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Seasonal Changes of Neutral -Glucosidase Activity in Human Semen

GESA MAAß

HANS-CHRISTIAN SCHUPPE

ANDREAS JUNG

WOLF-BERNHARD SCHILL

From the ^* Department of Urology, Friedrich Schiller University, Jena, Germany; and the Centre for Dermatology and Andrology, Justus Liebig University, Giessen, Germany.

Correspondence to: Dr Ralf Henkel, University of the Western Cape, Department of Medical Biosciences, Private Bag X17, Bellville, 7530, South Africa (e-mail: rhenkel{at}uwc.ac.za).

Received for publication May 10, 2005; accepted for publication July 14, 2005.

	Abstract

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Although there are contradictory reports, the biochemical evaluation of the seminal activity of neutral -glucosidase (NAG) has repeatedly been described as an important parameter to test epididymal patency and is recommended by the WHO. Because, for a number of diagnostic parameters, seasonal variations have been described even in the human, it was the aim of this study to investigate possible circannual changes of NAG. This is an important aspect of andrological diagnosis, as seasonal changes of specific diagnostic parameters might have an impact on the accuracy and predictive power of these parameters, which in turn might have an effect on the therapeutic concept for the patients. In a total of 473 patients, sperm concentration, volume of the ejaculate, total motility, progressive motility, pH value, number of peroxidase-positive cells, concentration of fructose, and NAG as functional markers of the seminal vesicles and the epididymis, respectively, were analyzed according to standard procedures. Seminal activity of NAG was significantly correlated with the sperm concentration (P < .0001), ejaculate volume (P < .0001), and the pH (P = .0025). Moreover, significant (P = .0008) seasonal changes in the activity of seminal NAG with the maximum in spring (76.87 mU/ejaculate) and the minimum in autumn (58.55 mU/ejaculate) were found. The incidence of low-ranged activity of the enzyme was 9.2% in spring, while it was 20.3% in autumn. Thus, our data clearly demonstrate circannual changes of the seminal activity of neutral -glucosidase. This in turn has clinical impact as the predictive power of the test system changes throughout the year.

     Key words: Human spermatozoa, seasonality, ejaculate, epididymal patency

Because, for a number of diagnostic parameters, like sperm concentration, motility, acrosin activity, or chromatin condensation, seasonal variations have been described even in the human (Henkel et al, 2001; Krause and Krause, 2002), it was the aim of this study to investigate possible circannual changes of other parameters. This is an important aspect of andrological diagnosis, as seasonal changes of specific diagnostic parameters might have an impact on the accuracy and predictive power of these parameters, which in turn might have an effect on the therapeutic concept for the patients.

	Materials and Methods

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In this retrospective study, a total of 473 consecutive patients attending the outpatient clinic for fertility problems between January 1998 and January 2000 (age: 17-66 years) were analyzed for sperm concentration, volume of the ejaculate, total motility, progressive motility, pH value, number of peroxidase-positive cells, concentration of fructose, and NAG as functional markers of the seminal vesicles and the epididymis, respectively. All parameters were assessed according to WHO guidelines (1999). For the assessment of seasonal variations, data were grouped according to the calendar month of obtaining the ejaculate and according to the season (spring: March, April, May; summer: June, July, August; autumn: September, October, November; winter: December, January, February) and averages were compared.

In brief, the concentration of fructose in seminal plasma was measured in duplicate after centrifuging the semen sample for 10 minutes at 1000 x g. The seminal plasma was removed and frozen at -20°C until analysis. After thawing, the samples were mixed and diluted (5 µL seminal plasma plus 50 µL distilled water) in an Eppendorf cup. Afterward, 12.5 µL 63 µM ZnSO₄ and 12.5 µL 0.1 M NaOH were added, mixed, incubated at room temperature for 15 minutes, and then centrifuged for 5 minutes at 8000 x g. Fifty microliters of the supernatant were removed, mixed with 50 µL indole reagent (2 µM indole in 16 µM benzoic acid), 500 µL 32% HCl added, covered with Parafilm, and heated for 20 minutes at 50°C in a water bath. After all, the samples were cooled in ice water for 15 minutes. Finally, 250 µL of the solution were pipetted into a 96-well microtiter plate and read at 470 nm in a spectrophotometer (EAR400 ATC, SLT LAB-Instruments, Vienna, Austria). Subsequently, the fructose concentrations were calculated.

For the determination of the activity of NAG, 300 µL ejaculate were centrifuged for 10 minutes at 1000 x g and the supernatant was frozen at -20°C until analysis. After thawing the sample, seminal plasma was diluted 1:5 with 100 mM phosphate buffer, pH 6.8, and centrifuged for 15 minutes at 10 000 x g. Then 10 µL diluted seminal plasma were placed into a microtiter plate well and 100 µL of 0.5% (w/v) 4-nitrophenyl--D-gluco-pyranoside (PNPG) and 1% (w/v) SDS in 100 mM phosphate buffer, pH 6.8, were added, mixed well, and incubated at 37°C for 4 hours. Finally, 100 µL of 0.2 M Na₂CO₃ were added to each well, including the blanks, and the samples were measured in duplicate against standards of p-nitrophenol (0, 10, 20, 40, 60, 80, 100 µM), which are equivalent to an activity of NAG (0, 4.37, 8.75, 17.5, 26.3, 35.0, 43.8 µU/mL), at 405 nm using an EAR microtiter plate reader (EAR 400 ATC).

All statistical calculations (mean, median, Spearman's rank correlation, Wilcoxon test, H test according to Kruskal-Wallis and Fisher's exact test) were performed after checking normal distribution of the data by means of the Kolmogorov-Smirnov test. For the receiving operating characteristic (ROC) curve analysis, azoospermia and -glucosidase (20 mU/ejaculate) have been used as discriminator, respectively. All calculations including ROC curve analysis were carried out with MedCalc (v7.5, MedCalc Software, Mariakerke, Belgium). A value of P < .05 was considered as significantly different.

	Results

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The summary statistics of all analyzed parameters are summarized in Table 1. The high variation of the values for sperm motility and concentration, which can be seen in the high standard deviation, is obvious, as is normal for these biological parameters. While strong significant positive correlations were observed for sperm concentration (r = .399; P < .0001) and the ejaculate volume (r = .497; P < .0001), there was a weaker inverse correlation with the pH (r = -.139; P = .0025). No correlation of NAG activity was found with the patient's age (H test: P = .1658), seminal concentration of fructose, the number of peroxidase-positive cells, and sperm motility (r = .083; P = .1589), though the significance level for progressive motility was close to significance (r = .110; P = .0621). As expected, significant differences in NAG activity were observed between normozoospermic, severe oligozoospermic (sperm count <5 million/mL) and azoospermic patients (Table 2). No differences in seminal NAG activity were observed between normozoospermia and oligozoospermia or oligozoospermia and severe oligozoospermia. For the azoospermic group of patients, both the mean (39.4 mU/ejaculate) and the median (27.6 mU/ejaculate) are higher than the cut-off value of 20 mU/ejaculate given by the WHO (1999).

View larger version (13K): [in this window] [in a new window]   Figure 1. Seasonal variation of seminal activity of the neutral -glucosidase in 473 patients in central Germany. A maximum of neutral -glucosidase (NAG) activity can be seen in April/May, while the nadir is in August/September.

View larger version (13K): [in this window] [in a new window]   Figure 2. Seasonal variation of seminal concentration of the neutral -glucosidase in 473 patients in central Germany. The maximum concentration of 23.16 mU/mL can be seen in April, while the minimum of 13.80 mU/mL is in August. The difference between these 2 months is significant (P = .0016).

With the cut-off value NAG activity (20 mU/ejaculate) given by the WHO (1999), 91.9% of the ejaculates with spermatozoa and 39.1% of the azoospermic semen samples included in this study could correctly be identified. The optimized calculations after ROC-curve analyses are depicted in Tables 3 and 4. While the statistical values (prevalence, sensitivity, specificity, positive predictive value, and negative predictive value) for the assessment of azoospermia with the activity of NAG as variable to be calculated were quite similar for all data groups (all data, spring and autumn), the calculated cut-off values for the seminal NAG activity differed strikingly (Table 3). The cut-off value for NAG activity in spring was 50.5 mU/ejaculate and that in autumn 24.5 mU/ejaculate.

View this table: [in this window] [in a new window]   Table 3. Calculation of the statistical parameters of the determination of -glucosidase (NAG) for azoospermia as differentiation criterion. For the calculation, a distinction was made between spring, autumn, and all data obtained throughout the year. The seasonal difference for the calculated cut-off value for NAG activity is obvious. The prevalence for summer and winter would be 25.9% and 26.5%, respectively

View this table: [in this window] [in a new window]   Table 4. Calculation of the statistical parameters of the determination of azoospermia for (NAG; -glucoside 20 mU/ejaculate) as differentiation criterion. For the calculation, a distinction was made between spring, autumn, and all data obtained throughout the year. The seasonal difference for the prevalence of azoospermia and the positive predictive value of the test is obvious. The prevalence for summer and winter would be 17.6% and 15.7%, respectively

For azoospermia as a variable to be calculated based on the cut-off for NAG activity (20 mU/ejaculate) given by the WHO (1999), all statistical values were quite similar, except for enormous low values for the prevalence (9.38%) and the positive predictive value (28.6%) in spring (Table 4). After calculation of the seminal NAG concentration (mU/mL) from its activity in the ejaculate (mU/ejaculate) and the ejaculate volume and subsequent ROC-curve analysis, a cut-off of 12.8 mU/mL for azoospermia was found. This value was 67.8% specific and 68.2% sensitive for the discrimination between ejaculates containing spermatozoa and those without. The positive and negative predictive values of this test were 39.3% and 87.5%, respectively.

	Discussion

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The epididymis is the main source of -glucosidase in human seminal plasma (approximately 80%; neutral isoform; NAG) (Paquin et al, 1984; Yeung et al, 1990; Peña et al, 2004), while the remaining 20% of the -glucosidase activity in human semen is attributable to the acidic isoform of -glucosidase that originates in the prostate (Paquin et al, 1984). The evaluation of NAG is regarded as a reliable parameter to examine epididymal patency (Guerin et al, 1986; Cooper et al, 1990). Therefore, the World Health Organization (WHO, 1999) considered seminal NAG activity a useful parameter to determine the cause of azoospermia, obstructive or nonobstructive, with testicular origin and suggested a cut-off value of 20 mU/ejaculate. In general, the NAG activities obtained in this study were in the range of those obtained by others (Mahmoud et al, 1998; Krause and Bohring, 1999; Zöpfgen et al, 2000; Comhaire et al, 2002). However, a significant relationship with sperm motility, which was observed by Viljoen et al (1990) and Elzanaty et al (2002), could not be confirmed in this study. Yet, the correlation between NAG activity and progressive motility showed at least a tendency (P = .0621).

In this study, we observed a distinct seasonality of the neutral isoform of -glucosidase with its maximum activity in spring (April/May) and a nadir in autumn (August/September). Minimum and maximum of seminal NAG activity coincided with the respective minima and maxima for sperm concentration (Henkel et al, 2001). Because the NAG activity is routinely used for andrological diagnosis in many centers, we performed ROC-curve analyses to assess the clinical value of the NAG determination as seasonal variations of the parameter might have an impact on its practical value. The calculated cut-off value for seminal NAG activity changed throughout the year, with higher values, reduced specificity, and increased sensitivity in spring. By setting the cut-off value of NAG activity in spring (55.5 mU/ejaculate) to the one of autumn (24.5 mU/ejaculate) or the one for all data (33.0 mU/ejaculate), specificity markedly increases (94.0 mU/ejaculate and 90.0 mU/ejaculate, respectively) and sensitivity decreases (39.3 mU/ejaculate and 46.4 mU/ejaculate, respectively). When applying the cut-off value of 20 mU/ejaculate given by the WHO (1999), the prevalence of azoospermia was markedly lower in spring than in autumn and is indicative of the generally elevated NAG levels in spring. Thus, our results indicate that the predictive power of the assay changes during the year, a fact of which clinicians should be aware of when counseling patients.

The reasons for the seasonality of seminal NAG activity, however, remain speculative. Though an influence of testosterone cannot be ruled out as the neutral isoform of -glucosidase is of epididymal origin and the epididymis is obligatory dependent on this male sex steroid (Orgebin-Crist, 1985; Cooper, 1990), decreased concentrations of the free testosterone will not only lead to morphological alterations of the epididymis but also to its dysfunction (Cooper, 1998). The assumption that the observed circannual variation of seminal NAG activity is due to seasonal changes of epididymal function is supported by the observation that the seminal concentration of NAG (mU/mL) shows the same pattern of variation but not the ejaculate volume.

Seasonality of reproduction is well known among most animals in the animal kingdom, including mammals. The reproductive phase is restricted to specific times of the year in which reproductive success is most likely; that is, when environmental factors like food and water availability or ambient temperature are given and, thus, the conditions are optimum for survival of the offspring. Even though the human is considered as showing no seasonality of reproduction, epidemiological studies demonstrated circannual variations in the birth rates (Becker, 1981; Levine et al, 1990). Even after adjusting for con-founding factors, fertilization rate, embryo quality, pregnancy rate, and birth rate success in in vitro fertilization show some seasonal variation (Stolwijk et al, 1994). Moreover, birth rates also seem to be dependent on the area where people live. For instance, while, in Northern Europe, its peak is in spring with the nadir in autumn, in the USA, most births occur in summer and the minimum is in spring (Lam and Miron, 1994).

Testosterone has repeatedly been shown to exhibit distinct seasonal variations (Abbaticchio et al, 1987; Reinberg et al, 1988; Dabbs, 1990). Our own data obtained in central Germany (not shown) show a maximum of the concentration of total testosterone in April/May and a minimum in autumn. On the other hand, the concentrations of the bioactive free testosterone decrease significantly while aging (Feldmann et al, 2002; Henkel et al, 2005), but no circannual changes could be found for seminal NAG activity. Alternatively, a direct influence of the photoperiod on epididymal function could be discussed, but such an effect has not been reported in the literature thus far. Methodological effects can be excluded because the technique is standardized and is performed under controlled temperature conditions. In addition, the maximum observed in spring does not support a methodological influence of room temperature on the assay, because the month with the highest temperatures is July.

With regard to sperm concentration and sperm motility and vitality, contradictory results have been published. While Mortimer et al (1983) and Ombelet et al (1996) could not find significant seasonal variations of these parameters, others clearly emphasize such an effect (Tjoa et al, 1982; Politoff et al, 1989; Saint Pol et al, 1989; Centola and Eberly, 1999; Andolz et al, 2001; Henkel et al, 2001). Seasonal variations in sperm concentration were clearly not attributable to differences in sexual abstinence (Gyllenborg et al, 1999). Moreover, there is also evidence of seasonal changes of functional sperm parameters, like acrosin activity (Krause and Krause, 2002) and chromatin condensation (Henkel et al, 2001), with maxima in springtime and winter, respectively. For chromatin condensation, inverse courses of variation on the Northern and Southern Hemispheres were observed (Henkel et al, 2001).

In conclusion, a significant seasonality of seminal NAG activity has been demonstrated in this report. Therefore, the predictive power of the test, which is routinely performed in many laboratories to distinguish between obstructive and testicular azoospermia, varies throughout the year. Because only a relatively small number of 470 patients in only 1 country have been examined thus far, the clinical consequences of this circannual variation cannot be estimated yet and more studies investigating this phenomenon should be performed. However, for the time being, clinicians should be aware of it when counseling patients.

	Acknowledgments

 
The authors wish to express their appreciation to Sharon Henkel for her excellent linguistic review.

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