精索静脉曲张患者精液质量及其不育患者手术前后精液变化的观察

目的观察精索静脉曲张（VC）患者的精液质量和精子形态学改变，以及VC不育患者手术前后精液的变化。方法121例VC患者精液按WHO标准常规分析并对精子形态学进行评价，23例健康男性精液检查结果作为对照。并对21例VC不育患者术前及术后的精液进行检测分析。结果121例VC患者的精子密度、（A＋B）级活动力精子（％）、活率、有效精子数、活动精子数、活力指数以及正常形态精子比例较对照组明显降低（P〈0．01）；畸形精子中小头、锥形头和无定形头精子数较对照组增多（P〈0．01）。21例VC不育患者手术后精子质量和精子形态学较术前明显改善。结论VC可以引起精液质量下降导致不育，精子形态学分析是判定VC患者精子受损的一个敏感指标，手术能有效地改善精液质量。     

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.     

《世界卫生组织人类精液分析实验室技术手册》与我国男科实验室现状

2010年出版的《世界卫生组织人类精液分析实验室技术手册》第5版是最为全面的一次修订。本文结合我国男科实验室精液分析的现状,从包括精子计数、精子活力、精子形态学、精子功能、抗精子抗体和精浆生化、以及精液分析的质量保证与质量控制等7个方面,对新出版的WHO手册进行了评论。笔者认为,该手册对精子浓度分析方法和参考值下调的推荐缺乏循证医学依据;精子活力分级标准的修订和精子形态学的严格标准和较低的4%的正常参考值与我国男科实验室目前使用的标准差距较大;精子功能指标尚未完善;抗精子抗体和精浆生化指标的检测方法显然不适合我国男科实验室现状;但精液分析的质量保证和质量控制对我国男科实验室有重要指导作用。需要指出的是,WHO手册中并没有涉及来自占世界人口1/5的中国人的任何临床数据和资料。因此,尽管WHO手册非常重要,它能否适应中国人精液分析实验室参考,仍需要评估。     

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。     

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.     

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.     

精液分析的外部质量控制研究

目的在严格遵循WHO第5版精液分析标准化方法培训的基础上,率先在国内男科学实验室尝试开展外部质量控制,探索精液分析外部质量控制的方法和模式,为在全国范围内开展精液分析的外部质量控制打下基础。方法本实验室根据《世界卫生组织人类精液检查与处理实验室手册》第5版（WH05）推荐的标准化精液分析方法,对来自全国23个实验室的技术人员进行理论与实际操作培训。各实验室技术人员带回质控样本,包括冷冻的混合精液、伊红一苯胺黑染色并封片的玻片、未经染色固定的形态学涂片,分别作为精子浓度、精子存活率和精子形态学评估的质控品。各实验室按要求采用血球计数板方法评估精子浓度;×100倍油镜评估精子存活率;巴氏染色法染色质控片并用×100倍油镜评估精子正常形态率;在规定日期内反馈结果。用WH05推荐方法对外部质控结果进行评估。结果于截止日期前21个实验室反馈了结果。对2个质控品进行精子浓度评估,各实验室间的变异系数（CV）分别为28．54％和27．11％。Youden图分析,15％实验室的2个质控品的检测结果均在95％可信区间内。对2个质控品进行精子存活率评估,各实验室问的CV分别为7．90％和9．59％。Youden图分析,42％的实验室2个质控品检测结果均在95％可信区间内。对2个质控品进行精子正常形态率评估,各实验室间的CV为52．96％和48．66％。Youden图分析,14％的实验室2个质控品检测结果均在95％可信区间内。结论结果表明,严格遵循WH05精液分析标准化方法培训后,除精子存活率评估各实验室间的CV较低、在控的实验室较多外,精子浓度评估和形态学分析CV、均较高、失控的实验室较多。精液分析的质量亟需提高、标准化培训亟待普及和推广,质量控制迫在眉睫。     

Ejakulatdiagnostik

Semen analysis plays a key role in the diagnostics of male infertility. Semen analysis has to be performed according to World Health Organisation (WHO) criteria. The updated version of the WHO manual was completed at the end of 2009 and published in 2010. Standard procedures in semen analysis include evaluation of sperm concentration, motility, morphology and vitality. In this new version particular attention has been paid to internal and external quality control, helping to identify and correct incidental and systematic errors both in routine analysis as well as in the field of research. The new manual describes all laboratory solutions, procedures and calculation formulas, and focuses on the definition of cryptozoospermia or azoospermia. A chapter concerning cryopreservation of spermatozoa has been newly integrated. The following overview presents the most important aspects of the updated WHO manual.     

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.     

Papanicolaou and Kruger assessment of sperm morphology: thresholds and agreement

The current World Health Organization guidelines (1992) suggest that the presence of ≥30% normal sperm forms (i.e. PAP criteria) is consistent with normal semen quality. Critical evaluation of sperm morphology (CE; Kruger classification) has shown an excellent correlation with human in vitro fertilization. Utilizing Kruger criteria, >14% normal sperm forms has been proposed as indicative of normal semen quality. We have performed a retrospective analysis on 261 individuals to assess the agreement between PAP and Kruger criteria for normal sperm morphology (NSM). When the threshold for NSM by PAP was set at 30%, a significant agreement was found between the percentage normal forms of both criteria (Kappa coefficient = 0.37; p  < 0.001). Sixty-seven (92%) of the 73 men found to have abnormal sperm morphology by PAP had abnormal semen by Kruger classification. When the threshold for NSM by PAP was established at 50%, the Kappa coefficient was 0.48 ( p  < 0.001). Sixty of the 72 samples (83%) classified as normal by PAP staining were normal by Kruger criteria. Interestingly, when NSM by PAP was between 30 and 50%, the specimen was just as likely to have normal or abnormal sperm morphology by Kruger (40 vs. 60%, respectively). These results strongly suggest that a high or low percentage of NSM by PAP is in agreement with the Kruger classification. The excellent agreement of Kruger and WHO criteria at the extremes (< 30% and >50%) may obviate the need for Kruger assessment. However, when WHO morphology is between 30 and 50%, the addition of Kruger evaluation may provide meaningful information to help better diagnose a patient and plan his treatment.     

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。     

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.     

Effects of microsurgical varicocelectomy on semen analysis and sperm function tests in patients with different grades of varicocele: Role of sperm functional tests in evaluation of treatments outcome

It seems that varicocele play a role in male infertility, as such, their prevalence increases from 15% in the normal population to 80% in secondary infertility subjects. Varicoceles may have negative effects on semen quality. Our goal was to assess the effects of microsurgical varicocelectomy on semen analysis and sperm functional tests in men with different grades of varicoceles. Thirty infertile men with different grades of varicoceles (grades 1 to 3) were enrolled in our study. Semen quality was assessed by semen analysis according to the WHO guideline (WHO, 1999) and four different sperm functional tests (aniline blue, toluidine blue, chromomycin A3 and TUNEL test) were carried out before and 3 months after microsurgical varicocelectomy (M‐varicocelectomy). When considered all three grades together, we showed that M‐varicocelectomy had statistically significant effects on all four types of sperm functional tests (p value<0.05). It also had positive effects on conventional semen parameters, although the effects were not statistically significant for some parameters (for example sperm count). When analysed separately (based on varicocele grades) the surgery, although caused improvements in semen quality, but may have more statistically significant effects on patients with varicocele of higher grade. In addition, in varicocele of lower grade (for example grade 2), sperm function test may be a better predictor of surgical success than the conventional semen analysis. Thus, we show that not only M‐varicocelectomy has significant positive effect on semen quality but also if sperm functional tests become more affordable in the future, because they yield more precise results, their use in daily practice may increase significantly in patients with varicoceles.     

因子分析方法在精液质量综合评价中的应用

目的:探讨因子分析方法在精液质量综合评价中的应用。方法:运用因子分析方法对正常生育力男性精液检测指标(精液量、精子浓度、精子总数、活动率、前向运动精子百分率、存活率和正常形态精子百分率、前向运动精子总数、有效前向运动精子总数、畸形精子指数(TZI)、精子畸形指数(SDI)、曲线速率(VCL)、直线速率(VSL)、平均路径速率(VAP)、精子头侧摆幅度(ALH)、直线性(LIN)、摆动性(WOB)、前向性(STR)、鞭打频率(BCF)和平均角位移(MAD)进行了分析,提取反映精液质量特征的公因子,综合评价精液质量。结果:依据累积方差贡献率达到85%的原则,共萃取6个特征值>0.8的公因子,累积方差贡献率为86.647%。6个公因子依次分别代表精子数量、运动速度、运动方向、畸形指数、精液量以及正常形态。因子综合得分的计算公式为:(36.243*F1+23.342*F2+8.910*F3+8.500*F4+5.538*F5+4.115*F6)/86.647,其中位数为-0.0180。以P5为界值,正常生育力男性各精液检测指标均达到WHO参考值范围的比例仅为79.2%;而因子综合得分达到界值的对象比例为95.1%,两者差异有统计学意义(P=0.000)。结论:在综合评价精液质量时,因子分析结果即因子综合得分指标对精液质量的判定更精确。     

Tests to measure the quality of spermatozoa at spermiation

This commentary is to critique the revised World Health Organization （WHO） semen analysis manual as it pertains to characteristics of a spermatozoon at spermiation. The aims of the revised WHO manual include improving the ＇quality of semen analysis＇ without any restriction to clinical use. Furthermore, the manual states that semen analysis may be useful for （a） ＇investigating male fertility status＇ and （b） ＇monitoring spermatogenesis during and following male fertility regula- tion.＇ However, if the analysis of ejaculated spermatozoa is intended for the purposes described in （b）, then cells that are abnormal at spermiation must be identified. This paper takes the position that the manual does not identify methods to estimate the quality of spermatozoa at spermiation. Instead, it uses a ＇gold standard＇ of sperm passing through the cervical mucus or arriving near the site of fertilization. Although this standard is appropriate for drawing conclusions regarding the probability that an individual could impregnate his partner, it is not appropriate for studying illness of the testes per se. Herein, the measures of sperm quality presented in the WHO manual are critiqued with respect to the detection of spermatozoa that were abnormal at spermiation vs. those that became abnormal subsequently. Quality assessments based on the percentage of motile or ＇viable＇ spermatozoa are meaningless. Alternative quality attributes defining spermatozoa at spermiation are presented in this paper. In conclusion, assessment of spermatozoal quality at spermiation, on the basis of quality attributes of individual ejaculated spermatozoa, is best achieved through application of （a） a new paradigm for the morphological evaluation of sperm quality and （b） modern analytical techniques to evaluate, in an adequate sample, several appropriate independent attributes in each spermatozoon in order to more accurately identify the proportion of abnormal spermatozoa.     

New WHO-reference limits--revolution or storm n a teapot？

Since release of the latest WHO manual with the new lower reference values of semen parameters, a lot of discussion has been raised about their usefulness and appropriateness for assessment of male fertility. As with the previous reference values the new limits do neither allow an andrological diagnosis based on nosological criteria nor clear-cut differentiation between fertility and sub-/infertility. Therefore, considering the fact that fertility is a continuum, the new lower reference limits should not be overestimated. Most probably, more sperm function tests, such as determination of DNA integrity, and-in the future-assessment of biomarkers, such as sperm proteomics will be included into andrological work-up, thus resulting in a more personalized approach of infertility management. On the other hand, the detailed instructions for standard and advanced semen analysis provided in the new manual are very much appreciated and should be adopted by each seriously working laboratory.     

精子功能检测与男性不育诊治的新进展

传统的精液常规分析是用于判断男性生育力的最基本临床指标,但是,只依靠精液分析的结果来预测男性生育状况仍是很不准确的。精子功能正常与否,对临床选择IVF还是ICSI治疗不育症极为重要。因为IVF需要功能完全正常的精子才能受精,而ICSI的受精只需要精子的正常核DNA,不需要其它的精子功能。在发明ICSI以前,患者IVF受精失败或低下(<30%)发生率很高(20%~35%)。研究证明,这些IVF受精失败的患者主要与精子功能障碍有关。常见的是少精子症,弱精子症和畸形精子症。但是有很多患者,精液分析结果仍正常。为了提高临床对精子功能测定的准确性,文献里有很多新的精子功能试验的研究报导,比如丫啶橙(AO)测定精子DNA、精子与透明带结合和穿透、顶体诱发精子顶体反应和精子与透明质酸结合试验。精子形态测定是常规精液分析中最重要的临床指标之一。但精子形态又是最难测定准确和稳定。IVF/ICSI受精失败的人卵可以用来测定精子功能。人卵透明带选择性地与正常形态和顶体完整的精子结合,透明带诱发的顶体反应与精子穿透明带的能力有很强的相关性。在不明原因的男性不育患者中,由于透明带诱发顶体反应障碍所导致的不育症占25%左右。少精子症(精子计数<2×106/ml)和严重精子形态畸形症(严格正常形态<5%)的男性不育患者,精子-透明带结合反应缺陷的发生率很高(>70%)。这类患者用IVF治疗受精率会很低,因此只能用ICSI治疗。精子与透明质酸结合试验与精子活力和形态有很强的相关性,但它不是很有用的精子功能试验。AO测定精子DNA对预测ART的受精和妊娠率的临床意义目前还没有肯定的结论,需要进一步研究。总之,在常规精液分析时,增加一些新的精子功能试验,在临床ART中对男性不育患者的诊治会有很大的帮助。     

Bias to routine semen analysis by uncontrolled changes in laboratory environment—detection by long-term sampling of monthly means for quality control

To detect systematic bias in the results of routine semen analysis over time, monthly means of semen parameters determined by the recommended WHO methods were computed. The analysis was based on a total sample size of 1784 ejaculates and included 18 months of observation. In addition to slight changes of morphology estimates caused by a change of laboratory staff, a major bias in the measurement of sperm motility could be detected. This observation triggered a search for changes in protocols not previously given the required attention. It revealed that the newly introduced use of polypropylene syringes with a mounted needle for accurate measurement of seminal volume impaired sperm motility. More detailed investigation by computerized sperm motion analysis in 10 semen samples treated simultaneously in different ways revealed that predominantly it was the needle which caused the drop in proportion of motile sperm (glass cylinder: 50.3 +/- 4.1% vs. syringe + needle: 26.6 +/- 5.3%; mean +/- SEM) and not the contact with the plastic material alone (syringe alone: 43.4 +/- 4.8%). Other motion parameters such as curvilinear velocity (36.0 +/- 1.6 microns/sec), linearity (78.5 +/- 8.4%) and lateral head displacement (3.8 +/- 0.9) were not influenced by the different methods of handling. The results indicate that long-term sampling of monthly means may serve as part of a quality control scheme in semen analysis.     

Clinical correlates of the biological variation of sperm DNA fragmentation in infertile men attending an andrology outpatient clinic

Determination of sperm DNA fragmentation, as assessed by the sperm chromatin structure assay (SCSA), has become an important tool for the evaluation of semen quality. The aim of the present study was to describe the biological variation of sperm DNA fragmentation in men attending an andrology clinic and to identify clinical correlates of the biological variation of sperm DNA fragmentation. For this study, two consecutive semen samples from 100 patients attending our andrology outpatient clinic were subjected to semen analysis, performed in parallel according to WHO guidelines and by SCSA. A good agreement between pairs of samples was found for SCSA-derived variables, as indicated by a significantly lower median coefficient of variation (CV) of the DNA Fragmentation Index (DFI) and the high DNA stainability (HDS) compared with WHO semen parameters. In half of the men attending our andrology clinic, however, the individual biological variation of DFI and HDS, expressed as CV of two samples, exceeded 10%. Dysregulation of spermatogenesis, as seen as testicular insufficiency or varicocele, was not associated with increased variability of DFI or HDS. A backward multiple linear regression analysis, however, indicated that the biological variation of DFI may be more profound in men with characteristics of normal spermatogenesis. In conclusion, we confirm previous reports that sperm DNA fragmentation has a lower biological variability than classical semen parameters. We hypothesize that the sperm chromatin structure may be more influenced in patients with normal spermatogenesis, whereas in men with disturbed spermatogenesis, the chromatin structure may be already so impaired that the effect of unidentified factors leading to variability of sperm DNA fragmentation in time may not be as profound.     

Kruger strict morphology and post-thaw progressive motility in cryopreserved human spermatozoa

The purpose of this prospective study was to evaluate Kruger strict morphology and conventional semen analysis in predicting cryosurvival and the progressive motility recovery rate of frozen spermatozoa. Our study included 56 semen samples with >10 million spermatozoa per ejaculate. The main outcome measures were conventional semen analysis, strict morphology analysis by the Kruger method, cryosurvival rate and post-thaw sperm motility. A significant reduction in sperm motility after cryopreservation was demonstrated. The freeze-thawing process caused a 66% reduction in rapid progressive motile spermatozoa, a 45% reduction in slow progressive motile spermatozoa and a 2% reduction in nonprogressive motile spermatozoa. The cryosurvival and progressive motility recovery rates were not correlated with parameters of conventional semen analysis, such as sperm concentration, motility, WHO morphology and total motile count, but the progressive motility recovery rate was significantly correlated with the percentage of spermatozoa exhibiting Kruger normal morphology (P = 0.028). The recovery rate of rapidly progressive motility was profoundly decreased compared with slow progressive motility following the frozen-thaw procedure of semen. Kruger strict morphology assessment was a better predictor of the progressive motility recovery rate following the freezing-thaw procedure than parameters of conventional semen analysis.