Assessing human sperm morphology： top models,underdogs or biometrics？

The assessment of the percentage of spermatozoa having an ＇ideal＇ morphology using so-called strict method is the method recommended in the latest edition of the World Health Organization （WHO） laboratory manual for semen analysis. This recommendation is a result of the statistical association between ＇ideal＇ sperm morphology and fertility, and of the current general belief that sperm morphology assessment should be used primarily as a fertility tool. The notion of an ＇ideal＇ sperm morphology has persisted despite the very low percentage of such spermatozoa in the semen of fertile men, a subject of intense controversy. The detailed categorization of each abnormal spermatozoon has thus, for a long time, been considered optional and partially redundant, an idea which is reflected in the earlier editions of the WHO manual. However, several recent studies have shown the importance of carefully assessing abnormal sperm morphology for use in the diagnosis ＆infertility, to determine fertility prognosis, and for basic or public health studies. One approach, which combines videomicroscopy and computer vision, and is the only approach able to assess the continuum of sperm biometrics, has been used successfully in several recent clinical, basic and toxicology studies. In summary, the visual assessment of detailed sperm morphology--including the categorization of anomalies allowing arithmetically derived indices of teratozoospermia--and the more modern computer-based approaches, although often considered to be redundant, are in fact complementary. The choice of the most appropriate method depends on the field of investigation （clinical, research, toxicology） and the problem being addressed. Each approach has advantages as well as certain limitations, which will be discussed briefly herein.     

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.     

2343例男科就诊患者精子动态参数分析

目的探讨精子动态参数在评价男性生育能力方面的应用价值。方法严格按照WHO技术规范，用计算机辅助精液分析（CASA）系统对本院2343例男科门诊就诊的患者精液进行常规指标和精子动态参数分析。结果①正常精液组和异常精液组各项精子动态参数比较，均存在统计学差异；②随着精子存活率的下降，曲线速度（VCL）、直线速度（VSL）、平均路径速度（VAP）、平均移动角度（MAD）、侧摆幅度（ALH）、前向性（STR）下降明显，鞭打频率（BCF）呈上升趋势，而直线性（LIN）、摆动性（WOB）差异无显著性；③与密度活力正常组比较：少精组除LIN、STR有所增加，MAD、BCF有所下降，差别有统计学意义外，其余五项参数差别无统计学意义；弱精组除MAD无统计学差异外，其余八项参数均有统计学差异；少弱精组除MAD和ALH无统计学差异外，其余七项参数均有统计学差异。结论精子动态参数是评估精液质量的重要指标，为男性不育的诊断、治疗和研究提供了充分的依据。     

DNA integrity in human spermatozoa: relationships with semen quality

The literature contains conflicting evidence regarding the existence of DNA damage in spermatozoa from infertile male patients. To examine this phenomenon, we have studied ejaculated spermatozoa from normozoospermic semen donors and from a group of the unselected male partners of couples attending an infertility clinic for initial investigation. Classical semen analysis according to World Health Organization (WHO) guidelines was undertaken with computer-assisted sperm analysis (CASA). Spermatozoa were prepared by sequential washing and centrifugation and were analyzed for DNA fragmentation using three assays: 1) a single-cell gel electrophoresis (comet) assay, 2) in situ nick translation with prior chemical decondensation (ISNT-decondensed), and 3) in situ nick translation without prior chemical decondensation (ISNT-condensed). In addition, reactive oxygen species (ROS) generation by spermatozoa was measured, and seminal plasma was analyzed for its total reactive antioxidant potential (TRAP). When the donor and patient groups were compared, the latter had lower levels of semen quality and higher levels of DNA damage, which was particularly apparent using the comet assay. Highly significant negative correlations were observed between DNA fragmentation, detected by all three assays, and semen quality, particularly sperm concentration. In addition, multiple regression analysis indicated that other attributes of semen quality, such as sperm movement and ROS generation, were also related to DNA damage. We conclude that a significant proportion of infertile men have elevated levels of DNA damage in their ejaculated spermatozoa.     

精索静脉曲张不育患者的精液质量和精子形态学观察

目的:观察不育伴精索静脉曲张(VC)患者的精液质量和精子形态学变化。方法:98例不育伴VC患者精液按WHO标准常规分析并对精子形态学进行评价。130例正常供精者精液检测结果作为对照。结果:VC患者正常形态精子和前向运动精子明显低于对照组(P<0.001),精子畸形的类型以梨形、锥形和不定型头部畸形为主。结论:VC可导致精子畸形率升高,后者可能是男性生育力受损的重要标志之一,经染色后的精子形态学分析是判定VC患者精子受损的一个敏感指标。     

连续射精对正常男性精液分析参数的影响

目的 :探讨连续射精对正常男性精液分析参数的影响。　方法 :8名正常男性逐日手淫取精 ,共 8d。采用世界卫生组织人类精液检验手册方法测定精液体积、pH值、精子密度、总精子数、存活率和活动率 ,进行统计学分析。　结果 :精液体积、总精子数逐日下降 ,从第 5d起至第 8d非常显著低于第 1d(P <0 .0 1)。精子密度亦呈逐渐下降趋势 ,但无显著性差异。精子存活率和精子活动率 (a +b级 )均呈逐渐上升趋势 ,但是仅第 7d显著高于第1d(P <0 .0 5 )。　结论 :人类精液体积、总精子数随射精频度增加而显著降低 ,精子存活率和活动率增加。频繁射精并不影响正常男性精液质量。     

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.     

精液常规参数初检合格后不同时间点再分析结果及其与精子DNA损伤的关系

目的:探讨完全液化且常规参数初检合格的精液标本,于不同时间再分析的结果差异,及精子DNA碎片化指数(DFI)与精子活动力改变的相关性。方法:选取127份符合纳入标准的精液标本,分别于取样后15、30、60 min时采用计算机辅助精液分析(CASA)系统进行分析。精子形态分析采用Shorr染色法,吖啶橙试验(AOT)检测DFI。结果:3个时间点精子浓度、a级和b级精子百分率均无统计学差异(P>0.05)。取样15 min时a+b和a+b+c级精子百分率显著高于30和60 min时的结果(P<0.05),后两者间无统计学差异(P>0.05)。不同时间精子活动力各项指标中,至少有1项由"正常组"变为"异常组"的发生率为25.2%,两组间DFI和形态学无统计学差异(P>0.05)。取样后15到60 min变化的精子活动力指标中,a、a+b、a+b+c级下降值与DFI存在正相关性(P<0.05)。结论:取样后15 min内完全液化并初查参数合格的精液标本,30～60 min内复查时,a级和b级精子百分率波动并无显著差异,而a+b级及a+b+c级精子则可能显著下降,精子活动力指标可能出现异常。故应至少进行2次精液分析,综合评估生育力。如2次结果差异较大,尤其是a级精子百分率下降幅度较大,则可能与精子DNA损伤有关,需进一步行精子DNA损伤检测。     

512例供精志愿者初次精液筛查结果分析

目的 :了解供精志愿者的精液质量状况。　方法 :用WHO推荐的标准化方法和计算机辅助精子分析(CASA)系统检查 5 12例供精志愿者首次精液样本的参数。　结果 :4 8% (2 4 6例 )具有潜在的生育能力 ,但其中只有2 8.5 % (14 6例 )达到卫生部供精质量标准 ,高达 5 2 % (2 6 6例 )未达到WHO的正常人参考值水平。　结论 :对未达到供精质量标准的志愿者 ,做纵向的精液分析追踪能提高精液合格率。供精人群的精液质量状况提示关注男性生殖健康是必要的。     

外周致密纤维1在人精子中的表达差异及其意义

目的:比较外周致密纤维1(ODF1)在健康男性和弱精子症患者精子中的表达差异。方法:根据WHO标准,收集正常男性和弱精子症患者的精液标本各20份,Percoll非连续梯度离心法分离精子,收集95%Percoll以下和57%与76%Percoll层之间的精子,以排除生精细胞和白细胞的污染;采用逆转录-多聚酶链式反应(RT-PCR)和Western印迹方法,从mRNA和蛋白水平检测ODF1的表达。结果:RT-PCR结果表明,与正常男性组相比,ODF1在弱精子症患者精子中的mRNA表达水平显著降低(2.79±0.28vs1.35±0.25,P<0.05);免疫印迹与RT-PCR结果一致,与正常男性组相比,弱精子症患者精子中的ODF1蛋白的表达亦显著降低(3.64±0.34vs1.44±0.26,P<0.05)。结论:ODF1在弱精子症患者精子中的表达显著降低,提示其可能与精子活动力低下有关。     

Apoptosis and kinematics of ejaculated spermatozoa in patients with varicocele

Increased DNA fragmentation is found in sperm from infertile men. Varicocele is an important cause of male infertility, even though it is present in 15% of men who father children. Semen analysis does not always identify infertility in these patients. Sperm motility is strongly correlated with male fertility potential. The goal of this study was to determine the correlation between apoptosis and kinematics in the ejaculated spermatozoa of patients affected by varicocele. Fresh semen samples were obtained from 30 patients with varicocele and 15 fertile controls. These samples were compared using computer-assisted semen analysis and were assayed to determine the degree of sperm apoptosis. The apoptotic index (AI) was calculated by dividing the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine-5'-triphosphate nick end labeling (TUNEL) stained spermatozoa by the total number of Hoechst 33258-stained sperm cells for 300 sperm. Five microscopic fields were analyzed to obtain 5 AIs for each individual. Results demonstrated no significant difference in semen quality and sperm motion characteristics; however, a significantly higher AI (23.05% +/- 4.07%: mean difference +/- SE, 95% CI, 15.06%-31.03%, P <.0001) was identified in the varicocele group than in the fertile controls. We concluded that sperm apoptosis does not seem to correlate with semen quality and sperm kinematics and that apoptosis is increased in ejaculated spermatozoa in patients with varicocele compared to normal fertile men.     

男性不育患者精液质量与精子顶体酶活性关系分析

目的:分析男性不育患者精液参数与精子顶体酶活性变化,探讨精液质量与顶体酶活性的关系。方法:对214例男性不育患者的精液作精子顶体酶活性、弹性蛋白酶、果糖、α葡糖苷酶、锌、酸性磷酸酶、精子尾部低渗肿胀试验检测,同时精子质量检测仪作精液分析。以检出精子顶体酶活性正常的(48.2~218.7μIU/106精子)111例作为对照组,与顶体酶活性异常(<48.2μIU/106精子)的103例精液参数作对比分析。结果:两组精液参数的精子密度、精子活动率、a+b级精子百分率、精子尾部低渗肿胀试验差异均有非常显著性(P<0.001)。弹性蛋白酶差异有显著性(P<0.05)。果糖、α葡糖苷酶差异亦有显著性(P<0.05)。精液量、锌、酸性磷酸酶差异无显著性(P>0.05)。结论:精子顶体酶活性与精液质量关系密切,精子顶体酶活性是反映精液质量的一项可靠指标。     

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.     

Relationship between morphological abnormalities in commercial bull frozen semen doses and conception rate

Commercial doses of frozen bull semen for artificial insemination may have a certain percentage of morphological defects, despite being subject to prior selection. The aims of this study were to determine the prevalence of morphological abnormalities in commercial doses (n = 55, r = 2) of dairy and beef bulls, from AI Centers and to determine the possible existence of differences between them, regarding the percentage of abnormal spermatozoa. At least 200 spermatozoa per sample were evaluated using Bengal Rose stain (3% m/v) and light microscopy (×1000 magnification). The mean percentage of abnormal sperm samples from dairy breeds was 7.19% ± 4.91% and from beef breeds was 15.83% ± 9.28%. Significant differences between biotypes were found in the proportion of abnormal spermatozoa, abnormal heads and abnormal midpieces; it could be due to different selection pressure. It was observed that the percentage of abnormal spermatozoa was not a good fertility level predictor for the commercial samples of frozen bovine semen used in this study. In both biotypes, the midpiece abnormalities were the most frequent, mainly its distal flexion (compensable defect). This could be as a result of the effects of freezing and thawing on spermatozoa.     

18～35岁男性军人精液质量调查

目的:了解部队男性官兵精液质量现况。方法:采用手工方法与计算机辅助精液分析对某部1054例基层官兵进行了精液参数10项分析,并根据年龄分为18～20岁,21～25岁,26～30岁和31～35岁4组,分析并比较各组精液参数。结果:1054例青壮年男性精液量为(2.6±1.4)ml、精子密度为(55.9±46.5)×106/ml、a+b级精子(47.1±19.0)%、精子存活率为(70.6±22.1)%、正常形态精子率为(84.7±10.2)%、顶体完整率为(86.1±7.2)%。18～20岁组精液量少、粘度大、pH值偏碱性,与其他3组相比,差异有显著性(P<0.05)。精液质量指标符合WHO标准的百分率为精液量73.5%、液化时间91.1%、pH值93.0%、a+b级精子45.5%、精子存活率86.7%、精子密度80.4%、正常形态精子率为98.2%、精子总数为78.0%,上述各项指标都符合WHO标准的对象仅占40.2%。结论:18～35岁部队基层官兵男性精液质量现况好于国内同类文献报道。在不同年龄分组研究中26～30岁年龄组人员精液质量好于其他年龄组。     

Dimensions of human ejaculated spermatozoa in Papanicolaou- stained seminal and swim-up smears obtained from the Integrated Semen Analysis System （ISAS）

Objective measurements are required for computer-aided sperm morphometric analysis （CASMA） machines to distinguish normal from abnormal sperm heads. The morphometric characteristics of spermatozoa in 72 samples of semen and of spermatozoa from 72 other semen samples after swim-up were quantified by the semi-automated Integrated Sperm Analysis System （ISAS） computer-aided system, which measured the sperm head parameters length （L）, width （W）, area （A）, perimeter （P）, acrosomal area （Ac）, and the derived values L/W and P/A. For each man a homogeneous population of distributions characterized seminal spermatozoa （7 942 cells： median values L 4.4 μm, W 2.8μm A 9.8 μm^2, P 12.5 μm, Ac 47.5%, L/W 1.57, P/A 1.27）, and there was no significant difference in within- and among-individual variation. Different men could have spermatozoa of significantly different dimensions. Head dimensions for swim-up spermatozoa from different men （4 812 cells） were similar to those in semen, differing only by 2%-5%. The values of L, Wand L/W fell within the limits given by the World Health Organization （WHO）. Although these samples were not biologically matched, linear mixed-effects statistical analyses permitted valid comparison of the groups. A subpopulation of 404 spermatozoa considered to fit the stringent criteria of WHO ‘normal＇ seminal spermatozoa from both semen and swim-up were characterized by median values （and 95% confidence intervals） of L, 4.3 μm （3.8-4.9）, W, 2.9 μm （2.6-3.3）, A, 10.2μm^2 （8.5-12.2）, P, 12.4μm （11.3-13.9）, Ac, 49% （36-60）, L/W, 1.49 （1.32-1.67） and P/A, 1.22 （1.11-1.35）. These median values fall within the 95th centile confidence limits given by WHO, but the confidence intervals for L and W were larger. Although these differences in head dimensions among men and after swim-up could be detected by CASMA, the small differences make it unlikely that technicians would be able to distinguish them. The values could be used as default sperm head values for the CASMA machine used here.     

Automated semen analysis by SQA Vision® versus the manual approach—A prospective double‐blind study

Next to clinical investigations, the evaluation of male fertility relies mainly on detailed sperm analyses, for example, cell counting, motility, cell morphology and vitality testing. The manual creation of a spermiogram is time‐ and material‐consuming. Therefore, reliable high‐throughput systems that may be substituted for manual methods are urgently needed. The present study aimed to compare conventional sperm analysis performed as per WHO 5th guidelines and semen analysis performed with the SQA Vision^® machine. SQA Vision^® is a commercial device for automated sperm analysis. Data obtained independently by both methods were compared by statistical analyses using Bland–Altman plots and Passing–Bablok regression analyses. The analyses revealed that the results for sperm concentration and total motility were comparable. The agreement for progressive motility was poor, and there were clear deviations in the determination of normal sperm morphology. Passing–Bablok regression analyses and the consideration of the 95% confidence intervals pointed out systematic and proportional differences between the manual semen analysis and the automated approach.     

Semen quality evaluation in a cohort of 28213 adult males from Sichuan area of south‐west China

The trends in semen quality are conflicting. Although many previous surveys on semen quality indicated a decline, the trends in semen quality in Sichuan area of south‐west China are not clear. We analysed the semen parameters in a cohort of 28 213 adult males close to general population in Sichuan between July 2007 and June 2012, and investigated the changes on semen quality. The semen parameters including pH, volume, concentration, motility, morphology were measured according to the World Health Organization (WHO) criteria. Kruskal–Wallis analysis of variance was used to examine the statistical differences of semen quality between groups. We found that the medians (5th and 95th percentiles) were 2.4 ml (1.0–5.0) for semen volume, 62.0 × 10⁶ ml^?1 (15.0–142.0) for semen concentration, 39% (18–60%) for sperm progressive motility and 10.5% (1.0–34.5%) for normal morphology. In these 5 years, sperm concentration and the percentage of sperm normal morphology were decreased from 66.0 × 10⁶ ml^?1 to 49.0 × 10⁶ ml^?1 and from 13.5% to 4.5%, respectively; among different reproductive history groups, sperm concentration and the percentage of sperm normal morphology were also decreased in these 5 years. And the incidence of azoospermia was increasing. These may imply that there is a decline in semen quality of adult males in Sichuan area.