Relationship between semen quality and the seminal plasma components carnitine, alpha-glucosidase, fructose, citrate and granulocyte elastase in infertile men compared with a normal population

A. Zöpfgen1, F. Priem2, F. Sudhoff1, K. Jung1, S. Lenk1,3, S.A. Loening1 and P. Sinha2

1 Department of Urology and 2 Institute of Laboratory Medicine and Pathobiochemistry, University Hospital Charité, Humboldt University, Berlin, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The seminal plasma components neutral {alpha}-glucosidase, carnitine, fructose, citrate, and polymorphonuclear granulocyte (PMN) elastase in 253 men were determined. The seminal plasma of 221 infertile men, a control group with proved fertility and 13 patients after vasectomy were investigated. The concentrations of free carnitine (212 versus 521 µmol/l, n = 219, P < 0.001), total carnitine (437 versus 743 µmol/l, n = 219, P < 0.001), and the activity of neutral {alpha}-glucosidase (15.1 versus 23.4 IU/l, n = 236, P < 0.05) were significantly reduced in the infertile patient group as compared to the fertile control group, the concentration of PMN elastase (102 versus 48 µg/l, n = 234, P < 0.05) being significantly increased in the infertile patients. In the patients after vasectomy the activity of neutral {alpha}-glucosidase was the only epididymal marker that was significantly reduced (4.3 versus 9.8 IU/l, n = 35, P = 0.002) in comparison with the patients with testicular azoospermia. At a limit of 6 IU/l the sensitivity of the method was 92% and the specificity was 72%. Altogether, the epididymal markers were reduced in subfertile patients compared with the control group. For the differential diagnosis of azoospermia only the determination of the neutral {alpha}-glucosidase activity is useful.

Key words: biochemical markers/carnitine/{alpha}-glucosidase/male infertility/seminal plasma


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In the seminal plasma, after separation of the cellular components, various chemical substances are to be found, which can be assigned to specific organs or compartments of the male genital system. Those substances can thus serve as diagnostic indicators [World Health Organization (WHO), 1993].

Neutral {alpha}-glucosidase, carnitine, and glycerolphosphocholine are markers of epididymal function (Cooper et al., 1990Go; WHO, 1993Go). Fructose and prostaglandins are mainly generated in the seminal vesicles, and the determination of citrate, zinc, and of the prostate-specific acid phosphatase enables the prostatic function to be evaluated (WHO, 1993Go). Polymorphonuclear granulocyte elastase (PMN elastase) in the seminal plasma, which is released by degranulation of polymorphonuclear granulocytes, can be used for diagnosing and observing the course of a clinically quiescent infection of the spermatic vessels (Jochum et al., 1986Go; Reinhardt et al., 1997Go).

Although clinical evaluations are available with respect to the individual parameters (Wetterauer, 1986Go; Cooper et al., 1988Go; Wolff et al., 1991Go; Reinhardt et al., 1997Go), these components were rarely compared or if so, the comparison was based on a small number of biochemical measurements. Therefore, it was the aim of the present study to compare the concentrations of neutral {alpha}-glucosidase, total carnitine and carnitine fractions, free carnitine and acetylated carnitine, fructose, citrate, and PMN elastase in seminal plasma of infertile patients with the respective values measured in a control group and thus to determine the diagnostic validity of these markers as additional tools of the classic spermiogram.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
Samples of seminal plasma of a total of 253 men with an average age of 33.2 years (18–59) were obtained. In all, 221 men presented at our department because they had failed to achieve conception in >2 years. In addition seminal plasma of 13 patients after vasectomy and of 19 patients with proven fertility was investigated. All 13 men after vasectomy had azoospermia (obstructive azoospermia). The spermiograms of the 19 fertile men in the control group were normal.

The 221 infertile men were subdivided according to their spermiograms (as recommended by WHO, 1993): normozoospermia, oligozoospermia, asthenozoospermia, teratozoospermia, OAT (oligoasthenoteratozoospermia) syndrome, azoospermia. Testicular azoospermia was proved by testicular biopsy in 22 of the patients with azoospermia.

Retrieval and preparation of ejaculates
Retrieval, analysis and classification of the ejaculates were performed according to the WHO recommendations (WHO, 1993Go).

Samples were obtained by masturbation into a sterile plastic vessel with a preparatory period of at least 3 days. After physical examination of the ejaculate (pH, volume, consistency, aspect), sperm motility after 60 min was determined microscopically at 400-fold magnification from the native sample and assigned to the categories (i) vividly motile, (ii) moderately motile, and (iii) immotile. Sperm count and concentration of round cells were determined in the counting chamber Thoma (depth 0.100 mm, volume 0.0025 mm3) after dilution with a bicarbonate, formalin, and gentian violet solution. The number of peroxidase positive cells was determined using a conventional staining technique with benzidine and cyanosine (Ludwig et al., 1996Go). The vitality of the spermatozoa was determined by staining with eosin. The differentiation between normal and pathological spermatozoa followed the WHO recommendations (WHO, 1993Go). Spermatozoa were considered normal if there were neither defects of the head (length 4.0–5.5 µm, breadth 2.5–3.3 µm, shape oval, ratio of length to breadth 1.5–1.75, acrosome easily distinguishable) nor defects of the neck, tail or centre part. One hundred spermatozoa were counted.

The seminal plasma was centrifuged at 3000 g for 15 min within 2 h after sampling. The supernatant was carefully removed and stored at –80°C before biochemical analysis.

Biochemical analysis
Neutral {alpha}-glucosidase, fructose, and citric acid were determined with commercially available test kits from Boehringer Mannheim GmbH (Mannheim, Germany). For determining neutral {alpha}-glucosidase, 4-nitrophenyl-{alpha}-D-glucopyranoside was converted into 4-nitrophenol and {alpha}-D-glucopyranoside, and 4-nitrophenol were photometrically measured. The acid isoenzyme was inhibited by addition of 1% sodium dodecyl sulphate and pH 6.8 phosphate buffer, so that only the neutral isoform was measured (Cooper et al., 1990Go). Fructose was determined according to the hexokinase method (Kunst et al., 1984Go) and citric acid with the ultraviolet method using the citrate lyase catalysed reaction (Möllering, 1985Go).

Granulocyte elastase was measured with the homogeneous immunoassay ECOLINE® PMN elastase (Merck, Darmstadt, Germany). For this purpose, seminal plasma was mixed with latex particles coated with antibody fragments [F(ab')2] against human PMN elastase, and after agglutination the opacity proportional to the elastase concentration was photometrically measured.

Free, acetylated and total carnitine were determined according to a radiometrical method (McGarry and Foster, 1976Go). With this technique, carnitine, after addition of [14C]acetylcoenzyme A and carnitine acetyltransferase, was completely transformed into acetylcarnitine. Subsequent treatment of the reaction mixture with anion-exchange resin Dowex 1-X10 removed the highly negatively charged [14C]acetyl-CoA. The positively charged [14C]acetylcarnitine remained in solution and was determined using an LKB-Wallac 1410 liquid scintillation isotope counter. A reference curve was used to obtain the carnitine content of the sample by calibration. Total carnitine was determined after previous saponification with 200 mmol/l NaOH.

Statistical evaluation
Data were statistically evaluated with the Statistics Package for Social Sciences (SSPS) 7.5 program for Windows (SPSS Inc., Chicago, USA). The Kolmogorov–Smirnov test was used to test the goodness of fit to the normal or non-normal distribution of values. The significance of the differences within the individual groups of the test population was evaluated with the Kruskal–Wallis test for non-normally distributed parameters and with the analysis of variance (ANOVA) test for normally distributed values. To answer one-sided questions, the one-sided significance was given. In the rest of the cases the two-sided significance was determined. For detecting a statistical correlation between two variables Spearman's coefficient of correlation of ranked data was calculated. Receiver operating characteristics (ROC) curve analysis was used to identify criterion values and to determine the discrimination power between two groups (Kairisto and Poola, 1995Go). The measured values were given as median values and interquartile range.


    Results
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 Materials and methods
 Results
 Discussion
 References
 
Biochemical parameters in seminal plasma of the control group and of infertile patients
Figure 1Go shows the data of the individual groups in the form of box plots. As not all patients in every subgroup could be measured for each parameter, the real number of patients investigated in each subgroup was given at the bottom of the figure. Total carnitine was not included in the figure because it behaved almost the same way as free carnitine. The results can be summarized as follows:

  1. When comparing infertile men having normozoospermia (n = 40) with the control group (n = 9), we found significantly reduced concentrations of free carnitine (295 versus 521 µmol/l, P < 0.001) as well as total carnitine (513 versus 743 µmol/l, P < 0.001) and significantly increased concentrations of PMN elastase (144 versus 48 µg/l, P < 0.05) were found in the infertile group (Figure 1Go). The rest of the parameters showed no differences. This was also true especially for the standard markers fructose and citrate.
  2. In the group of infertile patients, striking differences were detected only for neutral {alpha}-glucosidase, for total and for free carnitine, depending on the semen classification. In patients with oligozoospermia (n = 103) and azoospermia (n = 22) these parameters were significantly reduced (P < 0.01) compared with the values measured in patients with normozoospermia. It was striking that in the patients with asthenozoospermia (n = 17) significantly increased (P < 0.001) concentrations of {alpha}-glucosidase, total and free carnitine were measured in comparison with the rest of patients. In eight of 40 infertile patients with normozoospermia, PMN elastase concentrations >1000 µg/l were measured.



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Figure 1. Biochemical markers in the seminal plasma of controls and in the infertile men. The infertile men were classified into subgroups according to semen parameters. Symbols: contr = controls; nor = normozoospermia; ol = oligozoospermia; azo = azoospermia; ast = asthenozoospermia; ter = teratozoospermia; OAT = oligoasthenoteratozospermia syndrome; PMN = polymorphonuclear granulocyte. The central box in the box-and-whisker plot represents the interquartile range, the whiskers represent the ranges without the outliers and the middle line in the box represents the median.

 
The activity of neutral {alpha}-glucosidase was significantly reduced in the group with obstructive azoospermia (n = 13, patients after vasectomy) compared with the patients with testicular azoospermia (4.3 versus 9.8 IU/l, P < 0.01), whereas the rest of the parameters indicating the secretory capacity of the epididymis (free carnitine, total carnitine, acetylated carnitine) were not significantly reduced in concentration in the patients with obstructive azoospermia (Table IGo).


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Table I. Epididymal markers in cases of testicular and obstructive azoospermia. Values are given as medians and interquartile range in parentheses
 
Altogether, the epididymal markers were reduced in subfertile patients compared with the control group, the reduction of free carnitine and total carnitine being the most distinct. For the differential diagnosis of azoospermia, only the determination of the neutral {alpha}-glucosidase concentration was useful.

All patients in the group with obstructive azoospermia showed glucosidase activities <9 IU/l or 26 mIU/ejaculate. ROC curve analysis showed a maximum diagnostic efficiency at the threshold value of 6 IU/l to differentiate between testicular azoospermia and obstructive azoospermia. At this limit, the sensitivity was 92% and the specificity 72% for diagnosing an obstruction. The positive predictive value was 66% and the negative predictive value 94%.

Correlation between biochemical parameters and spermiogram findings
In Tables II and IIIGoGo, the correlations between the biochemical parameters and the quality criteria of the spermiogram are summarized.


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Table II. Rank correlations between the biochemical parameters in seminal plasma
 

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Table III. Rank correlations between semen parameters and the concentrations of free carnitine, total carnitine, neutral {alpha}-glucosidase, fructose, citrate, and PMN elastase in seminal plasma. The number of correlations is given in parentheses
 
Between the biochemical parameters of the epididymal function, free carnitine, total carnitine, and neutral {alpha}-glucosidase, a close statistical relationship with a correlation coefficient >0.5 was shown, while between the other parameters only single correlations of not more than 0.25 occurred (Table IIGo).

For the concentration of free carnitine the strongest positive correlations were demonstrated in the spermiogram to the number of spermatozoa as well as to motility, vitality, and morphology, whereas these correlations were less intensive for total carnitine and for the activity of neutral {alpha}-glucosidase (Table IIIGo).

The correlation between the concentration of PMN elastase in seminal plasma and the number of peroxidase-positive white blood cells in the ejaculate was clearly positive (r = 0.717, P < 0.001). There was no statistical relationship between the microbiological findings in the ejaculate and the PMN elastase concentration in seminal plasma.


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The biochemical parameters in the seminal plasma of infertile and of fertile men were seldom compared and, if so, had led to variant results. Kohengkul et al. (Kohengkul et al., 1977Go) were able to prove a reduced acetylcarnitine concentration and Soufir et al. (Soufir et al., 1984Go) demonstrated a reduced concentration of free carnitine in the seminal plasma of infertile men. Jeyendran et al. (Jeyendran et al., 1989Go) compared 18 different biochemical components in the seminal plasma of men after successful in-vitro fertilization and the seminal plasma of infertile men after therapy failure. The seminal plasma components did not reveal any significant differences between the groups, except for reduced glycerylphosphorylcholine. In another publication, higher concentrations of free carnitine, total {alpha}-glucosidase and citrate in infertile men with normozoospermia were described than for the values measured in fertile men (Cooper et al., 1991Go).

In this study, clearly reduced (P < 0.001) concentrations of free carnitine (212 versus 521 µmol/l) and total carnitine (437 versus 743 µmol/l) were demonstrated in infertile men, compared with controls. When only infertile men with normozoospermia were compared with the control group, those markers were still significantly reduced (P < 0.001). The activity of neutral {alpha}-glucosidase was lower in the infertile men than in the controls (15.1 versus 23.4 IU/l, P < 0.05). The reduction of neutral {alpha}-glucosidase in infertile men is to be attributed to the large proportion of patients with reduced sperm count and/or azoospermia in this group. Consequently, the comparison exclusively of patients with normozoospermia with controls showed no difference in the activity of the enzyme.

An association between a large number of leucocytes and limited male fertility has been described previously (Caldamone et al., 1980Go; Talbert et al., 1987Go). The present study revealed a slightly significant (P = 0.034) increase of the PMN elastase concentration in the presence of male sterility factor. Significantly increased values of PMN elastase (P = 0.015) were measured in patients with fertility problems and normozoospermia in comparison with the controls (48 versus 144 µg/l).

In eight of 40 infertile patients PMN elastase concentrations were >1000 µg/l, while in the control group no value >400 µg/l was measured. The results suggest that particularly in infertile men with normozoospermia, the reduced fertility may be due to clinically quiescent infections of the male genital tract. In these cases, antibiotic therapy would be indicated, with measurement of the PMN elastase concentration in the follow-up (Reinhardt et al., 1997Go).

The spectrum of clinical pictures involving reduced quantities of fructose and fertility problems ranges from androgen-dependent insufficiency of the seminal vesicles over general diseases, drug effects, infections of the male reproductive tract to pathological anatomical damage of the spermatic ducts (Schill, 1976Go). No differences in concentrations of fructose and citrate in infertile patients and controls were observed, nor were there any positive effects of these parameters on the semen quality. The results concur with the values measured by Lewis-Jones et al. (1996), who found no correlation between fructose and semen quality. The value of the markers of the accessory sexual glands has thus to be questioned and can be considered obsolete. Not to be confused with this is the use of fructose measurement in seminal plasma in individual cases, for instance for diagnosing dysfunction or hypoplasia of the seminal vesicles (Aumüller and Riva, 1992Go).

The importance of the determination of neutral {alpha}-glucosidase as a sensitive and non-invasive method for diagnosing an obstruction in patients with azoospermia was demonstrated by a number of examiners (Cooper et al., 1990Go; Garcia Diez et al., 1992Go; Mahmoud et al., 1998Go). The results of the current investigations were similar to theirs. Of the biochemical markers, only the activity of neutral {alpha}-glucosidase was significantly reduced in patients with azoospermia compared with patients with testicular azoospermia (P = 0.002), the sensitivity and specificity of measuring in the current study being comparable to that of Mahmoud et al. (Mahmoud et al., 1998Go).

The measurement of glucosidase proved to be useful for the differential diagnosis of azoospermia, whereas the determination of carnitine (free carnitine, total carnitine, acetylcarnitine) is unsuitable for differentiating between a testicular and an obstructive azoospermia.

Before starting medical reproductive measures, the reasons for male infertility must be precisely diagnosed. The measuring of PMN elastase concentrations (infection) and of the activity of neutral {alpha}-glucosidase (occlusion, testicular) in seminal plasma gives additional information.

From the results of this study, it is concluded that only {alpha}-glucosidase hints at an obstruction of the seminal ducts and that concealed inflammation of the male reproductive tract is often associated with male infertility. Therefore, in excluding an infection of the seminal ducts in subfertile men, measuring the PMN elastase concentration is useful only in addition to the spermiogram according to WHO criteria; in cases of azoospermia of unknown origin the concentration of {alpha}-glucosidase should be measured additionally.


    Acknowledgments
 
This work was supported in part by grants from the Fund of the German Chemical Industry (to K.J.; No. 400770). The study contains part of the doctoral thesis of A.Z.


    Notes
 
3 To whom correspondence should be addressed at: Department of Urology, University Hospital Charité, Humboldt University Berlin, Schumannstrasse 20/21, D-10117 Berlin, Germany Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Aumüller, G. and Riva, A. (1992) Morphology and function of the human seminal vesicle. Andrologia, 24, 183–196.[ISI][Medline]

Caldamone, A.A., Emilson, L.B.V., Al-Juburi, A. et al. (1980) Prostatitis: prostatic secretory dysfunction affecting fertility. Fertil. Steril., 34, 602–603.[ISI][Medline]

Cooper, T.G., Yeung, C.-H., Nashan, D. and Nieschlag, E. (1988) Epididymal markers in human infertility J. Androl., 9, 91–101[Abstract/Free Full Text]

Cooper, T.G., Yeung, C-H., Nashan, D. et al. (1990) Improvement in the assessment of human epididymal function by the use of inhibitors in the assay of alpha-glucosidase in seminal plasma. Int. J. Androl., 13, 297–305.[ISI][Medline]

Cooper, T.G., Jockenhövel, F. and Nieschlag, E. (1991) Variations in semen parameters from fathers. Hum. Reprod., 6, 859–866.[Abstract]

Garcia Diez, L.C., Esteban Ruiz, P.E., Villar, E. et al. (1992) Enzyme and hormonal markers in the differential diagnosis of human azoospermia. Arch. Androl., 28, 181–194.[ISI][Medline]

Jeyendran, R.S., van der Ven, H.H., Rosecrans, R. et al. (1989) Chemical constituents of human seminal plasma: relationship to fertility. Andrologia, 21, 423–428.[ISI][Medline]

Jochum, M., Pabst, W. and Schill, W.-B. (1986) Granulocyte elastase as a sensitive diagnostic parameter of silent male genital tract inflammation. Andrologia, 18, 413–419.[ISI][Medline]

Kairisto, V. and Poola, A. (1995) Software for illustrative presentation of basic clinical characteristics of laboratory tests – GraphROC for Windows. Scand. J. Clin. Lab. Invest., 55, 43–60.[ISI]

Kohengkul, S., Tanphaichitr, V., Muangnum, V. et al. (1977) Levels of L-carnitine and L-O-acetylcarnitine in normal and infertile human semen: A lower level of L-O-acetylcarnitine in infertile semen. Fertil. Steril., 28, 1333–1337.[ISI][Medline]

Kunst, A., Draeger, B. and Ziegenhorn, J. (1984) D-Glucose/D-fructose. In Bergmeyer H.U. (ed.), Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, 3rd edn, vol. VII, pp. 321–327.

Lewis-Jones, D.I., Aird, I.A., Biljan, M.M. et al. (1996) Effects of sperm activity on zinc and fructose concentration in seminal plasma. Hum. Reprod., 11, 2465–2467.[Abstract]

Ludwig, L., Frick, J. and Rovan, E. (1996) Praxis der Spermatologie. Springer, Berlin, pp. 42–43.

Mahmoud, M., Geslevich, J., Kint, J. et al. (1998) Seminal plasma alpha-glucosidase activity and male infertility. Hum. Reprod., 13, 591–595.[Abstract]

McGarry, D.J. and Foster, D.W. (1976) An improved and simplified radioisotopic assay for the determination of free and esterified carnitine. J. Lipid Res., 17, 277–281.[Abstract]

Möllering, H. (1985) Citrate. In Bergmeyer, H.U. (ed.), Methods of Enzymatic Analysis. Verlag Chemie, Weinheim, 3rd edn, vol. VII, pp. 2–12.

Reinhardt, A., Haidl, G. and Schill, W.-B. (1997) Granulocyte elastase indicates male genital tract inflammation and appropriate anti-inflammatory treatment. Andrologia, 24, 187–192.

Schill, W.B. (1976) Fructosebestimmung im Spermaplasma. Med. Klin., 24, 1031–1041.

Soufir, J.C., Doucot, B., Marson, J. et al. (1984) Levels of seminal free carnitine in fertile and infertile man. Int. J. Androl., 7, 188–197.[ISI][Medline]

Talbert, L.M., Hammond, M.G., Halme, J. et al. (1987) Semen parameters and fertilization of human oocytes in vitro: a multivariable analysis. Fertil. Steril., 48, 270–273.[ISI][Medline]

Wetterauer, U. (1986) Recommended biochemical parameters for routine semen analysis. Urol. Res., 14, 241–246.[ISI][Medline]

WHO (1993) In Rowe, P.-J., Comhaire, F.H., Hargreave, T.B. and Mellows, H.J. (eds), Manual for the Standardized Investigation and Diagnosis of the Infertile Couple. Cambridge University Press, Cambridge.

Wolff, H., Bezold, G., Zebhauser, M. et al. (1991) Impact of clinically silent inflammation on male genital tract organs as reflected by biochemical markers in semen. J. Androl., 12, 331–334.[Abstract]

Submitted on August 4, 1999; accepted on December 6, 1999.