Adenosine triphosphate (ATP) in human spermatozoa

The seminal adenosine triphosphate (ATP) content was determined by bioluminescence after treatment with trichloroacetic acid (TCA) in 81 semen samples 1.5 h after ejaculation obtained from men attending our fertility clinic, and selected to contain either 20% or less spermatozoa with good progressive motility (n = 22), or 60% or more spermatozoa with good progressive motility (n = 59) (Study I), and in 18 semen samples from fertile men 30 min and 3.5 h after ejaculation (Study II). The latter samples were divided into 2 equally large groups according to sperm motility. In Study I the mean sperm ATP concentration was significantly higher in the semen samples with bad motility (0.63 nmol per living spermatozoa x 10(-6)) than in semen samples with good motility (0.39 nmol per living spermatozoa x 10(-6); P less than 0.01). In Study II the ATP concentration per living spermatozoa was also lower in the group with the best motility in comparison with the spermatozoa with lower motility (P less than 0.01), both 30 min and 3.5 h after ejaculation. During the 3-5 h incubation the sperm ATP concentration decreased by 21% (P less than 0.01) in the former group of samples but remained unchanged in the latter group. The results indicate that, in semen samples with highly motile spermatozoa, the consumption of ATP is higher than in semen samples with impaired sperm motility. It is therefore essential that the time between ejaculation and ATP measurement is as short as possible to obtain comparable results. Repeated ATP measurements in combination with an analysis of the number of living spermatozoa, may provide further information on the fertilizing capacity of spermatozoa.     

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.     

Predictive value of semen parameters in in vitro fertilisation pregnancy outcome

This study investigated retrospectively the predictive value of routine semen analysis in pregnancy by in vitro fertilisation (IVF). The selected (n = 796) cycles were divided into two groups: pregnancy group (group 1; n = 264) and nonpregnancy group (group 2; n = 532), in which the female partners were normal or just had tube problems. No significant differences were found in the percentage of normal sperm morphology, sperm motility, sperm progressive motility, rapid progressive motility (rapid) and concentration between the two groups (P > 0.05). However, teratozoospermic index (TZI) and sperm deformity index (SDI) showed statistically significant differences between the two groups (P < 0.05). The number of retrieved eggs (P = 0.001), fertilisation rate (P = 0.000) and number of embryos transferred (P = 0.020) in group 1 were significantly higher than those in group 2, but no significant differences were noted in cleavage rates, and good quality embryo rates between the two groups (P > 0.05). Using receiver operating characteristics curve, we found that semen parameters (morphology, motility and concentration), fertilisation rate, TZI and SDI were not good indicators for pregnancy by IVF. Thus, the semen parameters evaluated according to criteria of the World Health Organization are no good predictors for accurately identifying the IVF outcome. However, TZI and SDI may be more informative than other semen parameters.     

Semen analysis and sperm function testing

Despite controversy regarding the clinical value of semen analysis, male fertility investigation still relies on a standardized analysis of the semen parameters. This is especially true for infertility clinics in both developing and developed countries. Other optional tests or sophisticated technologies have not been widely applied. The current review addresses important changes in the analysis of semen as described in the new World Health Organization (WHO) manual for semen analysis. The most important change in the manual is the use of evidence-based publications as references to determine cutoff values for normality. Apart from the above mentioned changes, the initial evaluation and handling methods remain, in most instances, the same as in previous editions. Furthermore, the review evaluates the importance of quality control in andrology with emphasis on the evaluation of sperm morphology. WHO sperm morphology training programmes for Sub-Saharan countries were initiated at Tygerberg Hospital in 1995. The external quality control programme has ensured that the majority of participants have maintained their morphological reading skills acquired during initial training. This review reports on current sperm functional tests, such as the induced acrosome reaction, and sperm-zona pellucida binding assays, as well as the impact of sperm quality in terms of DNA integrity, and the relationship of sperm function tests to sperm morphology.     

Multivariate discriminate analysis of the relationship between the hypo-osmotic swelling test and the in-vitro fertilizing capacity of human sperm

Multivariate discriminant analysis was used to evaluate the usefulness of routine semen parameters and the hypo-osmotic swelling test (HOST) as predictors of the in-vitro fertilizing capacity of human sperm as assessed by the zona-free hamster egg penetration assay (HEPA). Eighty-eight semen samples from untreated patients attending an infertility clinic were analysed. Semen samples were classified into the following three groups before statistical analysis: group 1--positive sperm penetration (greater than or equal to 10%, n = 39); group 2--borderline penetration rates for HEPA (greater than 0% but less than 10%, n = 39) and group 3--negative sperm penetration (0%, n = 10). The percentage of sperm with normal morphology and sperm count were found to be significant in discriminating between semen samples exhibiting different in-vitro fertilizing capacity. These two discriminating variables in combination gave an overall correct classification rate of 45.5%. The multivariate discriminant analysis was also performed after excluding the data of group 2 semen samples (n = 39), which exhibited borderline sperm penetration rates. As a result, three discriminating variables including semen volume, sperm count and the percentage of sperm with normal morphology were selected. These three variables in combination could accurately predict whether a semen sample would exhibit positive sperm penetration (group 1) or negative sperm penetration (group 3) with an overall accuracy of 75.5%. The percentage of swollen sperm after hypo-osmotic treatment was not related to the HEPA result, as determined by linear correlation and multiple regression analyses, and did not give additional information about the in-vitro fertilizing capacity of sperm as evaluated by multivariate discriminant analysis.     

Relationship between human sperm lipid peroxidation, comprehensive quality parameters and IVF outcome

The membranes of human spermatozoa contain an extremely high concentration of polyunsaturated fatty acids and are therefore susceptible to lipid peroxidation damage. The aim of this study was to retrospectively determine the association between the lipid peroxidation levels of washed spermatozoa, as indicated by thiobarbituric-acid-reactive substance concentration, and: (a) semen quality evaluated by basic routine, biochemical, cytological and quantitative ultramorphological analyses; (b) IVF fertilization rate. Semen samples from 45 male partners of couples who had been referred for IVF treatment due to a female infertility factor were evaluated for quality as well as for thiobarbituric-acid-reactive substance concentrations. The latter were found to have a negative correlation with total sperm count, semen volume, zinc/fructose ratio, and the integrity of sperm acrosome and axonema. It was suggested that lipid peroxidation has a deleterious effect on the ultramorphological status of the sperm cells and, thereby, on the male fertilization potential. The content of the seminal fluid, about 30% of which is produced by the prostate, may protect spermatozoa from this destructive process. A negative correlation was also found between thiobarbituric-acid-reactive substance concentrations and IVF fertilization rate. When the patients were subdivided into fertilizing (fertilization rate > 0%) and nonfertilizing (fertilization rate = 0%) subgroups (n = 33 and n = 12, respectively), the former exhibited significantly lower thiobarbituric-acid-reactive substance concentrations than the latter. A new IVF fertilization index based on the lipid peroxidation level was established. This index had a predictive power of 93% (94% sensitivity and 92% specificity). The clinical value of this index should be further verified.     

Redox potential in human semen: Validation and qualification of the MiOXsys assay

Seminal oxidative stress (OS) is a major contributing factor to male infertility. Semen analysis cannot identify reactive oxygen species (ROS), which can be measured using a chemiluminescence assay. Measurement of redox potential provides a more comprehensive assessment of OS, although the test has yet to be fully validated. This study aimed to validate the MiOX^sys analyser for measuring static oxidation–reduction potential (sORP). Results demonstrated that duplicate measurements must be taken, sensors must be batch tested, and sockets should be regularly changed to avoid inconsistency in measurement. Measurement of sORP using MiOX^sys exhibited good reproducibility across different operators (p = 0.469), analysers (p = 0.963) and days (p = 0.942). It is not affected by mechanical agitation (p = 0.522) or snap freezing and thawing (p = 0.823). The stability of sORP over time requires further verification, particularly in samples with high initial sORP. Measurement is temperature sensitive between 2 and 37°C, significantly increasing with increasing temperature (p = 0.0004). MiOX^sys is a more stable assay for assessing OS than chemiluminescence methods and permits greater flexibility for sample handling. MiOX^sys could be implemented to complement semen analysis as part of routine diagnostic testing for male infertility and may be useful in identifying contributing factors to idiopathic infertility.     

用传统方法与计算机辅助精液分析软件CRISMAS分析166名丹麦青年男性精液样本的结果比较

本研究以166名丹麦青年男性精液为样本,通过评估精于浓度和活力来比较传统精液分析与计算机辅助精液分析方法（CASA）（哥本哈根Rigshospitalet图像屋精子运动分析系统,CRISMAS软件4．6版本）。CRISMAS软件测定精子的浓度把精子活力分为三类。传统分析方法将精子活力分为四种状态。为了便于二者的比较,本文将传统的四种状态根据精子速度等级重新分为三个状态：rapidly progressive（A）,slowly progressive（B）和non-progressive（C＋D）。两种方法所研究的参数之间都有显著差异（P〈0．001）。与传统方法相比,CRISMAS高估了精子浓度以及快速运动精子的比例,因而低估了慢速运动和非运动精子。为分析研究结果是否会随精液分析时间而起伏变动,将精液分析结果按分析同期分为四个层次。结果表明CRISMAS对活力的分析结果比传统分析方法稳定,但两种方法都未表现出任何趋势。显然,无法比较CRISMAS CASA和传统分析方法在精子浓度和精子活力方面的分析结果。在临床上使用该软件时以及用其研究这些精子特性时需要说明这一点。     

Measurement and significance of sperm morphology

The measurement or evaluation and clinical significance of human sperm morphology has always been and still is a controversial aspect of the semen analysis for the determination of a male's fertility potential. In this review the background of the development of the evaluation criteria for sperm morphology will be discussed. Aspects of criticism on the strict criteria definition and use of the criteria for sperm morphology evaluation will be discussed as well as possible reasons for the decline in normal sperm morphology values and how we can compromise for this phenomenon resulting in the very low normal reference value as published in the 2010 WHO manual for the Examination and Processing of Human Semen. One of the possible solutions may be to give more attention to a limited number of abnormal sperm morphology categories and the inclusion of sperm morphology patterns. It is concluded in this review that if done correctly and with care and with strict application of existing guidelines as outlined in the 2010 WHO manual, sperm morphology measurement still has a very important role to play in the clinical evaluation of male fertility potential.     

Evaluation of human sperm motility by means of transmembrane migration method and computer assisted semen analysis: a comparison study

Comparing motility parameters with a trans-membrane migration method and a Hamilton-Thorn HTM-2000 computer assisted semen analyzer, we found that trans-membrane migration ratio (TMMR) correlated best with critical motility which indicated the fraction of fastest and straightest sperm in a semen sample. We also found that TMMR correlated better with progressive velocity than with track speed. It is concluded that nonprogressive sperm were not included in the estimation of TMMR and the trans-membrane migration method is most suitable for studying drug effect on straight and rapid sperm motility.     

Male factors and the likelihood of pregnancy in infertile couples. I. Study of sperm characteristics

A prospective study of 394 infertile men was conducted over 3 years following a primary semen analysis. The cumulative pregnancy rate was 43 and 64% after 1 and 3 years, respectively. The pregnancy rate was significantly higher in the secondary infertile group. The study of various sperm factors and the occurrence of pregnancy showed that they were not of equal significance in predicting male fertility potential. The percentage of pregnancies decreased significantly only when the sperm concentration was less than 5 x 10(6)/ml. The pregnancy rate increased significantly with the percentage of motile sperm. The percentage of sperm with normal morphology was also found to be significantly higher when a pregnancy occurred than when the couple remained infertile (43.6% vs 37.7%). In a detailed morphological analysis of the sperm, six abnormalities (microcephaly, double head, amorphous head, cytoplasmic droplet, bent tail and coiled tail) were found to be significantly more frequent when a pregnancy did not occur. The most predictive value was given by the Multiple Anomalies Index (MAI), which is the mean number of abnormalities observed per abnormal sperm. The pregnancy rate was significantly lower after both 1 and 3 years when the MAI was greater than 1.6. Multivariate analysis showed that the best prognostic indicator of fertility was given by the percentage of motile sperm and the MAI, particularly in patients with primary infertility.     

Clinical significance of the low normal sperm morphology value as proposed in the fifth edition of the WHO Laboratory Manual for the Examination and Processing of Human Semen

The very low cut-off value for sperm morphology of 4% morphologically normal spermatozoa, as proposed in the new edition of the World Health Organization （WHO） manual on semen analysis, is in agreement with recently published values and reflects the trend of a decline in reported mean values for normal sperm morphology. The reduced value for morphologically normal spermatozoa over the years may be due to several factors. The first is the introduction of strict criteria for the evaluation of sperm morphology. Other reasons may include the introduction of additional criteria for sperm morphology abnormalities and the suggested decrease in semen parameters because of increasing negative environmental influences. Although on its own the newly proposed very low normal value may not provide the strong predictive value for a males＇ fertility potential, as originally reported for sperm morphology evaluated according to strict criteria, a good predictive value can still be obtained if the holistic, strict approach for sperm morphology evaluation is followed together with additional sperm morphology parameters now available, because certain morphology patterns and sperm abnormalities are now known to be of strong prognostic value. In addition, better international standardization of the technical methodology, consensus on the interpretation of sperm morphology evaluation criteria and standardized international external quality control （EQC） schemes, are of utmost importance to maintain the good predictive value of sperm morphology.     

The diagnostic value of seminal adenosine triphosphate (ATP) in an in vitro fertilization (IVF) program: Der diagnostische Wert von ATP in einem IVF-Programm

The level of adenosine triphosphate (ATP) was quantitated in semen samples used for in vitro fertilization of human oocytes. Seminal ATP level correlated with the concentration and percentage motility of spermatozoa but not with the in vitro fertilization rate of human oocytes. Seminal ATP measurement appears to have little diagnostic value in predicting the fertilizing capacity of spermatozoa as evaluated by the multivariate stepwise discriminant analysis.     

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。     

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.     

精子质量参数分析的标准化与质量控制的研究进展

精液分析是评价男性生育能力的最基本测试。最近几年,对精液分析标准化的迫切需求已引起男科学家的广泛重视。本文对精子质量参数———精子密度、活动率和形态学分析的标准化及质量控制进行了综述。精子密度分析的关键是计数池的标准化,因此Cell-VU计数池应该是最佳的选择;精子活动率和精子形态学的分析由于主观性太强,CASA系统可能是其标准化的最终选择。精液质量参数分析的质量控制主要是质量控制材料的选择,以及在男科学实验室实施EQC和IQC项目,而一些监测质量控制的图表和计算方法应被相应地建立。     

精子质量参数分析的标准化与质量控制的研究进展

精液分析是评价男性生育能力的最基本测试。最近几年,对精液分析标准化的迫切需求已引起男科学家的广泛重视。本文对精子质量参数———精子密度、活动率和形态学分析的标准化及质量控制进行了综述。精子密度分析的关键是计数池的标准化,因此Cell-VU计数池应该是最佳的选择;精子活动率和精子形态学的分析由于主观性太强,CASA系统可能是其标准化的最终选择。精液质量参数分析的质量控制主要是质量控制材料的选择,以及在男科学实验室实施EQC和IQC项目,而一些监测质量控制的图表和计算方法应被相应地建立。     

Specialized sperm function tests in varicocele and the future of andrology laboratory

Varicocele is a common medical condition entangled with many controversies. Though it is highly prevalent in men with infertility, still it marks its presence in males who do have normal fertility. Determining which patients are negatively affected by varicocele would enable clinicians to better select those men who benefitted the most from surgery. Since conventional semen analysis has been limited in its ability to evaluate the negative effects of varicocele on fertility, a multitude of specialized laboratory tests have emerged. In this review, we examine the role and significance of specialized sperm function tests with regards to varicocele. Among the various tests, analysis of sperm DNA fragmentation and measurements of oxidative stress markers provide an independent measure of fertility in men with varicocele. These diagnostic modalities have both diagnostic and prognostic information complementary to, but distinct from conventional sperm parameters. Test results can guide management and aid in monitoring intervention outcomes. Proteomics, metabolomics, and genomics are areas; though still developing, holding promise to revolutionize our understanding of reproductive physiology, including varicocele.     

Determination of seminal oxidation–reduction potential (ORP) as an easy and cost‐effective clinical marker of male infertility

Oxidative stress (OS) is an important contributing factor to male infertility. While previous methods to measure seminal OS are time‐consuming and limited to the use of freshly produced semen, oxidation reduction potential (ORP) is easier and quicker to perform and can also be used in frozen semen. Therefore, this study evaluated the clinical utility of ORP as a potential marker of male infertility. ORP was measured in semen samples from 293 patients and 15 fertile controls and categorised according to WHO criteria as normozoospermic, oligozoospermic, asthenozoospermic, teratozoospermic and oligoasthenoteratozoospermic. Receiver operating characteristic (ROC) curves were generated to differentiate these categories. Semen parameters were significantly different when subjects were grouped as control and patients or between the patient and normozoospermic group for concentration and morphology. ORP levels were significantly different between the control and normozoospermic group. When subjects were grouped based on concentration, motility, morphology or a combination of these, the area under the ROC curve, sensitivity, specificity, positive predictive value and cut‐off values were significantly different. These differences were significant when combined with ORP and grouped with any two sperm abnormalities. In conclusion, ORP is a quick, easy, cost‐effective and reliable marker of semen quality as well as oxidative stress for use in a clinical setting.     

Famotidine does not affect human sperm fertility characteristics (motility,viability and DNA integrity) in vitro

Famotidine, a histamine‐2 receptor antagonist, is commonly used to relieve the acid‐related gastrointestinal diseases; however, its effect on human sperm parameters, and hence on sperm function, is still undetermined. Here, we intended to measure human sperm motility, viability, and DNA integrity of ejaculated human sperm in the presence of famotidine at 0, 0.1, 1 and 10 mM concentrations in vitro. Forty‐nine semen samples of normal count, motility, and morphology were included in this study. Sperm motility was assessed using Makler counting chamber and a phase contrast optics (200× magnification), whereas sperm viability was assessed using eosin–nigrosin staining procedure. The effect of famotidine on sperm DNA integrity was measured using flow cytometry. Famotidine at 0.1, 1 or 10 mM had insignificant effect on human sperm motility (progressive, p = .9594; and total, p = .8420), sperm viability (p = .6471), and content of DNA breaks in sperm (p > .05) compared with the control. In conclusion, famotidine at 0.1, 1 or 10 mM did not alter human sperm motility, viability or DNA integrity in vitro. Although, these findings indicate safety of famotidine in human sperm, further in vivo studies are required to establish the drug's safety.