双色荧光原位杂交（FISH）检测正常人精子中X精子与Y精子比率

本研究应用双色荧光原位杂交法检测正常人Ｘ精子、Ｙ精子及性比率，平均杂交效率为９９０５％±０４２％（９９４２％－９９９２％）对１３位正常健康男子总共计数８９３７５条精子，其中４４３４０条精子为Ｘ精子（４９６３％），４４１７６条为Ｙ精子，占４９４２％，Ｘ精子与Ｙ精子的比率接近１：１，与Ｘ、Ｙ精子各占５０％的理论值差别无显著性。     

精液白细胞对精液主要参数及精子功能的影响分析

目的探讨精液白细胞含量与精液主要参数和指标的关系。方法按照WHO人类精液实验室手册要求检测精液中的白细胞、主要参数、精子形态分析、精子顶体酶活性、精浆抗体（AsAb）、解脲支原体等，分析精液白细胞与男性不育相关因素的关系。结果238例男性不育患者中有75例（31．5％）精液中白细胞〉1×10^6个／ml，设为白细胞精子组；163例（68．5％）患者精液中白细胞≤1×10^6个／ml，设为非白细胞精子组。白细胞精子组的精子密度、精子活动率、a＋b级活力精子率、精子顶体酶阳性率均低于非白细胞精子组（P〈0．05）；而精子畸形率、精浆抗体（AsAb）、解脲支原体阳性率均高于非白细胞精子组（P〈0．05）。两组的精液量、pH值和液化时间差异无显著性。结论精液中白细胞含量与精液质量有密切的关系，是导致男性不育的重要原因。     

Klinefelter综合征临床资料分析

作者报告了８５例克氏综合征患者的临床资料。１异常体征主要是睾丸容积小于３ｍｌ，占９６５％。阴茎短小及阴毛稀少分别占１００％、８９４％、。２性功能状况：７２例已婚患者中，勃起不坚４８例，占６６７％，不能勃起７例，占９７％。夜间阴茎勃起硬度测量，器质性阳萎４４例，功能性阳萎１１例。３核型分析：４７，ＸＸＹ占８９％。４６，ＸＹ／４７，ＸＸＹ占８％。多Ｘ及多Ｘ嵌合型占２％。４内分泌：ＦＳＨ、ＬＨ远高于正常，Ｔ低于正常。ＰＲＬ略高于正常。Ｅ２略高于正常。５精液检查均为无精子。９例作抗精子抗体检查，４例ＩｇＧ阳性。综合内分泌及性功能调查资料表明：心理因素所致的功能性阳萎在患者中占有一定比例。抗精子抗体检测表明，克氏综合征的抗精子抗体的形成机制上，除隐蔽抗原释放所致外，还有其他的机制。     

标本采集时间对精液全自动分析各参数的影响

目的 为了了解精液分析中标本获取时间问隔对结果的影响，我们对44个健康受试者的精液进行了测试。方法 受试者48h内留取精液两次，采用伟力全自动精子分析仪对精液进行全自动分析。分别对已婚、未婚。24h、48h结果进行统计学分析。结果 两次取精中第二次精液量明显减少，且差异具有统计学意义。精子运动速度参数vsl、vd、vap，在已婚者中差异具有不同程度的统计学意义。精子密度、a级精子率两次差异均无统计学意义，而b级精子率和c级精子率两次差异均具有不同程度的统计学意义。结论 进行精液常规分析中应要求：取精间隔超过48h。并且最好每次检查前禁欲天数尽量保持一致，从而使检验结果具有可信和可比性。也有利于临床医生的诊断．及治疗效果的比较。     

绍兴地区281例男性婚检者精液常规检测结果分析

目的了解281例绍兴地区婚前体检人员男性精液质量状况。方法应用WLJY-9000伟力彩色精子质量分析系统对281例男性婚检人员精液的量、外观、粘稠度、pH值、液化时间、精子密度、活率、活动力进行分析。结果281例婚检者，精液量异常的为25例，占8．9％，液化异常31例，占11．0％，pH值异常36例，占12．8％，精予密度异常48例，占17．1％，精子活率异常29例，占10．3％，精子活动力a级〈25％或（a＋b）级〈50％共有56例，占19．9％。结论婚检人群的生殖健康知识知晓情况较差，全民应重视婚前检查，进一步提高人口素质及优生优育。     

冷冻干燥法对人精子DNA的影响

目的探讨冷冻干燥法用于人类精子保存的安全性。方法取健康志愿者合格精液40份，平均分为4组，其中3组分别加入不同的冻干保护剂（ETBS；ETBS＋海藻糖：ETBS＋海藻糖＋蛋黄）后给予冷冻干燥处理，在4℃冰箱中保存3周；1组作为新鲜精液对照组。对4组标本分别以原位缺口末端标记（TUNEL）法和彗星试验进行DNA断裂精子百分率检测。结果ETBS、ETBS＋海藻糖、ETBS＋海藻糖＋蛋黄组和新鲜精液组DNA断裂精子百分率以TUNEL法检测，分别为（6．39±1．46）％、（5．75±1．29）％、（5．20±1．38）％、（4．94±1．86）％；以彗星试验检测，分别为（6．48±1．58）％、（5．83±1．48）％、（5．28±1．42）％、（5．12±1．65）％。冷冻干燥保存后的各组与新鲜精液相比较，DNA断裂精子百分率差异均无统计学意义（P〉0．05）。结论以ETBS或ETBS加海藻糖、蛋黄为保护剂的冷冻干燥法对人精子DNA无明显损伤，可有效地保护人精子的DNA。     

Y染色体多态性的临床效应

本文对３０例Ｙ染色体多态与４种临床异常的相关性进行了分析。４组临床异常：自然流产，精液异常，生育异常患儿，智力低下的Ｙ染色体多发生率分别为１８．７５％，３０％，２５％，２０％，均明显高于正常人群，表明Ｙ染色体多态具有临床效应，３种Ｙ染色体多态中，大Ｙ最多，占７３．３％，Ｙｐ＾＋为２３．３％，Ｙｐ＾－仅１例。     

中山地区男性不育患者精液质量分析

目的对中山地区男性不育患者的精液进行常规分析,了解中山地区男性不育患者精液质量的现状。方法按照《世界卫生组织人类精液检查与处理实验室手册》（第5版）的标准,应用西班牙人类精液分析微机辅助智能系统（CASA）对2012年10月～2013年6月来中山市博爱医院生殖中心就诊的2224例不育男性患者的精液进行常规检测和采用改良巴氏染色法分析精子正常形态率。结果2224例不育男性精液中正常者1113例（31．61％）,异常者1521例（68．39％）,其中液化异常964例（43．35％）,pH值异常124例（5．58％）,精子总数异常1002例（45．05％）,精子浓度异常556例（25．0％）,精子存活率异常1031例（46．36％）,精子总活力（PR＋NP）异常1208例（54．31％）,无精子症372例（16．72％）,精子正常形态率异常1021例（45．91％）。结论精子总活力、存活率下降,精子总数减少,精子正常形态率下降和精液液化不良是引起中山地区男性患者不育的主要原因,临床上应加于重视。     

不育男性精液生殖细胞精蛋白的初步研究

在精液常规检查和巴氏染色的基础上按生殖细胞的种类和比例将６４例不育患者分为３型。分别提取生育与不育男性精液生殖细胞核内碱性蛋白，经氨基酸分析表明正常生育对照组合有约４７．４％的精蛋白成份。高效液相色谱分子筛分析生育与不育男性精液生殖细胞核碱性蛋白发现：１４例生育男性均存在精蛋白，其含量约占核碱性蛋白的３６．７８％～６４．４２％；５６例有精子和其它生精细胞的Ⅰ型和Ⅲ型不育患者中，精蛋白缺乏者５例，占８．９％。精蛋白含量减少者１１例，占１９．６％。精蛋白含量在正常生育男性范围内波动者４０例，占７１．４％；８例无精子的Ⅱ型不育患者均缺乏精蛋白。     

男性孕前精液质量检测结果分析

目的了解已婚未育孕前体检男性精液质量状况。方法应用WLJY-9000伟力彩色精子质量分析系统对576例已婚未育孕前体检男性精液量、液化时间、精子总数、精子密度、a级精子百分率、b级精子百分率、a＋b级精子百分率、精子活率等方面进行分析。结果已婚未育孕前体检男性的精液量、液化时间、精子总数、精子密度、b级精子百分率、a＋b级精子百分率、和精子活率与正常对照组之间差异无显著性（P〉0.05）;a级精子百分率显著降低（P〈0.05）结论随着全民教育和生活水平的提高,孕前人群的生殖健康知识认知情况很好,比较重视孕前检查。     

Reliability of estimates of diploid human spermatozoa using multicolour fluorescence in-situ hybridization [published erratum appears in Hum Reprod 1997 May;12(5):1118]

Multicolour fluorescence in-situ hybridization (FISH) analysis permits distinction between disomic and diploid spermatozoa. Thus estimates of the frequency of diploid spermatozoa can be obtained for human semen samples. The issue of the accuracy and reliability of these diploidy estimates has been addressed by analysing diploidy frequencies in 10 men using the same sperm sample to estimate diploidy twice-once during two-colour FISH analysis of disomy for chromosomes 1 and 12 and a second independent analysis of three-colour FISH for disomy estimates for chromosomes X and Y (with chromosome 1 used as the autosomal control). A minimum of 10,000 spermatozoa per hybridization per male was counted for a total of over 200,000 spermatozoa analysed. The mean frequency of diploid spermatozoa was 0.13% for the autosomal study and 0.14% for the sex chromosomal study, which were not significantly different. One donor had extremely divergent values of diploidy in the two studies. Analysis of values in the other nine donors demonstrated no significant difference in the two diploidy estimates. These results indicate that the FISH technique is an accurate and reliable method for determining diploid frequencies in human spermatozoa.      

Efficiency of sex pre-selection of spermatozoa by albumin separation method evaluated by double-labelled fluorescence in-situ hybridization

To evaluate the separation efficiency of Ericsson's two- and three- layer albumin separation methods, semen samples from 21 healthy males were studied. Seven patients already had two or more sons, another seven had two or more daughters and the other seven had primary infertility due to female factors. The semen samples were divided into three aliquots: one remained unprocessed initially, the other two aliquots went through two- and three-layer albumin separation methods respectively. All samples were then stained with X-Y double staining probes. In each group, four or five samples were processed at room temperature, and two or three at body temperature (37 degrees C). The labelling efficiency of X-Y double staining probe was over 99%. The X:Y sperm ratios were even in the original samples. The ratios of the X and Y spermatozoa were altered slightly but significantly after the two- layer (P < or = 0.05) or the three-layer (P < or = 0.005) separation. The alterations occurred only at room temperature. The X spermatozoa increased and the Y spermatozoa decreased, both to a small degree of difference (1.4-3.5%). Double fluorescence in-situ hybridization analysis therefore showed that albumin separation methods do not enrich Y spermatozoa.      

Efficiency of MicroSort flow cytometry for producing sperm populations enriched in X- or Y-chromosome haplotypes: a blind trial assessed by double and triple colour fluorescent in-situ hybridization

Using fluorescent in-situ hybridization (FISH) we have evaluated, on a blind basis, the efficiency of flow cytometry to separate human X- and Y-chromosome bearing spermatozoa. Our data demonstrate that human spermatozoa can be sorted to a purity of 80-90% for X spermatozoa and of 60-70% for Y spermatozoa. Our results using triple FISH fully agree with the sorting treatment used in each case and corroborate the efficiency of the flow sorting technique for sperm sex selection. In these limited samples (200-500 sperm/donor), the frequencies of disomic or diploid spermatozoa were not increased when comparing the sorted samples with unselected samples or with our control series.      

Comparison of Percoll, mini-Percoll and swim-up methods for sperm preparation from abnormal semen samples.

Two methods of density gradient centrifugation, Percoll (P) and mini-Percoll (MP), were compared with the swim-up technique for preparing spermatozoa from each of 40 abnormal semen samples. P and MP produced similar results with a mean recovery of spermatozoa with progressive motility which was significantly higher (18-19%) than that achieved with swim-up (5%). However, the swim-up method resulted in the recovery of spermatozoa with a higher mean motility (89 versus 58%), velocity (69 versus 56 microns/s), percentage with normal morphology (22 versus 16%) and intact acrosomes (61 versus 36%) than P and MP. The mean amplitude of lateral head displacement was the only characteristic of spermatozoa in semen which correlated with the recoveries of motile spermatozoa. Combining MP and swim-up methods for 10 samples produced a higher recovery (11 versus 6.9%) of spermatozoa with significantly better mean motility (94 versus 87%) than did swim-up alone. Although P and MP resulted in greater yields of motile spermatozoa than the swim-up preparation, the latter procedure selected higher proportions of spermatozoa with improved characteristics (velocity, intact acrosomes and normal morphology) which correlate with fertilization rates in vitro. It is concluded that P and MP are not superior to swim-up. However, sequential MP and swim-up preparation improves yields of high quality spermatozoa from some abnormal semen samples and therefore has potential for improving fertilization rates.     

Estimation of aneuploidy for chromosomes 3, 7, 16, X and Y in spermatozoa from 10 normospermic men using fluorescence in-situ hybridization

Fluorescence in-situ hybridization (FISH) is a fast and efficient method of estimating aneuploidy in human spermatozoa. In this study, we have estimated baseline disomy frequencies in spermatozoa from a group of 10 normospermic men, using stringent scoring criteria. A triple- probe FISH procedure was used for chromosomes 3, X and Y, while a double-probe FISH method was used for chromosomes 7 and 16. A total of 101273 spermatozoa were scored for chromosomes 3, X and Y, resulting in 97.83% haploidy (3X or 3Y), 0.39% disomy (33X, 33Y, 3XX, 3YY or 3XY) and 0.35% diploidy (33XX, 33YY or 33XY). A total of 100760 spermatozoa were scored for chromosomes 7 and 16, giving 98.9% haploidy (716), 0.11% disomy (7716 or 71616) and 0.27% diploidy (771616). Disomy frequencies for individual chromosomes differed (chromosome 3, 0.20%; chromosome 7, 0.05%, chromosome 16, 0.06%; X + Y, 0.19%). The frequency of disomy 3 was significantly higher than disomy 7 (P = 0.019) and disomy 16 (P = 0.022), while the frequency of sex chromosome disomy was significantly higher than disomy 7 (P = 0.0058) and disomy 16 (P = 0.0067), but not disomy 3 (P = 0.73). The disomy and diploidy (0.27- 0.35%) estimates obtained for this normospermic population were generally low and were similar to other recent reports.      

Sperm swim-up techniques and DNA fragmentation

BACKGROUND: Swim-up techniques for sperm separation may have detrimental effects on sperm DNA. We wished to determine whether the normal swim-up method with centrifugation used in our laboratory, which involves a centrifugation step, was harmful to sperm compared with swim-up without centrifugation. METHODS: Semen samples were obtained from patients undergoing IVF or andrology assessment. An aliquot was removed for fixation and subsequent DNA fragmentation determination. The remaining sample was divided into two equal parts, which were subjected to swim-up either with (normal swim-up) or without (direct-swim-up) centrifugation. Semen analysis was performed both before and after swim-up. DNA fragmentation, in spermatozoa previously fixed in 4% paraformaldehyde, was assessed by the terminal transferase-mediated DNA end-labelling procedure (TUNEL). The percentage of spermatozoa with DNA damage after each swim-up technique was compared with that in the original semen sample. RESULTS: DNA damage was <5% in most samples. No significant change in DNA fragmentation was observed between the two swim-up procedures, although the 'normal' swim-up sample had significantly less DNA fragmentation than the pre-swim-up sample. CONCLUSIONS: We conclude that our normal swim-up technique caused no more DNA damage to spermatozoa from normal semen samples than a direct swim-up technique that involved no centrifugation step.     

Fluorescence in-situ hybridization analysis of chromosomal constitution in spermatozoa from a mosaic 47,XYY/46,XY male

Sex-chromosome mosaicism in spermatozoa from a mosaic 47,XYY[20%]/46, XY[80%] male with fertility problems was assessed using triple-probe fluorescence in-situ hybridization (FISH) studies. Chromosome-specific probes for X, Y and 18 were used, and the possible outcomes were deduced. In normal haploid spermatozoa of the patient and a normal 46,XY male control, the X:Y ratio was close to 1:1. There was a significant difference in the total incidence of karyotypically abnormal spermatozoa between the patient and the 46, XY male control (2.31% versus 1.46%, P < 0.0001). The incidence of some types of disomic spermatozoa X+Y+18 (24,XY) and X+18+18 (24,X, +18), or diploid X+Y+18+18 (46,XY) spermatozoa was significantly increased in the patient's semen sample. There was, however, no significant difference in the incidence of disomic Y+Y+18 (24,YY) spermatozoa. Because the majority of the patient's spermatozoa was karyotypically normal, the aetiology of his fertility problems was unclear. These results add to the growing body of information regarding chromosome abnormalities in spermatozoa from men who are mosaic for sex chromosome abnormalities. In these men, FISH analysis of spermatozoa may be warranted to determine the relative percentages of abnormal cells, and to determine if in-vitro fertilization with preimplantation genetic diagnosis may increase the likelihood of a successful pregnancy.     

Assessment of sex chromosome ratio and aneuploidy rate in motile spermatozoa selected by three different methods

Using three-colour fluorescence in-situ hybridization, sex chromosome ratios and frequencies of diploidy and disomy for chromosomes X, Y and 18 were compared in spermatozoa of good and poor motility after separation by swim-up, glass-wool and two-layer discontinuous Percoll methods. Semen samples were collected from seven normal males aged 26- 31 years. A minimum of 6000 sperm nuclei per sample were evaluated for each chromosome for a total of 308,432 sperm nuclei. Hybridization efficiency was 99.8%. A slight change in the ratio of X- to Y-bearing spermatozoa was noted after Percoll separation (from 49.3:49.5 to 50.0:48.9; P = 0.036 and P = 0.046), but not after separation by the other two methods. We did not observe significant differences in the disomy rates for sex chromosomes or chromosome 18 or in the diploidy rate between spermatozoa with good and poor motility after separation by any of the three methods. Our data indicate that separation of motile spermatozoa does not alter the ratio of X- to Y-bearing spermatozoa to a degree that represents sex chromosome selection.      

Comparison of different hypo-osmotic swelling solutions to select viable immotile spermatozoa for potential use in intracytoplasmic sperm injection

The hypo-osmotic swelling test, originally developed as a diagnosticsperm test, is used to discriminate viable from non-viable spermatozoafor intracytoplasmic sperm injection (ICSI) in cases of completeasthenozoospermia. In the present study, three hypo-osmoticsolutions were compared, i.e. (A) Jeyendran solution containingsodium citrate and fructose; (B) a mixture of 50% culture mediumand 50% milli-Q water; and milli-Q water. While both the percentageof swelling and vitality assessed by eosin Y remained unchangedafter 5-30 min of sperm exposure to solutions A and B, incubationin water for only 5 min was in itself detrimental. Ten frozen-thaweddonor samples and 10 asthenozoospermic patient samples wereexposed to the three solutions for B and C, but only weaklycorrelated for solution A. Percentage viability was furtherassessed by eosin Y and motility of spermatozoa after 2 h and24 h exposure to the three solutions was compared with unexposedcontrol spermatozoa. While a significant decrease in both parameterswas observed for all three solutions in comparison with thecontrol, sperm quality was significantly higher after exposureto solution B than after exposure to solutions A and C. It maybe concluded that solution B (composed of 50% culture mediumand 50% water) is to be preferred for the selection of viableimmotile spermatozoa for ICSI.     

Differential sperm performance as judged by the zona-free hamster egg penetration test relative to differing sperm penetration techniques.

A prospective study on 61 unselected semen samples from infertile patients was conducted to evaluate the effects of sperm preparation techniques on the outcomes of the zona-free hamster egg penetration test (HEPT) to assess the in-vitro fertilizing capacity of spermatozoa. Each semen sample was divided into two equal portions before separation of the spermatozoa from seminal plasma either by the single-tube swim-up method, or using a two-layer discontinuous Percoll gradient. Spermatozoa were incubated overnight for initiation of capacitation after which HEPT was performed. The swim-up spermatozoa were further divided into two subgroups before HEPT as follows: with or without (control) treatment for 20 min with 50% (v/v) pooled, human follicular fluid (hFF) which had not been heat-inactivated. It was demonstrated (P less than 0.05) that the Percoll-separated spermatozoa exhibited higher penetration scores (percentage penetration rate and penetration index) than the control or the hFF-treated swim-up spermatozoa. A short exposure (20 min) to hFF significantly increased the penetration scores in HEPT for swim-up spermatozoa (P less than 0.05) but the average results were still significantly lower (P less than 0.05) than those of the Percoll separated spermatozoa, which had received no hFF treatment. Based on these findings, we conclude that the Percoll separation technique is better than the centrifugal pelleting and single-tube swim-up technique for reducing the false-negative results in HEPT. In addition, the use of hFF can significantly improve the performance of the swim-up sperm samples in HEPT.(ABSTRACT TRUNCATED AT 250 WORDS)