Reappraisal of the hypo-osmotic swelling test to improve assessment of seminal fertility status

The hypo-osmotic swelling (HOS) test has been proposed as a useful assay for evaluation of the functional competence of the human sperm membranes. To assess this further, the HOS-test was evaluated in 187 semen samples collected from fertile men and from male patients consulting for infertility. These samples were classified as normal, oligo-, astheno- or oligoasthenozoospermic on the basis of their standard semen variables. The percentage of total sperm tail swelling and of sperm exhibiting different tail swelling patterns was recorded. In the fertile men and in the group of patients with normal semen variables, significantly more (P less than 0.001) HOS-reactive sperm were observed after hypo-osmotic treatment in comparison with those groups exhibiting abnormal semen parameters. Swelling of the sperm in a hypo-osmotic medium was highly correlated with both progressive motility (r = 0.62, P less than 0.001) and sperm viability (r = 0.65, P less than 0.001). A weak positive correlation was also observed between sperm swelling and sperm morphological features (r = 0.31, P less than 0.005) and between sperm swelling and sperm concentration (r = 0.31, P less than 0.005). No significant correlation was observed between sperm swelling and in-vitro sperm fertilizing capacity as assessed by the zona-free hamster oocyte penetration assay. However the majority of the semen samples (87.3%) showing a normal penetration rate (greater than or equal to 10%) also exhibited a 60% (or higher) reaction in the HOS-test.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Multivariate discriminate analysis of the relationship between the hypo-osmotic swelling test and the in-vitro fertilizing capacity of human sperm

Multivariate discriminant analysis was used to evaluate the usefulness of routine semen parameters and the hypo-osmotic swelling test (HOST) as predictors of the in-vitro fertilizing capacity of human sperm as assessed by the zona-free hamster egg penetration assay (HEPA). Eighty-eight semen samples from untreated patients attending an infertility clinic were analysed. Semen samples were classified into the following three groups before statistical analysis: group 1--positive sperm penetration (greater than or equal to 10%, n = 39); group 2--borderline penetration rates for HEPA (greater than 0% but less than 10%, n = 39) and group 3--negative sperm penetration (0%, n = 10). The percentage of sperm with normal morphology and sperm count were found to be significant in discriminating between semen samples exhibiting different in-vitro fertilizing capacity. These two discriminating variables in combination gave an overall correct classification rate of 45.5%. The multivariate discriminant analysis was also performed after excluding the data of group 2 semen samples (n = 39), which exhibited borderline sperm penetration rates. As a result, three discriminating variables including semen volume, sperm count and the percentage of sperm with normal morphology were selected. These three variables in combination could accurately predict whether a semen sample would exhibit positive sperm penetration (group 1) or negative sperm penetration (group 3) with an overall accuracy of 75.5%. The percentage of swollen sperm after hypo-osmotic treatment was not related to the HEPA result, as determined by linear correlation and multiple regression analyses, and did not give additional information about the in-vitro fertilizing capacity of sperm as evaluated by multivariate discriminant analysis.     

Correlation between hypo-osmotic swelling test and 'water test' to assess human sperm membrane integrity

Summary. Fifty-nine men who requested vasectomy and 43 infertile patients had a semen analysis performed prior to surgery or during evaluation. A hypo-osmotic swelling test (HOS) and a new 'Water test' were performed simultaneously, in order to assess correlation between these two procedures. Our results showed that values obtained with the 'Water test' were significantly higher than those obtained with the HOS test ( P < 0.001). These findings suggest that it is necessary to determine normal values for this new test before introducing it in the routine semen analysis.     

Correlation of the hypo-osmotic swelling test, semen score and the zona-free hamster egg penetration assay in humans

The correlation between the hypo-osmotic swelling test, the modified Eliasson score for human semen analysis and the hamster egg penetration assay was examined. The results showed a weak but significant correlation between the group of subjects with swelling rates below 50% and the above 80% group. These groups showed hamster egg penetration assay rates of 9 +/- 14% and 39 +/- 29%, respectively (P less than 0.035). A significant correlation was also found between modified Eliasson scores (17 +/- 10 and 5 +/- 7, respectively) and swelling rates below 50% and above 70% (P less than 0.001). These results suggest that spermatozoa showing a low swelling rate have a poor fertilizing capacity. Therefore, the swelling tests and analysis of the patients semen can be used to discriminate sperm quality.     

Evaluation of the hypo-osmotic swelling test in relation with advanced methods of semen analysis

The hypo-osmotic swelling test was claimed to assess an independent functional characteristic of human spermatozoa bearing relevance to their fertilizing capacity. To test this claim, we have studied the relationship between the result of the hypo-osmotic swelling test with that of conventional semen analysis and sperm motility patterns, the semen content of adenosine triphosphate, the staining pattern to acidified aniline blue, and the zona-free hamster oocyte test. The result of the HOS test is significantly correlated with all sperm characteristics except for the aniline blue stainability and the hamster oocyte test. The capacity of spermatozoa to react in a hypo-osmotic environment expresses the same functional information as the viability test using eosine staining. It is concluded that the hypo-osmotic swelling test does not add relevant information to that obtained by routine sperm analysis with regards to the fertilizing potential of semen.     

Application of the hypo-osmotic swelling test to spermatozoa prepared by swim-up and discontinuous Percoll separation

The quality of spermatozoa prepared by washing and swim-up or by discontinuous Percoll centrifugation, was compared by applying the hypo-osmotic swelling (HOS) test to semen samples from 116 men of infertile couples. The HOS test performed on 95 normal semen samples showed that the percentage of swollen spermatozoa separated by both techniques was significantly higher than in the initial ejaculate ( p <0.001). The percentage of HOS-positive spermatozoa separated by the Percoll gradient technique was significantly higher than that separated by the swim-up technique ( p <0.001). On the contrary, in 21 abnormal semen samples, there was no significant difference in the percentage of spermatozoa which were positive in the HOS test between the Percoll gradient and the swim-up technique ( p =0.44). It is suggested that the Percoll gradient technique appears to be preferable to the swim-up technique when semen parameters are normal, but there is no significant difference between these two techniques in abnormal semen.     

Effects of phosphodiesterase-5 inhibitors on sperm parameters and fertilizing capacity

The aim of this review study is to elucidate the effects that phosphodiesterase 5 （PDE5） inhibitors exert on spermatozoa motility, capacitation process and on their ability to fertilize the oocyte. Second messenger systems such as the cAMP/adenylate cyclase （AC） system and the cGMP/guanylate cyclase （GC） system appear to regulate sperm functions. Increased levels of intracytosolic cAMP result in an enhancement of sperm motility and viability. The stimulation of GC by low doses of nitric oxide （NO） leads to an improvement or maintenance of sperm motility, whereas higher concentrations have an adverse effect on sperm parameters. Several in vivo and in vitro studies have been carried out in order to examine whether PDE5 inhibitors affect positively or negatively sperm parameters and sperm fertilizing capacity. The results of these studies are controversial. Some of these studies demonstrate no significant effects of PDE5 inhibitors on the motility, viability, and morphology of spermatozoa collected from men that have been treated with PDE5 inhibitors. On the other hand, several studies demonstrate a positive effect of PDE5 inhibitors on sperm motility both in vivo and in vitro. In vitro studies of sildenafil citrate demonstrate a stimulatory effect on sperm motility with an increase in intracellular cAMP suggesting an inhibitory action of sildenafil citrate on a PDE isoform other than the PDE5. On the other hand, tadalafil＇s actions appear to be associated with the inhibitory effect of this compound on PDE11. In vivo studies in men treated with vardenafil in a daily basis demonstrated a significantly larger total number of spermatozoa per ejaculate, quantitative sperm motility, and qualitative sperm motility; it has been suggested that vardenafil administration enhances the secretory function of the prostate and subsequently increases the qualitative and quantitative motility of spermatozoa. The effect that PDE5 inhibitors exert on sperm parameters may lead to the improvement of the outcome of assi     

Prevention of hypo-osmotic swelling by detergents provides clues to the membrane structure of rat sperm

The proportion of sperm from the caput epididymidis of the rat that swelled in hypo-osmotic media was reduced in a dose-dependent manner by detergents, with relative effectiveness as follows: CTAB greater than Digitonin greater than Hyamine 10-X greater than SDS greater than Triton X-100 greater than Deoxycholate. Polymyxin was a poor lytic agent, possibly because anionic lipids are in the inner leaflet of the membrane. Less digitonin was needed to lyse mature sperm from the distal cauda epididymidis, suggesting that there is less cholesterol in their membranes.     

Effect of milk-yolk on the fertilizing capacity of spermatozoa

Summary.  Pre-incubation of human sperm in egg yolk based medium (TEST-yolk) as well as milk have been shown to improve sperm fertilizing capacity. The purpose of this study was to compare a combination of these two media, called milk-yolk, against the two established media, in order to test for a possible synergistic effect(s) on sperm functions as measured by sperm motility, acrosome reaction and penetration assay. Statistically, no differences have been observed among the three media, except for SPA outcome in milk-yolk, being significantly lower than the corresponding values for the other two media. In conclusion the beneficial effects of egg yolk and milk appear to be eliminated once both media are combined, resulting in an antagonistic effect on the fertilizing capacity of the spermatozoa.     

Pregnancy following intracytoplasmic sperm injection of immotile spermatozoa selected by the hypo-osmotic swelling-test: a case report

Summary.  The success of intracytoplasmic sperm injection (ICSI) is poor when only immotile spermatozoa can be retrieved. In a couple with complete male asthenozoospermia the possible use of the hypo-osmotic swelling test to select spermatozoa for microinjection was examined. Following incubation in hypo-osmotic medium (Hypo 10, IVF Science, Göteborg, Sweden), 26% of immotile spermatozoa showed signs of sperm swelling (HOS-positive). After injection of HOS-positive spermatozoa, 5 out of 12 oocytes fertilized (41%) and after transfer of three embryos a healthy singleton pregnancy was achieved. In a previous ICSI cycle of this couple without preselection of spermatozoa by the HOS test, only 1 out of 10 oocytes fertilized. It is concluded that selection of spermatozoa by hypo-osmotic swelling-test prior to sperm microinjection seems to be a valuable tool to increase the fertilization rate in cases with complete asthenozoospermia.     

Evaluation of sperm cryoprotective media in respect to fertilizing capacity tested in vitro

Summary. Nine different cryoprotectant buffers were tested to measure their protective ability towards main sperm seminological parameters. These were: maintained sperm motility, progressive motility and sperm viability. Out of the nine tested buffers, medium E (TES-Tris without glycerol) and H (glycerol only) showed significantly lower ( P <0.001) values than the rest of the studied buffers in respect to all tested seminological features. The other media did not differ significantly in their cryoprotective abilities to sperm. Richardson's medium (A) preserved sperm viability significantly better ( P <0.001) than the other tested buffers, reaching 63.1% of viable spermatozoa in proportion to the fresh sperm sample before freezing. Three cryoprotectants, A (with egg yolk, no TEST buffer system), D (neither egg yolk nor TEST buffer system), F (TEST-egg yolk buffer system) were further studied for their ability to preserve sperm function in sperm-cervical mucus penetration (Penetrak) and sperm penetration assay (SPA). In our hands, neither supplementation of the buffer with egg yolk nor TEST-egg yolk buffer system promoted sperm capacity in functional tests. A,D,F buffers did not significantly differ among each other in applied functional assays, however, they all diminished ( P <0.001) sperm penetration ratios when compared with fresh sperm samples. Therefore enhancement of sperm capacity to fertilize after equilibration with TEST-egg yolk buffer system should be contested.
Sperm—     

Demonstration of acrosomal membranes using the hypoosmotic swelling test

Summary. The state of the acrosomal membranes in human spermatozoa was studied by means of the hypo-osmotic swelling test and indirect immunofluorescence using anti-boar outer acrosomal membrane antibodies. The swelling phenomenon observed in the acrosomal region was characterized by expansion of the plasma membrane without modification of the outer acrosomal membrane.     

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.     

Sperm-cell volumetric measurements as parameters in bull semen function evaluation: correlation with nonreturn rate

Sperm-cell volume, measured electronically by cell counter, is a parameter providing information about the state and integrity of the plasma membrane by determining cell osmotic reactivity (swelling level). Electronic volume measurement is a modification of the hypo-osmotic swelling test, based on the increase in sperm volume in response to hypo-osmotic stress. In this study the volumetric method was applied to bull ejaculates, and the relationships of volumetric parameters, osmolality of seminal plasma, and concentration of sodium and potassium ions in seminal plasma, with the nonreturn rate (NRR) were examined. Significant correlations were found between volumetric parameters, conventional spermatological parameters, and NRR. The relative volume shift of the mean volume correlated significantly with motility before and after thawing (P < 0.05). NRR correlated significantly with iso-osmotic cell volume (- 0.49; P < 0.05) and with the relative volume shift (0.51, P < 0.05). The prediction level of regression models was improved when volumetric parameters (iso-osmotic cell volume) were included in the multiple regression model. Therefore, using electronic volume measurement as a component for fast, correct and valid (up to 50,000 cells), recording sperm-cell population may help to evaluate ejaculate quality more precisely.     

Chromatin alterations induced by freeze-thawing influence the fertilizing ability of human sperm

A previous cytochemical study revealed that chromatin alterations could be induced in frozen-thawed human sperm. In order to evaluate the biological consequences of such chromatin alterations, we evaluated the relationship between these chromatin alterations and the drastic decrease or loss of sperm fertilizing ability after freeze-thawing. Using acridine orange staining and Feulgen-DNA quantitative microspectrophotometry, the nuclear variables of sperm were compared using samples which had either recovered, or lost their fertilizing ability after freezing-thawing during artificial inseminations, despite good post-thaw motility. There was a clear decrease in Feulgen-DNA content and nuclear surface values in sperm which exhibited a loss of fertilizing ability. Thus freeze-thawing may alter the DNA/nuclear protein relationships and impair the fertilizing ability of human sperm.     

Migration sedimentation technique as a predictive test for the fertilizing capacity of spermatozoa in an in-vitro fertilization programme

The migration-sedimentation technique (MST) has been proposed as a means of separating high quality motile spermatozoa. The present study was conducted in order to evaluate whether sperm performance following separation by MST predicts their fertilizing capacity in an in-vitro fertilization (IVF) programme. Ninety semen specimens were analysed for use in an IVF-embryo transfer (ET) programme. Each specimens was divided into two parts: one was processed in the IVF programme and was used after sperm swim-up separation for insemination of human ova. The other aliquot (0.2 ml) was separated by MST, and the sperm then characterized by their concentration, motility, degree of motility and morphology. Sperm characteristics after separation by MST were then correlated with the results of the IVF-fertilization rates. In 79 of 90 IVF-ET cycles, at least one oocyte was fertilized. All post-MST sperm characteristics were significantly higher in cycles with fertilizations compared to IVF cycles without fertilization. A larger percentage of the total motile spermatozoa were recovered after MST in semen specimens with fertilization, compared to semen specimens without fertilization (39.9 +/- 3.6 and 20.6 +/- 6.6%, respectively; P < 0.05). This value was correlated with the percentage of fertilized oocytes (r = 0.24; P < 0.02). More IVF cycles with fertilizations were recorded in cases in which the recovery of motile sperm was > 25% (P < 0.005), or when more than 1.5 x 10(6) motile spermatozoa were recovered after MST (P < 0.0001). As sperm characteristics after MST correlated significantly with their fertilizing capacity, the MST test could be used in evaluation of the fertilizing capacity of spermatozoa.     

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。     

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。