Sperm Viability: A Comparison of Analytical Methods

Two methods of measuring human sperm viability, the stain exclusion assay and the hypoosmotic swelling (HOS) test, were evaluated. Human sperm were pretreated with 2.0% glutaraldehyde or 0.1% Triton X-100 and compared to untreated controls. Approximately one half of the sperm were found to be viable in the control samples and nearly all sperm were non-viable in the Triton X-100 treated samples by both the stain exclusion and HOS assays. After glutaraldehyde pretreatment, presumably inactivating the spermatozoa, the HOS test revealed that most sperm were not viable, while the stain exclusion test found no difference between glutaraldehyde pretreated sperm and control sperm. Investigations with scanning and transmission electron microscopy demonstrated that the HOS test caused the membrane of the sperm tail to swell and the tail fibers to coil several times within the swollen membrane. It is concluded that the stain exclusion assay merely measures the structural integrity of the sperm membrane, whereas the HOS test also provides an indication of the physiological integrity of the sperm membrane.     

The direct effect of Hochuekki-To on human sperm fertilizability using the hypoosmotic swelling test.

The concentration and motility of human sperm increase is examined following the oral administration of a traditional herbal medicine, Hochuekki-To using the hypoosmotic swelling (HOS) test. After 1 h incubation in Hochuekki-To, the motility of sperm and the results of the HOS test improved, particularly for severely abnormal sperm. For both the original and incubated sperm, the results of the HOS test improved when Hochuekki-To was used in the swim-up medium for the swim-up washing method rather than when Whittingham's T6 solution was used. Hochuekki-To had a favorable, direct effect on human sperm functions, suggesting an effect on the physiological integrity of sperm membrane.     

The hypoosmotic swelling test: an update.

The hypoosmotic swelling (HOS) test is a relatively new assay used to evaluate the functional integrity of the sperm's plasma membrane. In fact, more studies have been published on the applicability of the HOS test than any other new sperm indicator. The assay is based on the fact that fluid transport occurs across an intact cell membrane under hypoosmotic conditions until equilibrium is reached. Due to the influx of fluid, the cell will expand and bulge, especially in the tail, and this change can be readily observed with a phase contrast microscope. Earlier studies have yielded some confusion regarding the interpretation of the data. This review is an attempt to clarify and update the usefulness of the HOS test as a tool to evaluate the sperm function.     

Evaluation of human sperm membrane integrity using the water test and the hypoosmotic test

The objective of the study was to compare the water test and the hypoosmotic test (HOS) in the assessment of the human sperm membrane. A total of 686 semen samples from human male donors were subjected to water and HOS tests after routine semen evaluation. The mean percentage of swollen spermatozoa was 71.8 +/- 9.6% in the HOS test and 67.8 +/- 9.4% for the water test; these values were not statistically different. The correlation of coefficients between the water test and the HOS test was highly significant whether the values for the HOS test were higher or lower than 60% (P < 0.001). A poor correlation was obtained when the two tests were compared for sperm counts either higher or lower than 20 x 6 ml-1 and when the results for both tests were compared with the percentage of eosin-Y staining spermatozoa. A poor correlation was also obtained when the results of each test were compared with eosin-Y staining spermatozoa in normal and abnormal semen samples. The coefficient of regression between the two tests showed a high correlation (P < 0.001). In conclusion, even though a high correlation between the HOS test and water test was observed in this study, it is not possible to recommend assessment of sperm membrane integrity using the water test and the consequent replacement of the HOS test in routine practice. Further studies are necessary to establish the best test for sperm vitality.     

Combination of hypoosmotic swelling/eosin Y test for sperm membrane integrity evaluation: correlations with other sperm parameters to predict ICSI cycles

The objective of our study was to evaluate the accuracy of the combination of hypoosmotic swelling (HOS) and eosin Y (Ey) exclusion tests to predict the ICSI cycles' outcome and its correlations with other sperm parameters. The functional and structural integrity of sperm membrane was evaluated with the combined HOS/Ey test in 95 ICSI cycles and the results were correlated with other sperm parameters, including concentration, motility, strict morphology, and total motile sperm count. The combined HOS/Ey test was evaluated for the prediction of the ICSI cycles' outcome parameters including fertilization, cleavage, and pregnancy rates. The HOS/Ey test presented significant relationships with concentration, motility, and strict morphology (p < 0,0001) but it couldn't predict the fertilization, cleavage, and pregnancy outcomes of ICSI cycles. The combined HOS/Ey test has strong correlations with motility and strict morphology parameters of sperm samples but is not sufficiently sensitive to estimate the outcome of ICSI cycles.     

Influence of enterococci on human sperm membrane in vitro

Aim： To study the influence of enterococci on human sperm membrane in vitro. Methods： Ejaculated human sperm were artificially infected with β-hemolytic or non-β-hemolytic enterococci at the bacteria： sperm ratio of 50：1 at 37℃. Sperm membrane integrity was examined after incubation for 1, 3 and 5 h by hypoosmotic swelling （HOS） test and electron microscopy. Results： Sperm infected with β-hemolytic enterococci had lower HOS scores compared with non-β-hemolytic strains or uninfected control （P 〈 0.01）. The HOS test scores of sperm infected with β-hemolytic enterococci increased in the presence of phosphatidylcholine, an inhibitor of hemolysin. Non-β-hemolytic strains showed no significant difference in swelling rate, compared with the control group （P 〉 0.05）. It was shown by electron microscopy that β-hemolytic enterococci caused significant rupture of human sperm membrane. Conclusion： β-hemolytic enterococci caused human sperm membrane injury, and might be mediated by the hemolysin of enterococci.     

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。     

精子尾部低渗膨胀试验的相关因子及染色法

共检测110例男性不育患者的精液标本,确证精子尾部低渗膨胀(HOS)试验与精子活动率有较显著的正相关(r=0.273,n=102,P<0.01);与精子膜表面麦芽凝集素(WGA)受体亦成正相关(r=0.251,n=72,P<0.05);但与精子膜表面附着的抗精抗体免疫微珠(IB)试验无关(r=0.027,n=62,P>0.05);与精液有无支原体感染无关(t=0.543,df=32,P>0.05);精液中圆细胞数量对 HOS 试验无影响(F=0.413,方差自由度为f_1=3,f_2=100,P>0.05)。故认为以上试验互不能取代,只能互相参考。我们用简便而可靠的铁苏木精染色法改进了 HOS 试验技术,以便于这种确有实用价值的精子功能检测技术在临床上推广。     

Sperm plasma membrane integrity in fertile and infertile men

Sperm plasma membrane characteristics were analysed by a combined method, the HOS-eosine test (HOS-E test), that consists of the hypoosmotic swelling test (HOS test), and the eosine-Y staining. Semen samples were categorized into four groups (Normo-, Oligo-, Astheno-, and Oligoasthenozoospermic) and subjected to the standard analysis (spermiogram), HOS test, eosine-nigrosine test (reflecting sperm viability); and HOS-E test. HOS-E test makes it possible to distinguish four groups of spermatozoa: type 1, HOS+/eosine-; type 2, HOS-/eosine-; type 3, HOS-/eosine+; and type 4, HOS+/eosine+. Normozoospermic samples showed 61.2 +/- 1.4% type 1, 9.2 +/- 0.8% type 2, 22.6 +/- 1.1% type 3, and 6.8 +/- 0.6% type 4 spermatozoa. Oligozoospermic samples showed no significant differences in these values, whereas asthenozoospermic samples showed a higher percentage of types 3 and 4 and a lower percentage of type 1. Oligoasthenozoospermic samples showed high percentages of types 2, 3, and 4 and a low percentage of type 1. Sperm plasma membrane integrity is a necessary condition for motility and fertilization. So, it is not surprising that semen samples with abnormal motility showed a HOS-E result indicative of a defective plasma membrane.     

Detrimental effects of cryopreservation on the structural and functional integrity of the sperm membrane.

Many centers have been disappointed with the pregnancy rate following the insemination of cryopreserved-thawed sperm, despite the maintenance of an adequate motile density. The possibility exists that damage to the sperm membrane might occur despite preservation of other semen parameters. Simple measurements of structural integrity (viability) and functional integrity (hypoosmotic swelling test) were performed on thawed specimens. In each instance, both the viability and HOS scores were less than the critical 50% level. Specimens from three different commercial centers had very poor HOS and viability scores from two of the centers, and, though the scores were generally greater than or equal to 50% from the third center, this was achieved by eliminating 11 of 12 donors. Reducing the glycerol concentration from 12 to 7% and switching from Nunc vials to plastic embryo straws did not improve the poor sperm membrane tests. The possibility exists that if modification of the cryopreservation technique leads to improved HOS and viability scores, perhaps improved pregnancy results will be realized.     

Sperm motility should be assessed in fresh sperm and after a sperm washing procedure.

A prospective study was carried out on semen samples from 118 consecutive unselected men attending our infertility clinic to determine whether sperm motility may be affected by seminal plasma. The incidence of asthenozoospermia as defined by fewer than 50% of spermatozoa with forward progressive motility in the untreated semen was 37.4%. This value was significantly reduced to 23% after washing and removing seminal plasma. Men with asthenozoospermia in untreated semen but normal in the washed sample had a percentage of normal sperm morphology and a percentage of swollen tails in the HOS test similar to those of controls, and higher than those of asthenozoospermics in both the untreated and washed sample. Sperm velocity was also significantly improved after the washing procedure. Spermatozoa selected by swim-down procedure and applied to a seminal plasma medium reduced sperm motility affecting negatively the HOS test. Sperm motility should be assessed after a sperm washing procedure, since seminal plasma contains constituents that decrease sperm motility without affecting membrane integrity.     

Hypoosmotic swelling test in the prediction of male fertility

The hypoosmotic swelling test (HOS) is a simple test to measure the functional competence of human sperm membranes. The question is, does a relationship exist between this competence and the fertilization potential of human spermatozoa? In this study the strongest correlation (r = 0.76) was obtained between sperm swelling and sperm viability (supravital staining). Only a moderate correlation (r = 0.50) was obtained with normal sperm morphology; weaker correlations were also obtained with the sperm penetration assay (r = 0.42) and human IVF (r = 0.24). The results, therefore, indicate that the HOS test has a limited predictive value. Notwithstanding this low concordance between sperm swelling and fertilizing potential, a less than 50% HOS test threshold was seen to be a definite indicator of a male factor.     

非阻塞性输精管滤过装置节育术的精浆中性α-糖苷酶的检测

本文采用ＷＨＯ推荐的改良精浆中性α－糖苷酶的测定方法测定输精管滤过装置节育术（ＩＶＤ组）和输精管结扎术（结扎组）的精浆中性α－糖苷酶活性。术前两组精浆均测出中性α－糖苷酶活性：ＩＶＤ组为４３．５０±２９．０１ｍＵ／每次射精（ｘ±ｓ）；结扎组为４７．８１±３１．２０（ｘ±ｓ）ｍＵ／每次射精；两组间无显著差异（Ｐ＞０．０５）。术后６、１２个月ＩＶＤ组分别有９１．５７％和７９．１７％的精浆测出中性α－糖苷酶活性；而结扎组仅有４．４８％和４．２４％的精浆测出中性α－糖苷酶活性；两组间均有非常显著的差异（Ｐ＜０．００１）。结果提示：ＩＶＤ组术后一年内其大部分受试对象的精浆中仍有附睾液存在。     

去透明带地鼠卵穿透试验与精子DNA荧光染色、精子尾部低渗肿胀试验相关性分析

本研究对６６例不明原因的原发不育及３４例继发不育男性病人的精液标本同时进行去透明带地鼠卵穿透试验（ＨＯＰ）、精子尾部低渗肿胀试验（ＨＯＳ）与ＤＮＡ荧光染色的有效精子计数（ＥＳＣ）的检测和统计分析。结果在１００例中ＨＯＳ和ＨＯＰ以及ＥＳＣ与ＨＯＰ的符合率分别达到７８％与７４％；ＨＯＳ与ＥＳＣ与ＨＯＰ三者的符合率亦达到６４％，表明ＨＯＳ、ＥＳＣ与ＨＯＰ一样也有反映精子受精能力的作用，并认为ＨＯＳ与ＥＳＣ的标准分别采用≥６０％与≥２０×１０￣６较为合适。此外，继发不育组的ＨＯＳ与ＨＯＰ符合率、ＥＳＣ与ＨＯＰ符合率以及ＨＯＳ与ＥＳＣ与ＨＯＰ三者符合率还明显高于原发不育组。     

Do antisperm antibodies cause functional impairment of the sperm membrane as manifested by a low hypoosmotic swelling test score?

Low hypoosmotic swelling (HOS) test scores were found to be associated with lower pregnancy rates. The mechanism seems to be related not so much to impaired fertilization but to inhibition of implantation. The defect may be present in males with normal or subnormal semen specimens. However, anecdotal experience suggested that the subset of males with antisperm antibodies (ASAs) have a higher frequency of low HOS scores. The possibility exists that ASAs may impair the functional integrity of the sperm membrane. The study presented herein, artificially added ASAs to sperm to see if this could lower the HOS score. The study would also determine if chymotrypsin, a protein digestive enzyme, could improve HOS scores, if, in fact, they were lowered by the addition of ASAs. The results did not show a reduction in the HOS scores following the addition of ASAs. Thus, it would appear that there is no cause and effect with the simultaneous presence of low HOS scores and ASAs. Possibly, however, longer exposure or a higher concentration of antibodies is needed to lower HOS scores.     

Effect of carboplatin on the functional integrity of the human sperm membrane in vitro

Although it is well known that carboplatin is a drug that binds directly to DNA, causing DNA-DNA and DNA-protein cross-links, which is the presumptive method for killing cells, the whole mechanism of action of carboplatin on spermatozoa is unclear. There are no published data in peer-reviewed journals focused on the interaction between carboplatin and cell membranes. Therefore, the purpose of this study was to investigate the minimal concentration of carboplatin that would affect the functional integrity of the human sperm membrane in an in vitro model. Human-ejaculated spermatozoa were obtained from 20 healthy normozoospermic donors. Solutions (SOL) of 0.5 mL of the semen samples and 0.5 mL NaCl (0.9%) containing increasing concentrations (7.5, 15, 30, and 60 ng) of carboplatin per 1 mL of SOL were prepared. Then, the hypoosmotic-swelling (HOS) test and the eosin test were performed on these samples and compared with the control (no carboplatin) group. Significant damage to the plasma membrane in the head region (eosin test positive) and in the tail region of spermatozoa, as assessed by the HOS test, was observed in concentrations of 30 and 60 ng carboplatin per 1 mL of SOL in comparison to the values evaluated in the control group. The results demonstrate that a minimal carboplatin concentration of 30 ng/mL causes significant damage to membrane integrity of spermatozoa in healthy volunteers.     

冷冻精子受精能力的综合评价

目的：观察不同冷冻期冷冻精子的受精能力。方法：应用精液常规分析（ＳＦＡ）、精子尾部低渗肿胀试验（ＨＯＳ）、去透明带地鼠卵穿透试验（ＨＯＰ）及冷冻精液人工授精（ＡＩＤ）对冷冻前后及不同冻存期的精液标本进行综合性检测。结果：１０份正常精液标本的冷冻精子活动率、ＨＯＳ值、ＨＯＰ值均明显低于冷冻前（Ｐ＜０．００１），而不同冻存期的精液行ＡＩＤ术后的总妊娠率与新鲜精液ＡＩＤ术的总妊娠率比较，则基本相同。结论：精子细胞膜虽在冷冻复温过程中受到一定的损伤，使精子活动率和受精能力下降。但是，精液的长期冻存及精子细胞膜部分改变并不完全影响精子的受精能力。     

Smoking influence on sperm vitality,DNA fragmentation,reactive oxygen species and zinc in oligoasthenoteratozoospermic men with varicocele

This study aimed to assess the influence of smoking duration and intensity on sperm vitality, sperm DNA fragmentation, reactive oxygen species (ROS) and zinc (Zn) levels in oligoasthenoteratozoospermic (OAT) men with varicocele (Vx). A total of 246 men were investigated who were divided into OAT nonsmokers, OAT smokers, OAT nonsmokers and OAT smokers with Vx. They were subjected to history taking, clinical examination and semen analysis. In their semen, sperm hypo‐osmotic swelling (HOS) test, sperm DNA fragmentation test, seminal ROS and seminal Zn were assessed. The results demonstrated significantly decreased HOS test, seminal Zn level and significantly increased sperm DNA fragmentation, seminal ROS levels in OAT smokers with Vx more than OAT smokers compared with OAT nonsmokers. Smoking intensity, smoking duration and Vx grade demonstrated significant negative correlations with sperm motility, HOS test percentage and significant positive correlations with sperm DNA fragmentation, seminal ROS level. It is concluded that smoking has a negative impact on sperm progressive motility, HOS test, seminal Zn and positive impact on sperm DNA fragmentation, semen ROS level that are exaggerated if Vx is associated being correlated with smoking intensity, smoking duration and Vx grade.     

正常男性附睾中精子功能的初步研究

目的　检测正常成年男性附睾中不同部位的精子指标 ,为男性附睾精子功能的研究提供实验资料。方法　将附睾切断分成附睾头、附睾尾和附睾体 ,分别纵向剖开 ,置于含有 10 %小牛血清的F10 溶液中 ,37℃孵育 30min ,孵育液作精子常规分析、低渗肿胀试验 (HOS)、精子 -宫颈粘液穿透试验。结果　附睾不同部位精子的形态和功能存在明显差异 ,尾部和体部精子的活率、活动力、HOS试验和CM穿透试验结果明显高于头部精子 ,差异有显著性 (P <0 .0 5或 0 .0 1) ,以尾部精子的各项指标最高 ,与正常排出体外的精子无显著差异 (P >0 .0 5 )。头部附睾未成熟精子、畸形精子比例明显高于尾部和体部附睾的精子 (P <0 .0 1和 0 .0 5 )。结论　附睾精子可应用于辅助生育技术 ,作为治疗梗阻性无精子症的最好选择。     

Predictive value of hypo-osmotic swelling test to identify viable non-motile sperm

Aim: To determine the predictive value of the hypo-osmotic swelling (HOS) test to identify viable, non-motile sperm. Methods: Semen samples from 20 men with severe asthenozoospermia underwent traditional seminal analysis, eosin-nigrosin (EN) staining and the HOS test. A further EN stain was then pexformed on a HOS pre-treated aliquot and a total of 2000 further sperm examined. Results: The median sperm density was 5.1 million/mL (IQR 4.3-13.1) and the median motility was 3.0 % (IQR 0-7). Seven samples showed complete asthenozoospermia. Initial EN staining showed 59 % viability (range 48-69) despite the poor standard parameters and 47 % (range 33-61) in thecomplete asthenozoospermia subgroup. The HOS test showed 49.9 % reacted overall (range 40-59) and 41.7 %(range 22-61) in the complete asthenozoospermia subgroup. The combined HOSfEN stain showed the positive pre-dictive value of the HOS test to identify viable sperm was 84.2 % overall and 79.7 % in the complete asthenozoospermia subgroup. Conclusion: The HOS test can effectively predict sperm viability in patients with severe and complete asthenozoospermia. ( Asian J Andro12003 Sep; 5: 209-212)