''Water-test'': a simple method to assess sperm-membrane integrity

The functional integrity of the membrane of sperm from semen samples collected from 10 fertile men (Group A) and from 50 infertile men (Group B) was assessed by studying the swelling reaction of sperm when suspended in a medium of distilled water ('Water-test'). The results were correlated with routine semen-analysis and with the results of Eosin-Y staining. The mean values for the 'Water-test' were significantly different (P less than 0.01) between Groups A and B (84.8 +/- 3.5 versus 70.9 +/- 13.3, respectively). No significant correlations were observed in either Group A or B, between values for the 'Water-test' and values for the sperm count, the percentage of sperm with normal morphology and the percentage of motile sperm. There was a good correlation in both Group A (r = 0.86, P less than 0.01) and Group B (r = 0.92, P less than 0.01) between values for the 'Water-test' and those for the Eosin-Y test. These results indicate that the 'Water-test' is a simple and reliable test for evaluating sperm membrane integrity.     

The HOS test and its relationship to fertility in the stallion

The hypo-osmotic test has been used successfully on equine semen and was considered to be a simple and accessible method which could be a useful addition to routine equine semen analysis. It was therefore of interest to determine whether the hypo-osmotic test is significantly correlated to proposed criteria of fertility. The stallions were divided into two groups on the basis of threshold levels of fertility. A significant difference (P<0.05) was found between the two groups for the following parameters: progressive motility, morphologically normal spermatozoa, percentage of swelling with the hypo-osmotic test, percentage of pregnant mares and number of services per pregnancy. The hypo-osmotic test provided a simple evaluation of membrane function and the results obtained show stallions with low swelling scores (<40%) to be of doubtful fertility. The hypo-osmotic test was not correlated with percentage of pregnant mares but showed a tendency to correlate with the number of services per pregnancy, therefore it could be an additional method for evaluating stallion fertility. Further studies are needed to confirm this observation.     

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.     

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)     

不同获能时相对人精子膜完整性的影响

本研究用人精子低渗肿胀试验（ＨＯＳ－ｔｅｓｔ）分析了１２例正常育龄男子的精液标本。精子分别用常规洗涤法、上游法和加入钙离子载体（Ａ２３１８７）诱导获能等三种不同方法处理后，分别在液化后、上游１ｈ后、获能３ｈ、６ｈ、２４ｈ后测定精子的低渗肿胀率，结果显示经不同方法处理、在不同获能时相之后的精子，在２４ｈ之内其肿胀率保持在８７．８±１４．６％～７３．３±１１．９％之间，统计学分析表明各标本组肿胀率之间无明显差异。本研究提示在２４ｈ内精子膜的完整性不受获能时相及不同处理方法的影响。     

Addition of caffeine to equine thawed sperm increases motility and decreases nitrite concentration

The aim of this study was to improve the quality of frozen-thawed equine sperm by the addition of caffeine to it. Semen from nine stallions was frozen and different concentrations of caffeine (3, 5 and 7.5 mM) were added to frozen-thawed semen. The sperm kinetic parameters, membrane functionality and integrity, and acrosome integrity and spontaneous acrosome reacted sperm were evaluated with a computer-assisted sperm analysis, a hypoosmotic swelling test and epifluorescent microscopy, respectively. Nitrite and hydroperoxide concentrations of frozen-thawed semen were measured using spectrophotometry. Sperm fertility was evaluated by artificial insemination (AI) of 16 mares with thawed ejaculates (control and 5 mM caffeine-treated groups). Compared to that in the control, the addition of 5 mM caffeine induced an increase in sperm motility (38.9 ± 2.8 versus 32.6 ± 3.4%), and a decrease in nitrite concentration (11.4 ± 2.1 versus 12.8 ± 2.9 µM/µg protein, p < .05). Moreover, the pregnancy rate from AI in the caffeine group was significantly higher (62.5%) than that in the control group (12.5%). These data suggest that caffeine reduced the nitrite concentration and enhanced sperm motility in thawed equine sperm, thus increasing the fertility rate in mares inseminated with caffeine-treated equine semen.     

Impact of using a fast‐freezing technique and different thawing protocols on viability and fertility of frozen equine spermatozoa

The effects of freezing technique and thawing protocol on thawed semen viability and fertility were studied. Ejaculates from 5 stallions (n = 25) were frozen by conventional or a fast‐freezing technique. Frozen semen was thawed by two thawing protocols (37 °C 30 s^?1 or 75 °C 7 s^?1). Thawed semen was evaluated by progressive motility, vigour, morphology and plasma membrane integrity. Mares (n = 25) were inseminated with 300 (n = 11) or 150 (n = 14) million spermatozoa. A greater (P < 0.05) vigour and progressively motile spermatozoa were detected, respectively, at thawing and after 20 min post‐thawing in the fast‐freezing technique than in the conventional one. Plasma membrane integrity was also greater (P < 0.05) in semen frozen with the fast‐freezing technique. Semen viability was not affected by thawing protocol. Pregnancy rate using the fast‐freezing technique was 76% (19/25), and did not differ (P > 0.05) between insemination doses. We concluded that the 150 million progressively motile spermatozoa per dose using a deep‐horn insemination maximises the use of equine semen. The fast‐freezing technique, as compared to the conventional one, efficiently preserves the viability and fertilising capacity of spermatozoa, indicating a new method to improve the fertility of frozen equine semen.     

Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa

Infertility remains a major problem in society, with recent data suggesting its presence in one of four couples. The objective of the present study was to evaluate the impact of nicotine (0.25, 0.5 and 0.75 mm), as a major component of cigarette smoke, in vitro, on sperm membrane [by spermatocrit and lipoperoxidation (LPO) tests], DNA integrity (by Comet assay), and viability of spermatozoa (by eosin staining) from normozoospermic men. Sperm samples were washed and diluted with phosphate-buffered saline. A drop in spermatocrit values and an increase in thiobarbituric acid-reactive substances/LPO rate was observed with the addition of nicotine, predominantly at a concentration of 0.75 mm, indicating a deleterious effect of nicotine on sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage viable sperm cell (r = -0.990). Data obtained from Comet assay technique revealed that nicotine could induce double-stranded DNA breaks (11% in 0.75 mm concentration) in the sperm nuclei. The value of r between LPO rate and percentage Comets was found to be +0.976. Taken together, nicotine proved to be a potential oxidant agent in the category of environmental factors to the integrity of sperm plasma membrane and DNA.     

The spermatozoal volume as indicative of the plasma membrane integrity (modification of the hypoosmotic swelling test). I. Methods

Summary. The aim of the present study was to establish a more differentiating indicator of plasma membrane integrity of spermatozoa than the classic version of the hypoosmotic swelling test according to Jeyendraan. Spermatozoa were prepared by density gradient centrifugation (90% Percoll) to select 'fertilization competent' spermatozoa only. After a second washing procedure sufficiently pure sperm cell suspensions were obtained. The volume distributions of these sperm cells were measured with a Coulter Counter at 25 °C after adaptation in 300 mosmolar NaCl solution resp. 150 mosmolar NaCl solution for 5 min. These volume distributions showed significantly different patterns for the isotonic and hypotonic stress situation in the simple salt solution. Moreover, the comparison of the response to hypoosmotic stress showed more than four reproducible characteristic patterns, promising well differentiated results for different sperm populations. The new method for the detection of hypoosmotic swelling effect might be a real and valuable functional parameter.     

硝苯地平对人精子膜功能完整性及压力敏感性Ca~(2+)内流影响的实验研究

目的:探讨模拟正常血药浓度下硝苯地平体外对人精子膜功能完整性及压力敏感性Ca2+内流的影响。方法:将10例生育男性的精液经上游优化处理后,分别与20、100、20×103ng/ml的硝苯地平在体外共同孵育60min,同时设立对照组进行低渗肿胀(HOS)试验及测定压力导致的精子细胞内钙离子浓度的变化。结果:20×103ng/ml组的精子HOS百分率明显低于对照组,差异具有显著性(P<0.01)。20、100、20×103ng/ml组的精子细胞内Ca2+荧光差值均明显低于对照组,差异具有显著性(P<0.01)。结论:硝苯地平在体外可以改变精子膜功能完整性,抑制压力敏感性Ca2+内流。治疗剂量的硝苯地平在体外可影响精子质量。     

The spermatozoal volume as indicative of the plasma membrane integrity (modification of the hypoosmotic swelling test). II. Diagnostic approach

Summary. If using a method of statistical sifting based on the Kolmogoroff-Smirnov test for differentiation of volume distribution curves of a modified HOS-test, different curve types are obtained under isotonic as well as under hypotonic conditions. Arrangement of spermatological parameters of the relevant ejaculates according to the individual curve types reveals significant differences for motility. No correlation can be seen with the results of the microscopic HOS-test. Consequences for the use of the modified HOS-test in medical practice are discussed.     

Comparison of different mathematical models to assess seasonal variations in the longevity of DNA integrity of cooled-stored stallion sperm

Dynamic assessment of sperm DNA fragmentation (SDF) has shown to give fuller understanding of stallion semen quality; however, there have been limited attempts to use this parameter to investigate seasonal changes in productive functions. The aims of this study were to: (a) establish a reliable mathematical model to describe the longevity of cooled-stored sperm DNA integrity; (b) to examine the effect of seasonal variations on SDF. Ejaculates were cooled to 5°C, and SDF was analysed after 0, 6 and 24 hr of storage. The coefficient of determination (R²) was calculated after fine-tuning linear (LIN), exponential (EXP) and second order polynomial (POL) models. R² was significantly higher (p < .001) for POL than for LIN and EXP. The rate of DNA degradation was calculated using the slopes of POL equations. After assessing the rate of change of the POL functions, significant differences between the acceleration of DNA fragmentation were found (p < .01) among seasons, being higher for winter and summer than spring and autumn. In conclusion, DNA analysis of stallion sperm fits better to a second order polynomial mathematical model, being spring the best season to collect and process cooled stallion semen in order to maintain the DNA integrity of the stallion sperm.     

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.     

An evaluation of the hypo-osmotic sperm swelling test as a predictor of fertilizing capacity in vitro

The ability of sperm to swell in hypo-osmotic conditions was examined in 211 semen samples from the partners of patients about to undergo oocyte retrieval for in-vitro fertilization (IVF). The test was performed using aliquots of semen, the remainder of which was then prepared for IVF. No significant difference was found, in either the percentage of swollen sperm or the type of swelling response, between samples that achieved fertilization in vitro and those that did not, or between any of the diagnostic categories of infertility (tubal damage, unexplained infertility, oligospermia). In samples which achieved fertilization in vitro there were correlations between sperm swelling and sperm motility (r = -0.51) and abnormal morphology (r = 0.33), but no such correlations were demonstrated in samples that failed to achieve fertilization. Moreover, there was no significant difference between the percentage of swollen sperm in semen (mean motility 64%), in samples immediately after preparation for IVF (mean motility 96%) or in capacitated sperm 24 h after preparation (mean motility 91%). These results demonstrate that the hypo-osmotic sperm swelling test does not assist in the prediction of the fertilizing capacity of human sperm in vitro.     

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.     

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.     

Cryodamage to plasma membrane integrity in head and tail regions of human sperm

Aim: To investigate the effect of cryopreservation on the plasma membrane integrity in the head and tail regions ofindividual sperm, and the relationship between intact cryopreserved sperm and its motility and zona-free hamster oocytepenetration rate. Methods: The eosin Y exclusion and the hypoosmotic swelling tests were combined to form a sin-gle test (HOS-EY test) to identify the spermatozoa with four types of membrane integrity. Results: After cryop-reservation, there was a marked decline in the percentage of spermatozoa with Type Ⅳ membrane integrity (head mem-brane intact/tail membrane intact), and a significant increase in those with Type Ⅰ (head membrane damaged/tail mem-brane damaged) and Type Ⅲ (head membrane damaged/tail membrane intact) membrane integrity (n = 50, P <0.01). The value of Type Ⅲ integrity had a wide range of variability, whereas Type Ⅱ (head membrane intact/tailmembrane damaged) was uncommon after thawing. A high correlation was observed between the percentage of Type Ⅳin     

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.     

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.