Practical semen analysis： from A to Z

Accurate semen analysis is critical for decisions about patient care, as well as for studies addressing overall changes in semen quality, contraceptive efficacy and effects of toxicant exposure. The standardization of semen analysis is very difficult for many reasons, including the use of subjective techniques with no standards for comparison, poor technician training, problems with proficiency testing and a reluctance to change techniques. The World Health Organization （WHO） Semen handbook （2010） offers a vastly improved set of standardized procedures, all at a level of detail that will preclude most misinterpretations. However, there is a limit to what can be learned from words and pictures alone. A WHO- produced DVD that offers complete demonstrations of each technique along with quality assurance standards for motility, morphology and concentration assessments would enhance the effectiveness of the manual. However, neither the manual nor a DVD will help unless there is general acknowledgement of the critical need to standardize techniques and rigorously pursue quality control to ensure that laboratories actually perform techniques ＇according to WHO＇ instead of merely reporting that they have done so. Unless improvements are made, patient results will continue to be compromised and comparison between studies and laboratories will have limited merit.     

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。     

Semen analysis performed by different laboratory teams: an intervariation study

Some recent studies have indicated that sperm concentration has decreased during the last 50 years. However, comparisons between laboratories have revealed that geographical differences seem to exist and that any decrease may not be global. One point of criticism concerning comparison of results from different laboratories has been that some of the discrepancies detected could reflect the lack of standardized methods used in the different laboratories. Four teams, each consisting of one physician and one technician from groups which have recently published data on semen quality, met in order to evaluate the variability between their laboratories on semen analysis. Twenty-six fresh semen samples from unselected men were analysed. The groups analysed the samples according to the normal practice in their laboratories, using their own equipment. The variation between laboratories was estimated through a random effects model. For sperm concentration and semen volume assessment a remarkable consistency between laboratories was detected, in contrast to the very considerable inter-individual variation. For sperm motility and morphology assessments interlaboratory consistency was much poorer. In conclusion, evaluation of sperm motility and morphology characteristics requires further standardization in order to achieve comparable data from different laboratories. However, semen volume and sperm concentration are characteristics which can be compared reliably between laboratories, when similar methodologies are used.     

Spontaneous improvement in semen quality: regression towards the mean

Regression towards the mean was demonstrated in semen analyses from 216 donors for artificial insemination by selecting those with low sperm concentration (less than 40 X 10(6)/ml), low motility (less than 50%) or low motility index (less than 120) in the first test. For each characteristic, the result was significantly higher in the second test, the means for first and second tests being, for sperm concentration 28 and 56 million per ml (n = 14, P less than 0.01), for motility 42 and 55% (n = 14, P less than 0.01) and for the motility index 95 and 131 (n = 24, P less than 0.001). Regression towards the mean occurs whenever extremes of the range of a variable and selected for re-measurement. Many of the current treatments for oligospermia and asthenospermia have been promoted because semen quality improved during uncontrolled studies in which this phenomenon may have been operative. Using means of multiple samples before and following therapeutic intervention will not prevent regression towards the mean, only reduce its magnitude. Thus, placebos are essential in the evaluation of treatments for male infertility.     

Semen analysis and external quality control schemes for semen analysis need global standardization

One semen analysis laboratory [the Institute of Reproductive Medicine (IRM), Münster, Germany] was enrolled in three external quality assurance programmes in Europe (United Kingdom External Quality Assurance Scheme, European Academy of Andrology, European Society of Human Reproduction) that control for the assessment of sperm concentration, sperm motility and sperm morphology. Agreement between the participating laboratory and the sperm concentrations designated by all three programmes was good. Disagreement between two quality control (QC) programmes providing motility assessment was particularly noticeable in the distinction between motility grades a and b. For the identification of normal sperm morphology, marked differences between the standards set by different QC programmes were apparent. One programme was stricter in its categorization of normal forms, such that an overestimation of normal forms was diagnosed at IRM, whereas agreement with the other programmes was achieved. Variation of results from technicians in the IRM internal QC programme was <13%. The discrepancies between external quality control (EQC) centres demonstrated here are challenging problems to be overcome partly by the andrology laboratories and partly by the providers of EQC services. The introduction of objective, computer-aided sperm assessment in providing designated values may help to rectify this situation. Until this is achieved, EQC programmes should develop an internal programme to monitor their materials and methods for assessment.     

爱活尿通促进精液液化的体外实验研究

目的 通过对爱活尿通处理精液前后液化时间的比较,研究爱活尿通在体外对液化异常精液的影响.方法 50例液化时间延长的精液标本每例分成3份,1份加爱活尿通提取液为实验组,还有2份为对照组,对照组1加糜蛋自酶,对照组2不作任何处理,观察不同方法处理后精液液化的时间及处理前后精子活力.结果 爱活尿通提取液和糜蛋白酶均可使液化延长的精液明显缩短液化时间(P＜0.05),19.爱活尿通处理后的精液液化时间较糜蛋白酶处理后精液液化时间更短,差异有统计学意义(P＜0.05),但精子活力及活率的差异没有统计学意义(P＞0.05).结论 爱活尿通提取液和糜蛋白酶均可以有效地改善精液液化时间,但爱活尿改善精液液化时间的效果优于糜蛋白酶.     

Automation of human semen analysis using a novel artificial intelligence optical microscopic technology

Current semen analysis still commonly depends on a manual microscopy method in clinical laboratories worldwide. However, some of the major disadvantages of this technique are that it is labour‐intensive, subjective, laboratory‐based and time‐consuming. Although computer‐assisted semen analysers (CASAs) have enabled partial automation of routine semen analysis, they lack wider acceptance due to their complicated operation. Therefore, the development of an accessible, rapid and standardised method for semen analysis is urgently needed. Here, we describe the development and clinical testing of a novel, automated, artificial intelligence optical microscopic (AIOM)‐based technology, LensHooke? X1 PRO (X1 PRO), designed for the quantitative measurement of sperm concentration, motility and seminal pH. We observed high degree of correlation in the results of concentration, progressive motility and progressively motile sperm concentration between the X1 PRO semen analyser and manual method using 135 clinical semen samples. In addition, the seminal pH results obtained by X1 PRO and manual methods were comparable (p = .12). In summary, our results showed that new X1 PRO semen analyser is a reliable diagnostic tool for routine semen analysis providing clinically acceptable results based on World Health Organization (WHO) 5th Edition guidelines.     

Critical appraisal of conventional semen analysis in the context of varicocele

Varicocele is present in approximately 15% of men, and, although it is the most commonly diagnosed cause of male infertility, nearly two-thirds of men with varicoceles remain fertile. It was decided to make use of the current evidence obtained from the previous meta-analyses between 2004 and 2015 as well as available articles covering this field, preferably randomized controlled articles dealing with the topic of semen analysis before and after repair. Two important meta-analyses were discussed as well as other articles dealing with the topic of semen analysis before and after varicocelectomy. The evidence suggests that all semen parameters improve after varicocele repair. Based on the available evidence, it is clear that there is a benefit in treating men with a palpable varicocele. One can expect that all semen parameters will improve within 3 months after repair.     

Reassessment of the accuracy of traditional sperm characteristics and adenosine triphosphate (ATP) in estimating the fertilizing potential of human semen in vivo

Receiver operating characteristic curves and accuracy parameters were computed for traditional sperm characteristics (concentration, motility, morphology) and the number of peroxidase negative cells, and the concentration of adenosine triphosphate (ATP) in semen from populations of fertile and infertile men, and men who achieved a pregnancy after varicocele treatment. The percentage and concentration per millilitre of spermatozoa with rapid linear progressive motility, and the ATP concentration, provided the best discrimination between fertile and treated fertile from infertile men. The misclassification rate was higher for sperm morphology, total progressive motility and viability, whereas sperm concentration and the total sperm count per ejaculate had the worst discriminating power. The number of peroxidase negative cells per 100 spermatozoa was highly specific in identifying men who achieved pregnancy after varicocele treatment. The lower limit of normality of sperm characteristics was remarkably different between fertile men and men achieving pregnancy after treatment or during infertility work-up.     

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。     

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.     

Fatty acids,food groups and semen variables in men referring to an Italian Fertility Clinic: Cross-sectional analysis of a prospective cohort study

Diet has been suggested as a factor affecting sperm quality, both in healthy and infertile men. To study whether specific food groups and fatty acids (FA) intake were associated with sperm parameters of men from couples referring to an Italian Fertility Clinic, we conducted a cross-sectional analysis. A semen sample was collected and analysed before proceeding with assisted reproduction. To evaluate food groups and fatty acids intake, we used a questionnaire of food frequency. We calculated odds ratios (OR) and 95% confidence intervals (CI) for abnormal semen parameters. Among 323 men, 19% had semen volume (SV) <1.5 ml, 31% sperm concentration (SC) <15 mil/mL, 33% total sperm count (TSC) <39 mil and 27% sperm progressive motility <32%. Low SC was more frequent in men with higher saturated FA (SFA) and polyunsaturated FA (PUFA) intake. Better SC and TSC were related to higher monounsaturated FA (MUFA)/SFA ratio. Monounsaturated FA and linoleic/linolenic acid ratio were not related to any considered parameters. Low TSC was associated with low vegetable intake. In conclusion, normal sperm parameters were more frequent in men with low intake of SFA and PUFA, and high vegetable intake. Higher MUFA/SFA ratio was associated with better sperm quality parameters.     

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。     

Possible factors influencing post‐ejaculatory changes of the osmolality of human semen in vitro

The human ejaculate is made up of secretions from the different accessory sex glands that empty in sequence at ejaculation. However, the different secretions only mix completely in vitro when the entire ejaculate is collected in a container and handled in the laboratory. At ejaculation, proteins from the seminal vesicles form a gel in the ejaculate and semen cannot be properly analysed and processed until the gel is liquefied. During and after liquefaction, there is continuous enzymatic activities and an ongoing increase in osmolality. The aim of this study was to investigate possible factors that influence the increase in semen osmolality in vitro. Osmolality was measured by freezing‐point depression. Prostatic secretion was measured as zinc concentration. The presence of spermatozoa neither influenced the actual measurement of semen osmolality, nor the increase in osmolality. Enzymatic inhibitors reduced the increase in osmolality, and semen dilution prevented any increase in semen osmolality. However, the increase in osmolality covaried with the seminal zinc concentration, indicating that the observed increase was related to factors of prostatic origin. A simple and convenient procedure to reduce the risk for osmotic challenges for spermatozoa during handling for assisted reproductive technologies might be early dilution of semen.     

Utilisation of sperm‐binding assay combined with computer‐assisted sperm analysis to evaluate frozen‐thawed bull semen

Due to homologies between the chicken egg perivitelline membrane with mammalian zona pellucida proteins, spermatozoa of several species are able to bind to this membrane. However, adequate standardisation is required to attest possible applications of this technique for semen evaluation of a given species. Therefore, we thawed and divided cryopreserved semen samples into two aliquotes, one kept in water bath at 37 °C (thawed) and the other submitted to snap‐freezing to damage sperm cells (dead spermatozoa). Aliquotes were mixed into different ratios of thawed:dead cells and analysed for motility, membrane and acrosomal integrity, and mitochondrial activity. In parallel, chicken egg perivitelline membranes were inseminated with these ratios, and the number of spermatozoa bound per mm² of membrane was assessed by conventional microscopy (CM) and computer‐assisted sperm analysis (CASA). Linear regression showed high correlation between thawed:dead sperm ratio and number of spermatozoa bound to the membrane (CM: r² = 0.91 and CASA: r² = 0.92 respectively). Additionally, positive correlations were found between the number of spermatozoa bound to the membrane and acrosomal integrity, membrane integrity, mitochondrial activity and motility. These findings indicate that sperm‐egg‐binding assay associated with CASA is a reliable, practical and inexpensive method for examining the fertilising capacity of cryopreserved bull semen.     

Computer-aided semen analysis: sperm concentration assessment by the Strömberg-Mika system

Summary.  The scope of this study was to evaluate the accuracy, precision and specificity of the sperm concentration measurements by the Strömberg-Mika Cell Motion Analyser (SM-CMA). Our data show that the instrument generally underscores the sperm concentration and therefore the uncorrected measurements must be corrected by the operator using the 'mouse'-driven option. In terms of precision, the system appears to have an excellent internal precision whereas its repeatability is influenced by the sperm concentration, the sample's homogeneity and the correction of the raw data. In order to increase the system's repeatability, we suggest that sperm counts should be carried out in various fields of the counting chamber, and the mean of the corrected values be taken as representative of the sperm concentration in the ejaculate if the various measurements show a homogenous (poissonian) distribution. The correction of the raw data with the 'mouse'-driven correction option was also shown to improve the system's reproducibility. Concerning specificity, our data evidenced that, without technical correction, the instrument failed to correctly classify certain spermatozoa as such, thereby grossly underscoring sperm counts. This finding was more evident at low sperm counts. Overall, the SM-CMA requires additional laboratory time but the corrected sperm counts are comparable to manual counts and semi-automated counts with the added option that it provides the andrologists with various motility characteristics not possible with the latter methodologies.     

全自动精子分析中各运动性参数间相关性研究

目的　考察全自动精子分析中各运动性参数间的相关性。方法　进行了 190份健康受试者精液全自动分析 ,并对其各运动性参数 :直线速度 (vsl)、曲线速度 (vcl)、平均路径速度 (vap)、直线性 (lin)、前向性 (str)、摆动性 (wob)、侧摆幅度 (alh)、平均移动角度 (mad)、鞭打频率 (bcf)、精子密度、精子活动率间进行相关性分析。结果　从相关性分析中可确定运动性参数具有代表意义的是vsl、str、mad。结论　全自动精子分析的众多参数中 ,可通过vsl、str、mad三参数了解精子活动性 ,从而达到简便、快速、准确诊断的目的     

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.     

Improvements in semen parameters in men treated with clomiphene citrate—A retrospective analysis

Clomiphene citrate (CC) is commonly used off‐label for the treatment of male infertility, yet there is limited data to guide patient selection. To identify a subset of patients more likely to benefit from CC, we aimed to define predictors of improvement in semen parameters among men receiving CC. We retrospectively analysed 151 men treated with at least 25 mg CC daily for male infertility and/or hypogonadism at two institutions between 2004 and 2014. Men previously on testosterone were excluded. The primary outcome was change in semen parameters. Variables included baseline patient characteristics, pre‐treatment hormone profiles and pre‐treatment semen analyses. A total of 77 men met inclusion criteria. Median length of therapy was 2.8 months. There was significant improvement in sperm concentration (14–21 million/ml; p = 0.002) and total motile count (TMC; 13–28 million; p = 0.04). One third of patients who began with fewer than 5 million motile spermatozoon improved to a TMC > 5 million, increasing reproductive options to include intrauterine insemination. Patient characteristics, pre‐treatment hormone profile and degree of oligozoospermia did not predict treatment response. While no predictors of improvement were identified, clinically useful response rates are described for use in shared decision‐making.