The effect of colloid osmotic pressure in human spermatozoa exposed to hypoosmotic conditions

The use of a protein source such as serum and albumin had been extensively employed as supplements of culture media for handling and culture of gametes and embryos. Protein molecules behave as colloids in solution and contribute to the osmotic pressure of fluids. The interaction of proteins in solution and spermatozoa needs to be assessed in order to determine their possible role in osmoregulation. The aim of this study was to assess possible osmoregulatory mechanisms of protein supplementation against exposure to hypoosmotic conditions by assessing the sperm's response to those environments. A stock hypoosmotic solution (HOS) was prepared by using a mixture of fructose and sodium citrate and adjusted to an osmotic pressure of 150 mOsm l-1. Another stock solution was prepared by diluting a preparation of synthetic serum supplement [SSS; 6% (v/v) total protein] with distilled water to obtain an osmotic pressure of 150 mOsm l-1 (hypoosmotic SSS or H-SSS). Three additional solutions were prepared by mixing the stock HOS and H-SSS solutions in the following proportions (v/v): (i) 75% H-SSS/25% HOS, (ii) 50% H-SSS/50% HOS and (iii) 25% H-SSS/75% HOS. Aliquots of washed spermatozoa from 18 men were diluted 1 : 10 (v/v) with each of the testing solutions and incubated for 60 min. Specimens were assessed on wet mounts for total and specific swelling patterns. Swelling patterns were classified as maximal (>50% tail length swollen) and minimal (<50% tail length swollen) swelling with or without associated sperm motility. The major finding of this study was that increasing the concentration of protein supplementation resulted in a decrease in the proportion of maximal sperm tail swelling patterns and an increase in the proportion of minimal tail swelling patterns. A proportion of spermatozoa which exhibited minimal swelling patterns were still motile in all solutions tested, and the percentage of those spermatozoa increased as the protein supplementation was also increased in the testing solutions. Incorporation of protein supplementation as described in this study delays the effect of sperm swelling in hypoosmotic conditions.     

Correlation between human sperm swelling in hypoosmotic medium (hypoosmotic swelling test) and in vitro fertilization

Human ejaculates (n = 83) were analyzed for standard sperm parameters (concentration, motility, and morphology), as well as for the ability of the spermatozoa to react (swell) in a hypoosmotic medium (Jeyendran et al, 1984). Subsequently, the fertilizing capacity of the spermatozoa was tested by their ability to fertilize human oocytes in vitro. Although the sperm concentration was adjusted for in vitro fertilization, no adjustments were made for sperm motility and morphology. Correlation of the in vitro fertilizing capacity of the spermatozoa with the hypoosmotic swelling test (r = 0.56) was much higher than with standard sperm parameters (r varied from -0.04 to 0.25). Complete overlap was noted with standard semen parameters whether the ejaculate did or did not fertilize oocytes and ranged from very low to very high values in both cases. By contrast, all the semen samples that fertilized oocytes showed a 60% or higher reaction in the hypoosmotic swelling test, whereas the majority of the "infertile" semen samples showed less than 60% swelling. It therefore appears that, under the conditions of our studies, the hypoosmotic swelling test is a more accurate predictor of successful in vitro fertilization outcome than the conventional semen parameters. A combination of all parameters, however, is likely to be most useful. The hypoosmotic swelling test is simple and economical, and it is recommended that this test be further scrutinized for its value as an additional tool in the assessment of the in vivo fertilizing capacity of ejaculated spermatozoa.     

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。     

Does the hypoosmotic swelling test predict human sperm viability?

Human sperm viability is essential for successful fertilization. Eosin Y is the usually accepted method for sperm viability assessment, though the hypoosmotic swelling test has been proposed for the selection of viable spermatozoa in procedures such as intracytoplasmic sperm injection. The present study was designed to determine the value of hypoosmotic swelling test in the prediction of sperm viability. For this purpose, hypoosmotic swelling and eosin Y were performed in parallel and in combination, on both fresh and freeze-thawed semen. Rates for eosin Y were significantly higher than for the hypoosmotic swelling test in fresh semen, with a weak, though significant correlation between the two tests (r = 0.47, p < 0.05). When both tests were performed in succession (hypoosmotic swelling test followed by eosin Y), 14.6% of swollen sperm incorporated the dye. Following exposure to hypoosmotic conditions, sperm viability decreased by 35%. When sperm were killed by freezing, hypoosmotic swelling test rates were higher than eosin Y. Results indicate that these two tests cannot be used interchangeably, since 15% of the swollen sperm apparently died, suggesting that plasma membrane integrity is lost before the capacity to maintain osmotic equilibrium.     

蒸馏水在精子计数和低渗肿胀试验中的应用

目的:探讨蒸馏水在精子计数和低渗肿胀试验中的应用。方法:收集37份精液标本,每份标本分别用蒸馏水和碳酸氢钠-甲醛稀释液作1∶20稀释,然后用血细胞计数池进行计数并比较,同时记录蒸馏水稀释时精子肿胀百分率。另收集26份精液标本用于低渗肿胀试验结果的比较。结果:蒸馏水作为计数稀释液与碳酸氢钠-甲醛液相比,精子计数结果差异无显著性(P>0.05)。蒸馏水稀释时所得的精子肿胀百分率与WHO推荐的低渗肿胀试验结果亦无差异(P>0.05)。结论:蒸馏水不仅可取代碳酸氢钠-甲醛稀释液作为精子计数稀释液,亦可取代低渗肿胀液用于低渗肿胀试验分析。     

Diagnostic value of sperm function tests and routine semen analyses in fertile and infertile men

The results of routine semen analyses, the zona-free hamster oocyte penetration test, the hypoosmotic swelling test, and semen adenosine triphosphate levels were studied in 66 fertile and 130 infertile men. Multivariate discriminant analysis demonstrated that routine semen parameters including semen volume, sperm count, percent sperm motility, and percent normal spermatozoa in combination could predict the fertility of these patients with 70.4% accuracy. Of the three sperm function tests evaluated, the zona-free hamster oocyte penetration test and the hypoosmotic swelling test were selected by the multivariate discriminant analysis as variables capable of providing significant information on the fertility status of the patients. However, the addition of the results of these two tests to the routine semen analysis did not significantly improve the predictability of fertility. The overall correct prediction rate was 77.6% after incorporation of the results of these two sperm function tests. In this group of subjects, the presently available sperm function tests did not predict the fertility status of a patient with a high degree of accuracy.     

Relationship between hypoosmotic swelling test, semen analysis, and zona-free hamster ovum test

The hypoosmotic swelling test is a simple test for measuring the functional competence of the human sperm membrane. Fifty-four patients with idiopathic infertility were assessed by hypoosmotic swelling test and the results were compared with those of routine semen analysis and zona-free hamster ovum human sperm penetration test (ZSPT). Semen samples with abnormal semen parameters had lower percentage of swollen sperm in comparison with those with normal semen parameters. A positive correlation was observed between sperm concentration and sperm swelling (r = .50, p less than .05). A strong positive correlation was observed between the percentage of sperm motility and the percentage of swollen sperm (r = 0.60, p less than .01), and between motile sperm concentration and sperm swelling (r = .62, p less than .01). On the other hand, sperm swelling correlated only weakly with the percentage of sperm penetration. The results indicate that the hypoosmotic swelling test appears to evaluate different functional qualities of sperm than ZSPT.     

Evaluation of computerized analysis of sperm movement characteristics and differential sperm tail swelling patterns in predicting human sperm in vitro fertilizing capacity

Multivariate discriminant analysis was used to evaluate the usefulness of computer image analysis of sperm movement characteristics and differential patterns of sperm tail swelling after hypoosmotic treatment for predicting the human sperm in vitro fertilizing capacity assessed by the zona-free hamster egg penetration assay. Fifty-five semen samples, mostly normospermic, from untreated infertility clinic patients were analyzed. The % normal sperm morphology, linearity of seminal sperm movement, seminal sperm head beat frequency, mean and maximum amplitudes of lateral head displacement, and hypoosmotic sperm tail swelling patterns c, d and f were selected by multivariate discriminant analysis to be capable of discriminating the samples exhibiting the presence or the absence of sperm in vitro fertilizing capacity. The % total sperm tail swelling did not give additional information about in vitro fertilizing capacity. These preliminary data suggest that computer image analysis of sperm movement characteristics and differential evaluation of hypoosmotic sperm tail swelling might be useful for the prediction of human sperm fertility. Further prospective studies are necessary to validate their predictive functions.     

Hypoosmotic swelling test in patients with varicocele.

The hypoosmotic swelling test is a simple test for measuring the functional competence of human sperm membrane. Thirty-five patients with varicocele were assessed by hypoosmotic swelling test and the results were compared with those in patients with idiopathic infertility. Percentage of swollen sperm in varicocele tended to be lower than that in idiopathic infertility, especially in sperm concentration between 30 x 10(6)/mL and 40 x 10(6)/mL. Varicocele with sperm motility of 40% or more revealed lower percentage of swollen sperm than idiopathic infertility did. The results indicate that varicocele may particularly injure sperm membrane function because hypoosmotic swelling test is a test for measuring functional ability of human sperm membrane. The percentage of swollen sperm recovered after the operation earlier than did the other sperm parameters such as sperm concentration or motility, which indicates that impaired sperm membrane injury by varicocele is reversible.     

Comparisons of sperm quality, morphometry and function among human sperm populations recovered via SpermPrep™ II filtration, swim-up and Percoll density gradient methods

Summary. The purpose of this study was to compare the morphology/morphometry and fertilizing capacity of human spermatozoa recovered via swim-up method, Percoll density gradient method, and SpermPrep™ II filtration method. Thirty-three ejaculates were equally divided into 2 aliquots. Aliquot 1 was processed via the direct swim-up method, whereas aliquot 2 was filtered via a SpermPrep™ II column. The Percoll density gradient method was compared with the SpermPrep™ II method in a similar protocol using 43 ejaculates. Sperm populations recovered via the SpermPrep™ II filtration method showed significantly higher hypoosmotic swelling test results, acrosin profiles, and percentage of hyperactivated spermatozoa than sperm fractions recovered by the swim-up method. Furthermore, significant differences were found in most of sperm morpho-metric parameters between the above sperm populations. However, sperm fractions recovered via the SpermPrep™ II method did not show significantly different values for these same tests and for most of sperm morphometric parameters compared to the Percoll density gradient method. These results suggest that the SpermPrep™ II filtration and Percoll density gradient method are equally efficient in isolating sperm subpopulations with better functional parameters than the swim-up method.     

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.     

含雌激素的冷冻保护剂对少弱精子玻璃化冻存效果的研究

目的 比较添加雌激素的冷冻保护剂对少弱精子的冻存效果.方法 将40例少弱精子患者的精液标本分别采用甘油、甘油-卵黄-柠檬酸钠（GYC）、甘油＋雌激素（甘油＋ E2）、甘油-卵黄-柠檬酸钠＋雌激素（GYC＋ E2）四种冷冻保护剂（CPM）冷冻,比较冻融前后精子活率、活力（a＋b级）、正常精子形态百分率、精尾低渗肿胀率（HOSR）.结果 四种CPM对少弱精子的冷冻效果存在差别,精子活率、活力、正常形态百分率、精子尾部低渗肿胀率在冻后都有不同程度下降（P〈0.05）,但甘油＋E2、GYC＋ E2 能显著提高冻存后精子的活率及活力（a＋b级）.结论玻璃化冻存对精子存在损伤,含雌激素的冷冻保护剂可以提高低质量精液的冻存效果.     

Oocyte Penetration and Acrosome Reactions of Human Sperms II: Correlation with other Seminal Parameters

Washed sperm suspensions of fertile men and men consulting for infertility were evaluated for their ability to penetrate zona-free hamster eggs and for their ability to exhibit an acrosome reaction in vitro. Furthermore, the swelling of the spermatozoa under hypoosmotic conditions as indication of their membrane integrity was determined. In the group of fertile men and in the group of patients with normal spermiogram, significantly more acrosome reactions were observed than in the group of infertile men with abnormal spermiogram parameters (p less than 0.05). This difference was still more significant when men with a positive hamster penetration test (H.O.P. test) and men with a negative H.O.P. test were compared (p less than 0.005). However, within the groups the level of acrosome reactions after incubation appeared to be highly variable. In a second series of experiments, working with semen obtained during our in vitro fertilization program, we found that the fertilization of human ova does not seem to be dependent on a strong progress of the acrosome reaction. Finally, swelling of the spermatozoa in a hypoosmotic medium was weakly correlated (r = 0.70, p less than 0.01; n = 73) with trypan blue exclusion. No significant correlations with other semen parameters, hamster ova test included, were found.     

Osmotic tolerance limits and properties of rhesus monkey (Macaca mulatta) spermatozoa

Fundamental cryobiological characteristics of rhesus spermatozoa must be determined for successful cryopreservation techniques to be established. The main objectives of the present study were to determine the osmotic behavior and osmotic tolerance limits of rhesus macaque spermatozoa. Cell volume changes over anisotonic conditions were assessed using an electronic particle counter and sperm motility was evaluated with a computer-assisted sperm analysis system. Analysis of membrane integrity and mitochondrial membrane potential was performed using flow cytometry. Rhesus monkey spermatozoa behave as linear osmometers in the osmotic range tested (75-900 mOsmol kg(-1)), as shown by the Boyle van't Hoff plot (r(2) =.99). Rhesus spermatozoa have a mean cell volume of 36.8 +/- 0.5 micro m(3) at 22 degrees C, with 77.2% of the intracellular volume being osmotically inactive. Results regarding sperm tolerance to osmotic stress showed that sperm motility was more sensitive than membrane integrity to deviations from isotonicity and, in addition, that rhesus sperm motility and membrane integrity were more sensitive to hypertonic than hypotonic conditions. Mitochondrial membrane potential did not explain the lack of sperm motility observed under anisosmolal conditions in our study. Although most spermatozoa were able to recover initial volume after osmotic stress, they were not able to recover initial motility.     

Intracytoplasmic sperm injection with motile and immotile frozen-thawed testicular spermatozoa (the Hungarian experience)

The authors summarize their experience in 75 in vitro fertilization cycles, where frozen-thawed testicular spermatozoa were used for intracytoplasmic sperm injection. In 32 cases, motile spermatozoa could be observed in the frozen-thawed sample. In 34 cases, motility could be induced by pentoxifylline and in nine cases immotile spermatozoa, selected with hypoosmotic swelling test, were used for fertilization. The fertilization rates obtained with motile and immotile spermatozoa (66.1% versus 52.3%) were not significantly different. Our data demonstrate that freezing of testicular spermatozoa opened new possibilities for the treatment of azoospermic men. The clinical pregnancy rate per embryo transfer (ET) (21.87%) was comparable with previous results use of fresh testicular spermatozoa (27.7%). The quality and number of transferred embryos had the most significant impact on the pregnancy rate. The fertilization rate and frequency distribution of good-quality embryos were lower in the case of immotile spermatozoa, and pregnancies were only achieved when motile spermatozoa had been used.     

Effects of putative epididymal osmolytes on sperm volume regulation of fertile and infertile c-ros transgenic Mice

Volume regulation by spermatozoa has been demonstrated to be crucial in both mice and men for transport in the female tract. In order to determine the nature of osmolytes used by spermatozoa, they were released from the cauda epididymis of fertile c-ros heterozygous mice into incubation medium of uterine osmolality (representing an osmotic challenge), containing increasing concentrations of compounds that are major epididymal fluid components and known osmolytes in somatic cells. This should nullify the concentration gradients for osmolytes that mediate volume regulation, prevent osmolyte efflux, and lead to swelling. Of the osmolytes tested, K(+) caused the most rapid and extensive volume increases; glutamate, taurine, L-carnitine, and myo-inositol also were effective, but glycerophosphocholine was not. Such effects were not observed in cauda sperm from the infertile knockout mice, demonstrating a defect in normal volume regulation. K(+) concentrations in cauda epididymal fluid were 21 mM higher in the knockout than the heterozygous mice, but no differences were found in caudal fluid glutamate, carnitine, or myo-inositol. The carnitine content of cauda sperm from knockout males was not different from that of fertile males, but lower amounts of glutamate and inositol were found that could explain the poor volume regulation. In heterozygous mice, cauda but not caput sperm responded to the K(+) channel blocker quinine by swelling, demonstrating development of volume regulation during epididymal transit, whereas knockout cauda sperm showed no response, as with the osmolytes. Major epididymal secretions could serve as osmolytes in murine spermatozoa for volume regulation in response to physiological osmotic challenge in the normal fertile mice; the reduced sperm content of inositol and glutamate in the c-ros knockout mice might reflect maturational abnormalities in volume regulation.     

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。     

Treatment of male infertility: current situation and problems]

We evaluated the results of treatment for male infertility in 883 patients who visited our clinic. The efficacy rate and pregnancy rate after varicocele operation were higher than those after drug therapy. Drug therapy was effective only in patients with a sperm count of 10-40 x 10(6)/ml. The percentage of swollen sperm in hypoosmotic swelling test elevated after operation in varicocele patients. The hypoosmotic swelling test seems to be useful to show the improvement of sperm function by treatment.     

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.     

Relationship of cytoplasmic droplets to motility, migration in mucus, and volume regulation of human spermatozoa

A significantly greater percentage of motile than immotile spermatozoa bore droplets at the osmolality of semen and cervical mucus. The percentage of spermatozoa with droplets was not significantly correlated with the osmolality of semen or the extent of cell swelling in response to quinine in hypotonic medium. Droplets appeared slightly more frequently (ie, were more obvious) in the presence of quinine, which blocks regulatory volume decrease, indicating that they are the major site of volume expansion. There was no selection for or against droplet-bearing spermatozoa migrating through viscous surrogate mucus at high or low osmolality. Sperm swelling in response to quinine at mucus osmolality was significantly greater in fathers than in patients whose partners had no fertility problem. Therefore, cytoplasmic droplets are not deleterious to sperm motility and may be related to physiological volume regulation, which may be predictive of some forms of human male infertility.