The correlates and alleged biochemical background of the resazurin reduction test in semen

Reduction of the blue dye resazurin to pink resorufin is used to estimate the concentration of metabolically active spermatozoa in semen samples. In order to quantify the reduction of resazurin, a spectrophotometric method was developed measuring the change from blue to pink in the butanol extracted colour. The biochemical mechanisms involved in the reduction of resazurin by motile spermatozoa and seminal plasma were investigated. Addition of NADH + H⁺ to sperm suspension or seminal plasma increased the reduction of resazurin. The reduction reaction was inhibited by high concentrations of dicoumarol, a specific inhibitor of the diaphorase enzyme, in a dose-dependent manner. It is suggested that the sperm diaphorase enzyme transfers electrons from NADH + H⁺ to resazurin, reducing it to resorufin.
The degree of resazurin reduction was strongly correlated with the concentration of motile spermatozoa recovered from the 90% Percoll fraction ( r  = 0.98, p  < 0.001). A positive correlation was also found between the reducing capacity of seminal plasma ( n  = 62) on the one hand, and sperm concentration ( r  = 0.72, p  < 0.0001), progressive motility ( r  = 0.45, p  < 0.01), normal morphology ( r  = 0.50, p  < 0.01), and γ-glutamyltransferase ( r  = 0.36, p  < 0.05) on the other hand.
These findings, together with our previous observations that the reduction reaction is inhibited by reactive oxygen species and polymorphonuclear white blood cells, increase our understanding of the biochemical basis of the resazurin test and may provide better insight into the interpretation of this test.     

Spectrophotometric application of resazurin reduction assay to evaluate boar semen quality

The resazurin reduction assay depends on the ability of metabolically active cells to reduce the resazurin redox dye to resorufin. In the present study we applied and made a diagnostic evaluation of a spectrophotometric application of the resazurin reduction assay to assess the colour change of resazurin reduction in butanol extracted colour to evaluate boar semen quality. Forty-one samples of boar semen from various breeds were included in the study. The absorption peaks for resazurin and resorufin were found to be 610 and 575 nm, respectively. Absorbance at 610 nm, where the minimum overlap of the two peaks was observed, was used in further analysis. Spearman rank correlation analysis was used to determine the correlation between the resazurin reduction assay and various semen parameters. The highest correlations were observed with the concentration of motile spermatozoa (r = -0.841; p < 0.001), sperm concentration (r = -0.833; p < 0.001), sperm index (-0.826; p < 0.001) and concentration of viable spermatozoa (r = -0.763; p < 0.001). Sensitivity and specificity, at 94.1 and 91.7%, respectively, indicate that the present test is highly accurate in discriminating between the samples according to the sperm index. When motile sperm concentration was used to distinguish between good and poor samples, high sensitivity (93.6%) was also found, whereas the test was only moderately, 80%, specific. The stability of butanol extracts in terms of A610 at different times of measurement confirmed that the resazurin reduction could be spectrophotometrically measured within 7 days from the time of assay performance, making the assay much more useful. Based on these results, the assay could be used as an additional tool for evaluating the quality of boar semen.     

Determination of the diagnostic value of the resazurin reduction assay for evaluating boar semen by receiver operating characteristic analysis

Aim： To assess that metabolic status of spermatozoa could provide a useful tool for evaluation of semen quality. Methods： The accuracy of the spectrophotometric application of the resazurin reduction assay was assessed using receiver operating characteristic （ROC） analysis. Results： Areas under ROC curves （AUC） for motile sperm concentration and sperm index （SI） （sperm concentration multiplied by the square root of percentage sperm motility multiplied by the percentage normal sperm morphology） were 0.922. The best discrimination between poor and good semen samples according to the SI was achieved at a cut-off point of A610 = 0.209, where high sensitivity （94.1%） and specificity （91.7%） were calculated. The assay was less accurate when motile sperm concentration was used as the criterion value, yielding sensitivity of 88.2% and specificity of 87.5%, respectively. Likelihood ratios （LR） indicate that absorbances lower than 0.209 were at least 11.3 times as likely to be found in good semen samples than those in poor according to the SI, whereas in the case of motile sperm concentration, the LR was calculated to be 7.06. Conclusion： These results show that the resazurin reduction assay combined with spectrophotometry is an accurate method of assessing the quality of boar semen.     

刃天青还原分析法在用接收器特性法评估雄猪精液中的诊断意义

目的:确定精子代谢状态可以作为一个有用的精液质量分析工具。方法:用接收器特性法(ROC)评估刃天青还原法联合分光光度计法的精确度。结果:活动精子密度和精子指数(SI)(精子密度×精子活力百分比的平方根×形态正常精子百分比)的 ROC 曲线下面积为0.992。参考 SI 值,采集到的精液样本的质量高低区分点的吸光度为0.209(610nm 波长),该点的敏感性和特异性值分别为94.1%和91.7%。如果以活动精子密度为标准值,这种分析方法的精确度就会降低,敏感性和特异性值分别为88.2%和87.5%。似然比(LR)表明在质量良好的精液样本中测得的吸光度低于0.209,至少相当于在质量不佳的精液样本中测得的数值的11.3倍,但是就活动精子密度而言,计算出的 LR 值为7.06。结论:本研究结果表明刃天青还原分析法联合分光光度计法是一种精确的评估雄猪精液质量的方法。     

Flow cytometric evaluation of the acrosome reaction of human spermatozoa: a new method using a photoactivated supravitaI stain

A flow cytometric assay using a double-stain method for the measurement of the acrosome reaction of human spermatozoa is described. The use of a stable photoactivated stain, ethidium monoazide, allowed evaluation of the viability of spermatozoa. This stain was more stable in fixed samples than propidium iodide, which is not bound covalently to DNA and is therefore removed readily during the washing procedure. The permeabilized acrosome was labelled with Pisum sativum agglutinin conjugated with fluoroisothiocyanate. Since this lectin binds to the acrosome and acrosomal contents, a decrease in the fluorescence intensity allows the cytometric evaluation of the acrosome reaction.
Microscopic analysis and flow cytometric analysis were well correlated and cell sorting was performed to ensure the homogeneity of each different subpopulation encountered.     

Clinical importance of a micro-assay for the evaluation of sperm acrosome reaction using homologous zona pellucida

This study aimed to develop an acrosome reaction assay using microvolumes of solubilized human zonae pellucidae among 35 couples attending an in vitro fertilization programme. The sperm morphology of the men was classified as g-pattern (5-14% normal forms) and/or normal pattern (> 14% normal forms). All the couples had a history of repeated poor or failed in vitro fertilization rates from previous attempts. A zona-induced acrosome reaction test was performed using homologous 0.25 zona pellucida microl-1 incubated with spermatozoa to induce the acrosome reaction. Acrosome reactions were measured with FITC-PSA staining, and expressed as the difference between zona-induced and spontaneous acrosome reaction spermatozoa. The results indicated that microvolumes of solubilized human zona pellucida could successfully be used to determine the acrosome reaction status of spermatozoa. The results were compared with in vitro fertilization rates of metaphase II oocytes, and analysed with the receiver operating characteristics curve. Receiver operating characteristics analyses divided the patients into two groups: i.e. zona-induced acrosome reaction < 15% and > 15%. The sensitivity and specificity for zona-induced acrosome reaction results versus fertilization were 93% and 100%, respectively. The correlation coefficient between zona-induced acrosome reaction and in vitro fertilization was r = 0.94 (P < 0.0001). Zona-induced acrosome reaction data can be used as an indicator for fertilization failure, thus helping clinicians to refine the therapeutic approach for infertile couples prior to the onset of the treatment.     

Effect of freezing–thawing on the expression of mannose-ligand receptors on human spermatozoa: the impact on sperm capacitation and acrosome reaction

The aim of this study was to evaluate the change in the expression of mannose-ligand receptors following a freezing-thawing procedure, in order to assess its impact on sperm capacitation and acrosome reaction. Twenty semen samples were obtained from fertile donors. Sperm samples were divided into two equal volumes. One aliquot was cryopreserved and the other aliquot was incubated at 32 degrees C. After 2 h the frozen sample was thawed and both samples were further incubated at 32 degrees C to allow capacitation. Mannose receptors were examined following 4 and 22 h of incubation using a mannosylated-BSA-FITC probe. The expression of mannose-ligand receptors on the sperm plasma membrane was determined according to the fluorescence pattern: pattern I represents pre-capacitation, pattern II represents capacitated spermatozoa and pattern III represents acrosome-reacted spermatozoa. After 4 h incubation in capacitating medium, the percentages of patterns I, II and III were 90, 7 and 3% for fresh spermatozoa and 89, 8 and 3% for frozen-thawed spermatozoa, respectively (P > 0.05). Following 22 h of incubation, the percentages of patterns I, II and III were 84, 11 and 5 for fresh spermatozoa and 83, 11 and 6% for frozen-thawed spermatozoa, respectively (not significant at P > 0.05). The percentages of patterns II and III in fresh and frozen-thawed spermatozoa were increased by the same magnitude with longer incubation in the capacitating conditions. It was concluded that the freezing-thawing procedure for human spermatozoa does not affect the expression of mannose-ligand receptors and the dynamics of sperm pre-fertilization processes.     

Untersuchungen über die Änderung der Motilität menschlicher Spermatozoen in Abhängigkeit zur morphologischen Qualität/The Alteration of the Motility Rate of Human Spermatozoa and Relationships to Morphological Quality

Die Anzahl der Patienten mit im Normbereich liegenden Spermiogrammparameterwerten wurde durch normwertorientierte Untergruppenbildung herausgearbeitet. Zur weiteren statistischen Bearbeitung wurden Korrelationsanalysen und eine Kovarianz-Selektion durchgeführt. Letztere erlaubte mittels graphischer Darstellung einen näheren Einblick in die korrelative Struktur der Korrelationsmatrix und bestätigte die Ergebnisse der Korrelationsanalyse. Es ergab sich eine positive Korrelation zwischen gesteigertem Anteil pathologischer Spermatozoen und gesteigerter Motilitätsänderung, unter gleichsinniger Beteiligung der verschiedenen Fehlbildungsarten. Besonders groß war die Korrelation zu den Kopffehlbildungen. Die intraindividuelle Schwankungsbreite der einzelnen Spermiogrammparameter war am geringsten bei den verschiedenen Fehlbildungsformen der Spermatozoen, am größten bei der Spermatozoendichte. Die Schwankungsbreite der Initialfructose war, bei detaillierter Betrachtung, gering.
Summary: On the ejaculate of 715 patients with fertility disorders (one spermatogram) as well as on 90 patients (two spermatograms) the alteration of the motility rate comparing the morphological quality of the spermatozoa has been carried out. In some norm orientated subgroups a statistical evaluation (correlation analysis, covariance selection) were determined. We found a positive correlation between increased percentage of pathological spermatozoa and increased motility rate. A special graphic allows more information about the correlative structure of the correlation matrix: this confirms the results of the correlation analysis. On the different malformations of the spermatozoa the intraindividual fluctuation was in the least, but very high on the sperm density. The fluctuation of the fructose content (detailed observed) was very little.     

Properties of sperm separated using Percoll and IxaPrep density gradients. A comparison made using CASA, longevity, morphology and the acrosome reaction

Since the withdrawal of Percoll from use in human assisted reproduction techniques in 1996, alternative density gradients have been sought. IxaPrep is one candidate that has low toxicity and can separate spermatozoa. Fractions of spermatozoa from normozoospermic men, separated by Percoll and IxaPrep gradient centrifugation, were assessed for sperm recovery, motility, morphology and the ability to undergo the acrosome reaction. The Percoll fractions had a significantly higher sperm recovery, motile count, rapid motile count, VAP, VSL, and VCL than those obtained using IxaPrep, both immediately after separation and up to 24 h later. In contrast, ALH, level of abnormal sperm morphology and the ability to acrosome react were not statistically different in sperm fractions separated using either gradient forming material.     

Evaluation of the acrosome reaction and viability in buffalo spermatozoa using two staining methods: the effects of heparin and calcium ionophore A23187

In this study, we investigated the effect of heparin and calcium ionophore A23187 on in vitro induction of buffalo sperm acrosome reaction (AR). Two methods for detection of the AR and viability were employed. Fluorescein isothiocyanate-conjugated Arachis hypogea agglutinin (FITC-PNA) was used as a vital stain in combination with ethidium homodimer-1 (EthD-1) to determine the acrosome status of viable spermatozoa. In another experiment, trypan blue replaced EthD-1 to differentiate live and dead spermatozoa having undergone AR. The results from the two methods were significantly correlated (r > 0.9). Four different staining patterns were found in both methods. The FITC-PNA intensely labels the acrosome region of acrosome-intact spermatozoa. EthD-1 and trypan blue stained red and blue at the post-acrosomal region of dead spermatozoa, respectively. Spermatozoa incubated with heparin showed a significant increase ( p < 0.05) in the percentage of live acrosome-reacted sperm after 30 min incubation period. This trend continued and was significantly different over the entire incubation period when compared with the control group at the same interval. In the ionophore-treated group, the proportion of changes in live acrosome-intact and live acrosome-reacted spermatozoa was statistically significantly different ( p < 0.001) when compared with those treated with heparin at the same interval. The AR occurred sooner and to a greater extent when incubated with the ionophore but at 5 h of incubation the percentage of false acrosomal reaction was significantly higher than those in the control and heparin-treated groups. The results in this study indicated that the in vitro induction of AR by heparin and calcium ionophore evaluated by both methods could be used to assess sperm fertilizing capacity for in vitro fertilization of this species.     

A comparison of the bovine cervical mucus penetration test and the postcoital test

In 68 ejaculates an in vitro bovine cervical mucus penetration-test (Penetrak) was compared to the postcoital-test and the spermiogram. There was a positive correlation between the Penetrak-Test and concentration, motility, and morphology of the spermatozoa, however there was no correlation to the postcoital test. In connection with the PCT, the bovine mucus penetration-test seems to be a useful tool in the exploration of male infertility.     

Separation of human spermatozoa with hyaluronic acid induces, and Percoll® inhibits, the acrosome reaction

The effect on the acrosome reaction of human spermatozoa of two widely used sperm separation media, hyaluronic acid (Sperm Select®) and Percoll®, was studied. Viable and highly motile fractions of human spermatozoa were separated from seminal plasma using self-migration on a Percoll® gradient. After translocation of separated spermatozoa from the Percoll® solution to a culture medium, serum, Percoll® or hyaluronic acid (Sperm Select®) was added to aliquots of the spermatozoa containing culture medium. At increasing time intervals, the influx of ⁴⁵Ca²⁺ into spermatozoa was measured and the concentration of viable spermatozoa that had undergone the acrosome reaction was analysed using the triple stain technique. Serum was found to be necessary to support sperm motility and viability. Compared to culture medium with serum only, addition of hyaluronic acid induced influx of ⁴⁵Ca²⁺ and the acrosome reaction, whilst Percoll® inhibited both of these actions. Hyaluronic acid (Sperm Select®) added to spermatozoa separated by a 'swim-up' method induced, and the addition of Percoll® inhibited, influx of ⁴⁵Ca²⁺ when compared to the addition of culture medium with serum only. This study demonstrates that both hyaluronic acid (Sperm Select®) and Percoll® affect the acrosome reaction and the prerequisite for Ca²⁺ influx in human spermatozoa. These effects should be taken into consideration when using these media for preparation of spermatozoa for insemination or for fertilization in vitro.     

The progesterone‐induced sperm acrosome reaction is a good option for the prediction of fertilization in vitro compared with other sperm parameters

Progesterone (P₄) is crucial for the physiological function of spermatozoa. In the study, we investigated the correlation between P₄‐induced sperm acrosome reaction (AR) and parameters including sperm progressive motility, normal morphology and sperm DNA fragmentation (SDF), and compared the in vitro fertilization (IVF) predictive values of these indicators based on the multivariate regressions analysis and receiver operator characteristics (ROC) curve analyses. The results demonstrated a negative correlation between P₄‐induced sperm AR and the SDF, with the correlation ?9.05 (?17.25 to ?0.84), p<0.05, n = 47). No relationship was found between the sperm progressive motility, normal morphology and the induced AR. The P₄‐induced AR and SDF were both significantly correlated to the fertilization rate. ROC curve analyses indicated that P₄‐induced AR was a better prognostic predictor for the fertilization rate compared with the SDF, with the areas under the curve 0.729 (0.580–0.849), p<0.01 and 0.637 (0.484–0.772), p=0.16 respectively. The cut‐off value for P₄‐induced AR to predict “50% fertilization rate” was 23.4% with sensitivity and specificity of 63.3% and 88.2% respectively. The overall results indicated that the assessment of P₄‐induced AR seemed to be a more sensitive indicator for fertilization rate in vitro compared with other sperm parameters.     

Fluorescence Supravital Stain of Human Sperm: Correlation with Sperm Motility Measured by a Transmembrane Migration Method/Supravital-Fluoreszenz-Färbung von menschlichen Spermatozoen: Korrelation zur Spermatozoenmotilität (Messung mittels Transmembran-Migration-Methode)

We developed a supravital stain of human sperm with fluorescent dyes. Either Hoechst 33,258 or fluorescein isothiocyanate could be used, the former stained sperm head while the later stained the whole sperm. Sperm vitality assessed with any of these two fluorescent dyes correlated well with that determined by eosin-nigrosin counterstain. When sperm vitality was compared with sperm motility measured with a transmembrane migration method, we found that many vital sperm were immotile because sperm vitality was higher than sperm motility in tested samples.     

Small‐volume vitrification for human spermatozoa in the absence of cryoprotectants by using Cryotop

Cryotop is a carrier that has been used successfully in the cryopreservation of human spermatozoa. Here, we explored a novel method to vitrify human spermatozoa without cryoprotective agents (CPAs) using Cryotop. Spermatozoa from 21 Normozoospermic patients were collected and vitrified without CPAs or with sucrose in small volume using Cryotop. The sperm recovery rate, motility, viability, chromatin damage and DNA fragmentation were assessed. No significant difference was observed in the sperm recovery rate and motility rate between the spermatozoa cryopreserved without CPAs and with sucrose. The post‐thawed spermatozoa cryopreserved without CPAs had a higher viability and lower damage to sperm chromatin and DNA than those cryopreserved with sucrose. These results suggest that small numbers of human spermatozoa can be successfully vitrified without CPAs using Cryotop.