Semen analysis in 21st century medicine： the need for sperm function testing

Sperm function testing, once commonly performed for the infertile couple before employing assisted reproductive technology （ART）, has fallen out of favour in many reproductive medicine centers throughout the world. Indeed, the most recent addition of the ＇World Health Organisation （WHO） Laboratory Manual for the Examination and Processing of Hu- man Semen＇ now groups many of these procedures into a section termed Research Procedures. In large part, this reflects the current clinical practice of bypassing the in-depth evaluation of the male partner, while assuming that if a spermatozoon can be found for intracytoplasmic sperm injection （ICSI）, it must be a healthy cell capable of achieving fertilization. Never- theless, sperm function testing can provide valuable clinical insights into defects causing male infertility. Admittedly, in some cases, functional sperm deficiencies can be overcome using an ART. In other cases, couples will be empowered by the knowledge of the cause of their infertility, and for some couples, perhaps even the likelihood of ICSI success （relative to the spermatozoa）. The knowledge allows them to make truly informed reproductive decisions, including （perhaps） the de- cision to seek donor insemination, to adopt or to remain childless. Knowledge of the cause of their infertility may provide closure for couples and a sense of confidence regarding their choice of reproductive treatment.

Sperm function tests after vasovasostomy

Aim: To evaluate the sperm function after vasovasostomy. Methods: Semen samples from 42 subjects aftervasovasostomy (Group A: 1-6 months, Group B: 6-12 months; Group C: 12-18 months after vasectomy rever-sal) were investigated. Semen from 34 normal fertile men was used as controls. Sperm function tests, including hypo-osmotic swelling test (HOST), acridine orange (AO) fluorescence, acrosome reaction (triple-stain), cervical mucuspenetration test (CMPT), etc were done. Results: After vasectomy reversal, the percentage of HOST was signifi-cantly lower than that of the normal fertile men. In regard to AO, there were no significant differences between thethree vasovasostomy groups and between these 3 groups and the controls. With triple-stain, the percentage of normalacrosome reaction was significantly lower in Group A as compared with the controls, but not in Groups B and C. Therewere no significant differences in the results of CMPT between the vasovasostomy groups and the controls. However,the number of 'poor' type was significantly higher in Groups A and C than in the controls; the percentage of 'nega-tive' type were higher in Groups A and B than in the controls. Conclusion: After vasovasostomy a lower level ofHOST remained for one year and gradually recovered after one year. Six months after vasectomy reversal, the percent-age of acrosome reaction could be changed from lower level to normal range. The data of AO indicated that the geneticmaterial (double-stranded DNA) in spermatozoa was not affected by vasovasostomy. To evaluate the result of CMPTafter vasectomy reversal, not only the normal results but also the abnormal results ('poor' and 'negative' types)should also be considered.Aim: To evaluate the sperm function after vasovasostomy. Methods: Semen samples from 42 subjects aftervasovasostomy (Group A: 1-6 months, Group B: 6-12 months; Group C: 12-18 months after vasectomy rever-sal) were investigated. Semen from 34 normal fertile men was used as controls. Sperm function tests, including hypo-osmotic swelling test (HOST), acridine orange (AO) fluorescence, acrosome reaction (triple-stain), cervical mucuspenetration test (CMPT), etc were done. Results: After vasectomy reversal, the percentage of HOST was signifi-cantly lower than that of the normal fertile men. In regard to AO, there were no significant differences between thethree vasovasostomy groups and between these 3 groups and the controls. With triple-stain, the percentage of normalacrosome reaction was significantly lower in Group A as compared with the controls, but not in Groups B and C. Therewere no significant differences in the results of CMPT between the vasovasostomy groups and the controls. However,the number of 'poor' type was significantly higher in Groups A and C than in the controls; the percentage of 'nega-tive' type were higher in Groups A and B than in the controls. Conclusion: After vasovasostomy a lower level ofHOST remained for one year and gradually recovered after one year. Six months after vasectomy reversal, the percent-age of acrosome reaction could be changed from lower level to normal range. The data of AO indicated that the geneticmaterial (double-stranded DNA) in spermatozoa was not affected by vasovasostomy. To evaluate the result of CMPTafter vasectomy reversal, not only the normal results but also the abnormal results ('poor' and 'negative' types)should also be considered.Aim: To evaluate the sperm function after vasovasostomy. Methods: Semen samples from 42 subjects aftervasovasostomy (Group A: 1-6 months, Group B: 6-12 months; Group C: 12-18 months after vasectomy rever-sal) were investigated. Semen from 34 normal fertile men was used as controls. Sperm function tests, including hypo-osmotic swelling test (HOST), acridine orange (AO) fluorescence, acrosome reaction (triple-stain), cervical mucuspenetration test (CMPT), etc were done. Results: After vasectomy reversal, the percentage of HOST was signifi-cantly lower than that of the normal fertile men. In regard to AO, there were no significant differences between thethree vasov

Relationship of Naturally Occurring Antisperm Antibodies in Blood Serum and Seminal Plasma of Cattle Bulls with Sperm Function and Fertility Tests

The study was planned with an objective to assess the level of antisperm antibodies (ASA) in the blood serum and seminal plasma of breeding cow bulls and their relationship with sperm function and fertility tests. ASA was analyzed in blood serum and seminal plasma by SpermMar test, Immuno peroxidase assay (IPA) and Enzyme linked immunoabsorbant assay (ELISA). In SpermMar test, about 54% bulls were with >40% IgG in blood serum against sperm surface antigens, whereas none of the bulls were with >10% IgG in seminal plasma. More than 20% and >10% IgA against sperm surface antigens were detected in the blood serum and seminal plasma of 65.8% and 37% bulls, respectively. Out of 26 bulls, seminal plasma of 21 bulls reacted with spermatozoa both in IPA and IgA latex particles and that of only 12 bulls reacted with IgG. In IPA, about 50% of the bulls had >40% ASA against head surface antigens, whereas, there were 23% bulls with >10% ASA in seminal plasma. Also ELISA indicated a higher antibody titre in blood serum (3200 - 6400) and seminal plasma (40 - 80) of 50% and 42% bulls, respectively. There were 11 bulls with low values of HOST/in vitro acrosome reaction/cervical mucus penetration assay and higher level of either serum or seminal plasma ASA. Our study revealed that a significant level of ASA in serum or seminal plasma may have effect on the fertility of bulls by affecting the sperm function.

丹参提取液在精液体外处理中的作用研究

在精液体外处理中 ,加入丹参提取液 ,能有效的增加精子的活力 ,提高精子质量。与对照组比较 ,经统计学处理P <0 0 1,有显著性差异。提示丹参提取液在精液体外处理中 ,能有效的增加精子的活力 ,提高精子质量。

精子功能检测对选择IVF或ICSI治疗不育症的临床意义

本文评价了精子功能检测对选择体外受精(IVF)或卵胞浆精子注射(ICSI)治疗不育症的临床意义。精子功能缺陷所导致的精子与透明带结合反应(即透明带结合、透明带诱发顶体反应和透明带穿透)异常是IVF受精失败的主要原因。在常规精液分析中,精子形态学测定对预测精子受精能力最有价值,但精子形态学评估则是最难做准确和稳定。IVF受精失败的卵子是很有用的生物材料,可用来测定精子形态和顶体功能。人卵透明带有选择性地与正常形态和完整顶体的精子相结合。透明带诱发顶体反应与精子穿透明带的能力呈显著正相关。在不明原因不育症患者中,约25%的不育原因可能是透明带诱发顶体反应缺陷或低下所引起。在严重畸形精子症(正常形态率≤5%)、少精子症的不育病人中,精子与透明带结合异常比率很高。因此,临床上应把这些有精子缺陷,尤其是会影响精子与透明带结合的患者检测出来,采用ICSI治疗,避免这些病人用IVF治疗而导致很低的受精率。精子形态学分析和精子与透明带结合试验有助于临床选择IVF或ICSI治疗不育患者。

Evaluation and assessment of semen for IVF/ICSI

Evaluation and assessment of semen is very important for both diagnosis of male infertility and selection of patients for treatment with IVF or ICSI. In standard IVF, sperm function is essential for normal fertilization: sperm must be able to bind to zona pellucida (ZP), undergo the acrosome reaction and penetrate the ZP and fuse with the oolemma before fertilization takes place. In contrast, most sperm functions are not required for fertilization in ICSI since sperm bypass the ZP and oolemma by injection of a single sperm directly into cytoplasm of oocyte. Therefore, the clinical decision on treatment of patients with either IVF or ICSI is mostly dependent on results of sperm tests. However, conventional semen analyses do not provide accurate information about sperm fertilizing ability since many patients with subtle sperm defects can not be detected. More advanced sperm function tests are required to detect sperm defects that may lead to failure of fertilization in standard IVF. In the last 15 years we performed extensive studies on relationship between sperm functions and fertilization rates by logistic regression analysis in large numbers of IVF patients including 370 patients with zero fertilization rate by IVF. We confirmed sperm morphology assessed strictly was strongly related to fertilisation rate with standard IVF. Thus sperm morphology assessment is very useful for selection of patients for ICSI. We also developed a number of new tests including sperm-ZP binding, sperm-ZP penetration and the ZP-induced AR and evaluated the clinical value of these tests. Sperm-ZP binding and sperm-ZP penetration tests are the most powerful indicators for sperm fertilizing ability in vitro. The ZP-induced AR is highly correlated with sperm-ZP penetration. We discovered a condition we call disordered ZP-induced AR which causes serve infertility in up to 25% men with otherwise idiopathic infertility In conclusion, the combination of semen analysis with advanced sperm function tests provide important diagnostic and prognostic information for male infertility and is crucial for selection of patients for treatment with IVF or ICSI. (Asian J Androl 2002 Dec; 4: 281-285)

补中益气汤对精索静脉曲张大鼠睾丸生精功能和一氧化氮的影响

目的探讨补中益气汤对精索静脉曲张大鼠睾丸生精功能和腹主动脉血中一氧化氮(nitric oxide,NO)的影响。方法将30只SD成年雄性大鼠,按随机数字表法分为正常组、模型组和治疗组,共3组,每组10只。各组均在建立实验性精索静脉曲张大鼠模型7天后开始治疗,连续用药45天。实验结束后,抽取腹主动脉血,分离血清,检测血清NO;获取睾丸组织,做病理切片;处死大鼠。结果与正常组相比,模型组和组治疗组中左右两侧睾丸组织均出现病理性改变;与正常组相比,模型组和治疗组中NO含量显著升高(P<0.05),但模型组与治疗组相比NO含量无显著性差异(P>0.05)。结论补中益气汤颗粒剂能够能够减轻精索静脉曲张大鼠睾丸生精功能的损害,但不能降低腹主动脉血中NO的含量。

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〈2×10^6／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。

Kremer试验与伊红Y水试验在精子功能检查中的意义

本文比较作为精子功能检查的Kremer试验与伊红Y水试验的临床意义。以Kremer试验精子穿透CM高度分组,I组精子穿透高度<2.0cm/30min,Ⅱ组精子穿透高度2.0～3.9cm/30min,Ⅲ组精子穿透高度≥4.0cm/30min。伊红Y水试验Ⅰ组精子头部未着色同时尾部膨胀率为16.2±12.6％,Ⅱ组43.1±11.1％,Ⅲ组56.9±8.5％,Kremer试验与精液常规检查,Ⅰ组与Ⅱ、Ⅲ组比较均有非常显著差异,Ⅱ组与Ⅲ组比较只伊红Y水试验有非常显著差异,显示Kremer试验与伊红Y水试验临床意义的区别。两种方法各自在不同角度反映精子的功能状态。作为精子功能检查都具有方法简便、经济、结果有临床意义的优点,均可作为临床检查的常规手段。

水试验评价人精子膜功能完整性的观察

目的探讨水试验和精子尾部低渗肿胀试验在评价人精子膜功能完整性方面的相关性,以便为简便易行的水试验在精子膜功能的检测方面提供一些试验依据。方法将14例生育男性和16例不育男性的精液标本同时进行了水试验和低渗肿胀试验。结果生育组和不育组的精液常规分析指标、精子尾部肿胀率、g型精子百分率之间差异均存在统计学意义(P<0.05或0.01);无论是生育组还是不育组,水试验与低渗肿胀试验的尾部肿胀率之间均无显著性差异(P>0.2);无论是生育组还是不育组,水试验、HOS-test结果与精子存活率、精子活动率、正常形态精子之间均呈正相关(P<0.05或0.01)。结论水试验能客观准确地反映精子膜功能的完整性,且比低渗肿胀试验更为廉价、简便、快速。

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 workin~ laboratory.

21世纪医学的精液分析:精子功能检测的必要性

精子功能检测曾经是不孕夫妇进行辅助生殖技术(ART)前普遍采取的检查。如今世界各地的生殖医学中心已忽视了这些检查。精子功能检测操作步骤的确已分组纳入最新出版的《世界卫生组织人类精液分析实验室技术手册》的"研究步骤"章节。这很大程度上反映了此现状,即当前临床实践忽略了对不孕夫妇中男方因素的深入检查。认为如果能找到精子,一定就是健康、具有受精能力的、可进行胞浆内单精子注射(ICSI)的精子。然而,通过精子功能测试,可揭示造成男性不育的精子缺陷,为不孕夫妇提供指导。诚然,在某些情况下,ART可克服某些精子功能缺陷,但在其他情况下,夫妇有权了解不孕不育的原因,甚至ICSI的成功率(和精子有关)。知晓这些,有助于其真正清楚自己的生育状态,以证实是否寻求供精人工授精、领养孩子或维持无子女状态。了解不孕的真正原因,有助于说服不孕夫妇放弃艰难又毫无结果的自然妊娠,更加坚定地选择ART治疗不育。

伊红Y水试验法应用研究进展

基于体外活体染色技术、低渗肿胀试验和水试验原理建立的伊红Y水试验方法可同步检测精子头部和尾部、精子膜结构和功能的完整性,在中国已被临床广泛应用。伊红Y水试验方法学上的3个特点包括:1同时对精子头部和尾部进行检测,全面评估精子膜各部位损伤的内在联系,便于观察;2用蒸馏水代替常用的配方溶液,避免不同渗透压或不同比例糖和电解质溶液对精子膜有不同作用影响水分子通过精子膜,既简化了方法,又使反应标准化;3检测时间短,可以重复检测,适合治疗前后反复检测。本文综述伊红Y水试验法的基本方法及其改进,在精子功能检查、男性不育常规精液分析、睾丸穿刺精子质量评估、精液冷冻保存程序研究、受微波辐射男性精子膜完整性等相关研究方面的应用。

精子膜功能试剂盒的临床应用对比研究

目的：研究精子膜功能试剂盒的临床应用价值．方法：使用该试剂盒与精子尾部低渗膨胀试验和伊红Ｙ水试验对３３例正常生育男性和６５例不育症患者进行比较观察．结果：该试剂盒与精子尾部低渗膨胀试验和伊红Ｙ水试验检测结果均呈显著的相关性，三种方法精子膨胀率生育组分别为（６６．５５±４．８７）％ ， （６５．８２±４．６８）％ ， （６８．６９±５．５９）％ ；不育组分别为（４５．７４±１３．８７）％ ， （４６．８８±１３．６６）％ ，（４９．３８±１２．６２）％ ． 结论：精子膜功能试剂盒快速简便、结果准确，可代替精子尾部低渗膨胀试验和伊红Ｙ水试验检测精子膜功能并在临床推广应用．

不同浓度低渗肿胀试验结合伊红染色对精子质量的评估

本文应用不同浓度精子低渗肿胀试验结合伊红染色法，对８例生育男性和１０例不育男性的精子膜功能进行评估。结果表明：１．活精子尾部低渗肿胀率均达到９０％以上，生育组与不育组无显著性差异；２．在１５０ｍＯｓｍ低渗溶液的稀释倍数为３～７倍之间，精子头部膜的损害程度在生育组与不育组之间存在显著性差异；３．在不同浓度的低渗溶液中，精子头部膜与精子尾部膜对低渗溶液的顺应性并不一致。实验证明不同浓度低渗肿胀试验结合伊红染色可正确评价精子膜的整体功能。

某保健食品遗传毒性试验研究

目的对某保健食品的遗传毒性进行研究。方法采用Ames试验、小鼠骨髓嗜多染红细胞微核试验和小鼠精子畸形试验等对某保健食品进行安全性试验研究。结果Ames试验、微核试验和精子畸形试验结果均为阴性。结论在本次实验条件下,产品未见遗传毒性,产品是安全可靠的。

用有效活精指数评价精液质量

同时应用有效活精指数及精子尾部低渗肿胀试验、精子前向运动级别、毛细管穿透试验，精子顶体染色，对９０例正常男性和１２９例不育男性精液进行检测。结果显示：有效活精指数与精子总肿胀率（Ｔ），ｇ形精子率（Ｇ），前向运动级别（Ｐ），穿透力评分（Ｓ），顶体完整率（Ａ）之间，生育组呈显著正相关，不育组呈非常显著正相关。以上各项试验参数在两组间均有非常显著差异。不育男性结果表明，有效活精指数与Ｔ、Ｇ、Ｐ、Ａ的异常检出率之间均无显著差异，而仅与（Ｓ）的异常检出率有显著差异。