ＜０．０１），生育组ＳＨＯＴＩ分布范围为：０．０９６～０．３３６；不育组ＳＨＯＴＩ分布范围为０．０２５～０．２１５。结论：ＳＨＯＴＩ综合反映了精子的整体膜功能及受精能力，可作为评估男性生育力的一个新的指标。初步确定临床参考值为ＳＨＯＴＩ＜０．０９６，提示精子功能不良，不能生育；在０．０９６～０．２１５之间精子功能部分正常，有机会生育；在０．２１５～０．３４０之间预示精子功能正常，生育力良好。     

Towards the identification of reliable sperm biomarkers for male infertility: A sperm proteomic approach

Male infertility evaluation is mainly based on semen analysis. Thus, identification of additional diagnostic methods is valuable. The aim of this study was to analyse the sperm proteome of infertile men to identify the underlying mechanisms and reliable diagnostic biomarkers. This cross‐sectional study consisted of 16 infertile men and seven proven fertile men. An LC‐MS/MS approach was performed in five pooled samples of each group (proven fertile men, primary infertility and secondary infertility). Differentially expressed proteins were used for functional enrichment analyses, and the most central proteins involved in altered functions in both infertile groups and the testis‐specific proteins were validated using Western blotting and immunocytochemistry. In total, 1,305 sperm proteins were identified, of which 102 were underexpressed and 15 were overexpressed proteins in both infertile groups. Underexpressed proteins were mostly related to protein post‐translational modification and folding, especially BAG6, HSPA2 and SPA17. Validation analysis revealed an underexpression of BAG6 in infertile men, whereas HSPA2 and SPA17 expressions did not differ between the groups. No differences were observed in the sperm localisation of these proteins. An overexpression of HIST1H2BA—a testis‐specific protein—was observed in both proteomic approaches. Therefore, BAG6 and HIST1H2BA are potential candidates for male infertility biomarkers.     

Zinc in sperm chromatin and chromatin stability in fertile men and men in barren unions

The stability and the content of zinc of the chromatin were studied in spermatozoa from ten men with unexplained infertility, and in spermatozoa from five fertile donors. A positive relation was found between zinc in sperm nuclei (X-ray microanalysis) and the resistance of the chromatin to decondense in sodium dodecylsulfate (SDS). The infertile men had lower degree of sperm chromatin stability and lower sperm zinc content than the fertile donors. A subgroup of the infertile men, which all had minor clinical signs of prostatic inflammatory reaction, had the lowest content of zinc in the chromatin and the lowest degree of chromatin stability. A low content of nuclear zinc would impair the structural stability of the chromatin and thereby increase the vulnerability of the male genome. This mechanism may be one explanation for the reduced fertility of the men with minor inflammation of the prostate.     

Incidence of chromosome 8,10,X and Y aneuploidies in sperm nucleus of infertile men detected by FISH

We studied the frequency of aneuploidy in sperm nuclei of six infertile men with abnormal semen profile and normal karyotype, using fluorescence in situ hybridization (FISH) with DNA probes for chromosomes 8, 10, X and Y. The control group consisted of four healthy fertile men with normal karyotype and semen profiles. The purpose of this study was to determine whether there are differences between infertile male donors and control donors for: (1) the incidence of sex chromosome aneuploidy, and (2) the number of disomies for chromosomes 8, and 10 cosegregating with chromosomes X and Y. FISH analysis showed no significant differences of sex ratios of the sperm nuclei in and between infertile and control groups. The most significant abnormalities in the infertile group were clusters of sperm nuclei bearing XY and XYY. In addition, the incidence of disomic sperm nuclei for chromosomes 8 and 10 consegregating with sex chromosomes was not significantly different between the patient and control groups, nor within them. However, the total frequency of aneuploid sperm nuclei was significantly different between the infertile group and the control group. We observed a significant excess of sperm nuclei bearing chromosome 10 along with disomy for chromosome Y (10YY). In conclusion, our results from FISH analysis demonstrate a significantly increased frequency of aneuploidy for the sex chromosomes in sperm nuclei from infertile men. Therefore it may be concluded that infertility is a risk factor for sex chromosome aneuploidy in sperm nuclei.     

植物凝集素荧光标记法检测精子顶体结构的临床意义

目的　探寻评估人精子授精能力的简便有效方法。　方法　采用络合异硫氰酸荧光素的植物凝集素 (FITC PSA)进行精子顶体荧光标记 ,分别测定 5 8例不育患者及 4 6例正常生育男子的精子顶体完整率 ,并对其中 14例精液标本 (不育组及正常组各 7例 )进行人卵透明带诱发的精子顶体反应实验 ,对精子顶体完整率与发生顶体反应的百分率的相关关系进行分析。　结果　正常生育组顶体完整率及发生精子顶体反应百分率显著高于不育组 (P <0 .0 1) ,精子顶体完整率与发生精子顶体反应百分率呈正相关 (r =0 .74 68)。　结论　FITC PSA荧光标记法操作简便 ,技术结果可靠 ,特异性强 ,可作为评价人精子授精能力的有效指标之一。     

Heterologous ovum penetration test and seminal parameters in fertile and infertile men

Sperm penetration rates in the heterologous ovum penetration test were correlated with results of routine semen analysis in 30 fertile and 50 infertile men. There was no difference in penetration rates when comparing infertile men with normal and abnormal seminal parameters, nor was any difference seen between fertile (15-83%) and infertile men (8-83%). Of the 22 infertile men with normal seminal characteristics, seven had partners with no discernible reproductive dysfunction. The penetration rates of these men (38-81%) did not differ from those of fertile men. Stepwise regression analysis of seminal characteristics, with penetration as the dependent variable, indicated that sperm count and morphology are the most important seminal parameters for fertility assessment. Discrimination analysis revealed that sperm numbers and morphologic variables provide significant information for discriminating between fertile men and infertile men with normal or abnormal seminal characteristics. Sperm penetration and motility were not indicated as important factors. The present data suggest that, in unselected male patients seeking reproductive evaluation, the sperm penetration assay did not yield any additional information on the cause of infertility.     

Infertile men with varicocele show a high relative proportion of sperm cells with intense nuclear damage level, evidenced by the sperm chromatin dispersion test

The frequency of sperm cells with fragmented DNA was studied in a group of 18 infertile patients with varicocele and compared with those obtained in a group of 51 normozoospermic patients, 103 patients with abnormal standard semen parameters, and 22 fertile men. The spermatozoa were processed to discriminate different levels of DNA fragmentation using the Halosperm kit, an improved Sperm Chromatin Dispersion (SCD) test. In this technique, after an acid incubation and subsequent lysis, those sperm cells without DNA fragmentation show big or medium-sized halos of dispersion of DNA loops from the central nuclear core. Otherwise, those spermatozoa containing fragmented DNA either show a small halo, exhibit no halo with solid staining of the core, or show no halo and irregular or faint stain of the remaining core. The latter, that is, degraded type, corresponds to a much higher level of DNA-nuclear damage. The varicocele patients showed 32.4% +/- 22.3% of spermatozoa with fragmented DNA, significantly different from the group of fertile subjects (12.6% +/- 5.0%). Nevertheless, this was not different from that of normozoospermic patients (31.3% +/- 16.6%) (P = .83) and with abnormal semen parameters (36.6% +/- 15.5%) (P = .31). No significant differences were found between the normozoospermic patients and the patients with abnormal semen parameters. Strikingly, the proportion of the degraded cells in the total of sperm cells with fragmented DNA was 1 out of 4.2 (23.9% +/- 12.9%) in the case of varicocele patients, whereas it was 1 out of 8.2 to 9.7 in the normozoospermic patients (11.1% +/- 9.9%) in the patients with abnormal sperm parameters (12.2% +/- 8.3%) and in the fertile group (10.3% +/- 7.2%). Thus, whereas no differences in the percentage of sperm cells with fragmented DNA were evident with respect to other infertile patients, individuals with varicocele exhibit a higher yield of sperm cells with the greatest nuclear DNA damage level in the population with fragmented DNA. This finding illustrates the value of assessing different patterns of DNA-nuclear damage within each sperm cell and the particular ability of the Halosperm kit to reveal them.     

Comparison of chromatin assays for DNA fragmentation evaluation in human sperm

Sperm chromatin integrity is vital for successful pregnancy and transmission of genetic material to the offspring. We evaluated chromatin integrity in sperm from 60 infertile men and 7 fertile donors comparing the sperm chromatin structure assay (SCSA), TdT-mediated-dUTP nick end labeling (TUNEL), the sperm chromatin dispersion (SCD) test, and acridine orange staining technique (AOT). The TUNEL and SCD assays showed a strong relationship with the SCSA (r > .866; P < .001) for sperm DNA fragmentation, both in infertile men and donors of known fertility. AOT did not show any relationship with SCSA. The breakdown of the DNA fragmentation index (DFI) into 3 categories (< or =15%, >15%-<30%, and > or =30%) showed that the SCSA, TUNEL, and SCD test predict the same levels of DNA fragmentation. AOT consistently showed higher levels of DNA fragmentation for each DFI category. DNA fragmentation in sperm between infertile men and donor sperm was significantly different (P < .05) under SCSA (22.0 +/- 1.6 vs 11.8 +/- 1.4), TUNEL (19.5 +/- 1.3 vs 11.1 +/- 0.9) and SCD (20.4 +/- 1.3 vs 10.8 +/- 1.1), respectively. DNA fragmentation in sperm evaluated by AOT did not differ (P > .05) between infertile men (31.3 +/- 2.4) and donors (32.7 +/- 4.8). AOT showed extreme variations for sperm DNA fragmentation in semen from both infertile men and donors. The problems of indistinct colors, rapid fading, and the heterogeneous staining were also faced. In conclusion, SCSA, TUNEL, and SCD show similar predictive values for DNA fragmentation, and AOT shows variable and increased levels of DNA fragmentation, which makes it of questionable value in clinical practice.     

Effect of leukocytospermia on sperm DNA integrity: a negative effect in abnormal semen samples

Controversy exists over levels of DNA integrity in the sperm of fertile and infertile men. In addition, the effect of leukocytospermia on sperm DNA in these 2 groups is unclear. We decided to address these questions by collecting semen samples from men known or presumed to be fertile and men from infertile couples. Samples were analyzed and assessed for sperm concentration, motility, and morphology. Samples failing to meet World Health Organization (WHO) standards in one or more of these parameters were judged abnormal. Samples were then arbitrarily assigned normalized scores in each of the above parameters, and scores were summed to give a normalized value for overall sperm quality. DNA abnormality was determined by an in situ DNA denaturation test with acridine orange and expressed as a percentage of cells with abnormal DNA integrity (ADI). Assessment of 187 samples revealed a moderate inverse correlation between ADI and sperm quality (r =.58), although a large degree of ADI dispersion was observed in abnormal semen samples. The average ADI for normal and abnormal semen samples was 18% +/- 2.8% and 36% +/- 5.8%, respectively, with the threshold of 95% probability set at 30%. When sorted for leukocytospermia, the difference in ADI between normal and abnormal semen groups without leukocytospermia was much smaller (17% +/- 2.2% and 22% +/- 4.6%; P =.023). Leukocytospermia had no significant effect on ADI in the normal semen group (P = .46); however, ADI was more than double the ADI in the abnormal semen group (18% +/- 2.4% and 50% +/- 11%; P < .001). The results of our analysis show that at least 3 factors affect net DNA integrity in leukocytospermic samples that fail to meet WHO standards: 1) primary DNA damage, which is moderately inverse to sperm quality, in particular to sperm concentration; 2) effect of leukocytes increasing primary or provoking potential DNA damage in a cascade-like manner, particularly in sperm with poor morphology and motility; and 3) a decreasing proportion of cells with damaged DNA in semen with the worst quality.