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Advancement in biochemical assays in andrology

Wolf-Bernhard Schill, Ralf Henkel

Center of Dermatology and Andrology, Justus Liebig University, Giessen, Germany

Asian J Androl  1999 Jun; 1: 45-51

Keywords: male infertility; biological markers; spermatozoa; male genital diseases; sex gland secretion
Determination of markers of sperm function, accessory sex gland secretion and silent male genital tract inflammation is of considerable diagnostic value in the evaluation of male infertility. The introduction of biochemical tests into the analysis of male factor has the advantage that standardized assays with a coefficient of variation characteristic of clinical chemistry are performed, in contrast to biological test systems with a large variability. Biochemical parameters may be used in clinical practice to evaluate the sperm fertilizing capacity (acrosin, aniline blue, ROS), to characterize male accessory sex gland secretions (fructose, -glucosidase, PSA), and to identify men with silent genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG, ROS).

1 Introduction

The use of biochemical markers in the diagnosis of male infertility provides specific information about anatomical and functional disturbances at the level of the accessory sex glands and the epididymis, the occurrence of acute and chronic genital tract inflammation, and the fertilizing capacity of human spermatozoa. Thus, biochemical markers in andrology are important complementary tools for the proper diagnosis of fertility disorders. In contrast to bioassays, the use of marker substances has the advantage that these are objective methods and thus are subjective to common conditions of laboratory methods including quality control.

The following biochemical markers can be used in clinical andrology to determine and identify (I) sperm fertilizing capability (aniline blue, acrosin, reactive oxygen species [ROS]); (II) male genital tract inflammation (elastase, C'3 complement component, coeruloplasmin, IgA, IgG); (III) accessory sex gland and epididymal dysfunction and obstruction (fructose, -glucosidase, acidphosphatase, prostatic specific antigen [PSA]). Some of these markers are part of the routine program of an andrological laboratory, others are sophisticated methods which will only be performed in selected patients.

2 Results and Discussion

2.1 Sperm fertilizing capacity

Apart from the light microscopical determination of morphological malformations, the assessment of disturbed acrosomal function is considered to be an important diagnostic parameter to estimate the fertilizing capacity of a sperm population[1,2]. In addition, the following biochemical parameters have been found to be helpful in andrological diagnosis: (i) determination of acrosin activity; (ii) aniline blue staining, (iii) determination of reactive oxygen species.

2.1.1 Acrosin activity

Determination of acrosin, which is one of the best characterized sperm-specific enzymes, is a suitable approach to evaluate the fertilizing capacity of human spermatozoa. Acrosin is a trypsin-like serine proteinase which is exclusively located within the mammalian sperm acrosome; it is considered to be the major penetration enzyme required for zona penetration through limited proteolysis of zona proteins. Another important function is its ability to bind to the zona pellucida[3,4]. Acrosin is apparently also involved in capacitation and acrosome reaction[5,6]. In addition, it may act as a sperm-stimulating agent during intrauterine sperm migration when it is released from the acrosome of dead spermatozoa, since it is able to liberate kinins from kininogen. Kinins were demonstrated to enhance sperm metabolism and sperm motility in vitro[7].

Several methods have been described to assess the acrosin activity in human spermatozoa[7]. A very simple method is the determination of the proteolytic potential of spermatozoa on gelatine plates[8]. Acrosin is released by hyperosmolaric rupture of the acrosome and leads to halo formation during incubation in a humid chamber at 37. Halo formation is predominantly brought about by living spermatozoa, which is supported by correlation with the eosin test (r=0.619). The more dead spermatozoa are identified, the lower is the halo formation rate. No acrosin is available in case of globozoospermia[9]. The method of gelatinolysis is advantageous in that its equipment is simple and acrosin activity can be determined in individual spermatozoa (Figure 1). It shows good correlation with the biochemical assay[10].

Figure 1. Human spermatozoa on gelatin coated micro slides after 2 h incubation at 37 in a moist chamber. A: Spermatozoa showing a halo diameter >10 m. These spermatozoa have normal acrosin activity. B: Spermatozoa showing a halo diameter <10 m. These spermatozoa have significantly decreased acrosin activity. C: Spermatozoa showing no halo formation. These spermatozoa have no acrosin activity. Phase contrast (400); micro meter bar: 10 m.

Figure 2 shows a comparison of fertilization rates and acrosin activity index calculated from halo diameter and halo formation rate in an IVF program (110 patients). Normal acrosin activity indices are observed in men with high fertilization rates, whereas the halo diameters and halo formation rates are smaller in most cases of poor fertilization (<50%)[8]. Thus, the method may give information about the fertilizing potential of a sperm population. Patients showing normal acrosin activity index but low fertilization probably have defects other than impaired acrosin activity (eg, impaired acrosome reaction, impaired sperm-oolemma interaction or disturbance of chromatin decondensation) (Figure 3). This is a reason why statistical calculations show low sensitivity (26%), while high specificity (98%) and a high predictive value (positive predictive value 90%, negative predictive value 74%) exist for human IVF outcome[8]. The rate of false negative results of this assay is 3.5%.

Figure 2. Correlation between acrosin activity index (meanSEM) and fertilization rate in 110 patients attending the IVF program at the University of Giessen, Germany. The acrosin activity index is calculated from halo diameter and halo formation rate by multiplication of these two values and division by 100. The four groups differ significantly by Mann-Whitney test (P=0.0003).
Figure 3. Cumulative distribution of the acrosin activity index of 110 parients. There is a shift to the left for patients showing fertilization rates <50%. Patients with a normal acrosin activity index but low fertilization probably have fertilization disorders other than decreased acrosin activity.

A more sophisticated method is the spectrophotometric determination of acrosin after acid extraction from the sperm acrosome, performed in the presence of synthetic acrosin inhibitors to avoid autoactivation from proacrosin. Proacrosin is the zymogen form predominating in epididymal and freshly ejaculated spermatozoa. This method allows the determination of active, non-zymogen acrosin, proacrosin, and total acrosin activity[7]. In most sperm populations, acrosin activity shows normal values and a wide overlap of the range of acrosin levels. In contrast, significantly lower acrosin activity is observed in patients with severe teratozoospermia and polyzoospermia, the latter with an average of <60%[7]. By immunological methods it was shown that the acrosomal membrane integrity was severely disturbed in most spermatozoa from polyzoospermic men. Thus, polyzoospermic patients equal men with severe oligozoospermia showing reduced fertility compared to normozoospermic controls. Although contradictory results have been reported[11], the importance of assessing the acrosin activity in infertile men and its predictability for fertilization has repeatedly been emphasized[12,13], thus, supporting the concept that acrosin determination is a useful parameter to predict the fertilizing potential of spermatozoa[14].

In conclusion, assessment of acrosin should be considered in selected cases of teratozoospermia, particularly to confirm the diagnosis of globozoospermia. Acrosin activity should also be determined in polyzoospermic patients to recognize severe acrosomal dysfunction. In addition, the demonstration of sufficient amounts of acrosin in men from couples with idiopathic sterility, in cases of unknown male sterility factor and before in vitro fertilization may exclude severe disturbances of the sperm acrosome.

2.1.2 Aniline blue staining for determination of chromatin condensation

During spermiogenesis, lysine-rich histones are normally replaced by protamines. This process is prerequisite for the later occurring decondensation to form a male pronucleus during oocyte fertilization. In case of disturbed chromatin condensation, histones persist and can be identified by staining with acidic aniline blue[15]. Since nuclear proteins play a significant role in chromatin condensation, this method is an attempt to discriminate between fertile men and those suspected of being infertile[16-18] using nuclear maturity as a parameter. Disturbed chromatin condensation is often observed in combination with an increased number of acrosomal defects[19]. In case of >50% aniline blue-positive spermatozoa, a protamine gene defect has been discussed. According to studies by Dadoune et al[20] and Hofmann et al[19], a normal ejaculate should contain at least 75% unstained spermatozoa, which indicate normal chromatin condensation. This indicates that normal chromatin condensation is mandatory to induce fertilization. Thus, aniline blue staining is highly predictive and may be used as an easily performable laboratory test which should precede all methods of assisted reproduction. However, its value is apparently restricted to conventional IVF procedures, because studies assessing chromatin condensation in spermatozoa used for intracytoplasmic sperm injection failed to predict the outcome of fertilization by ICSI[21,22]. It should be mentioned in this connection that Henkel et al[23] showed glass wool filtration to have a selective capacity to enrich the number of normal chromatin condensed spermatozoa, suggesting its beneficial effect for the various procedures of assisted reproduction.

2.1.3 Reactive oxygen species (ROS)

Since the first report by McLeod[24] on the influence of ROS on human spermatozoa, it is now believed that oxidative stress is associated with male infertility[25,26]. Spermatozoa have a much higher content of polyunsaturated fatty acids in their membranes than somatic cells. Therefore, they are particularly susceptible to oxidation by ROS which cause lipid peroxidation. In extreme cases this might result in a dramatic loss of normal sperm function, eg, markedly reduced motility[27] and penetration in the zona-free hamster ovum penetration test[26,28] or impaired membrane integrity[29], thus indicating decreased fertilizing capability of spermatozoa. In addition, oxidative damage to spermatozoa is closely correlated with inflammatory processes in the genital tract and occurrence of leukocytes, particularly granulocytes, which generate at least 100 times more ROS than spermatozoa themselves[30]. In addition, a highly positive correlation has been found between reactive oxygen species, elastase, a specific parameter of inflammation, sperm concentration and motility[31]. (Table 1)

Table 1. Sperman rank correlations between reactive oxygen species and different semen parameters



Total number
of spermatozoa



Number of peroxidase-positive cells

Number of round cells






























Several authors revealed that 30-40% of ejaculates from infertile men generate excessive levels of ROS[32-34]. Especially oligozoospermic patients tend to have high ROS production of spermatozoa[31,35]. From a clinical view it is, therefore, important to determine semen samples that produce excessive amounts of ROS and to separate leukocytes and damaged spermatozoa from those sperm cells which do not yet show signs of lipid peroxidation. Because of the sensitivity of spermatozoa to oxidative damage, sperm separation should be performed very carefully, preferably by means of density gradient centrifugation or glass wool filtration. Using the SpermFertil® glass wool filtration columns (Mello Ltd, Exeter, UK) we were able to reduce leukocyte contamination in human ejaculates to an extent higher than 90%[36]. Moreover, with this technique it was possible to distinguish whether ROS production in ejaculates was produced by spermatozoa or leukocytes[34]. In addition, both density gradient centrifugation and glass wool filtration have been shown to maintain normal sperm function with regard to motility and penetration into zona-free hamster oocytes[26,27,37].

2.2 Male genital tract inflammation

From a clinical point of view, differential diagnosis of chronic male genital tract inflammations and non-inflammatory complaints such as vegetative urogenital syndrome or anogenital symptom complex is mandatory. Chronic inflammatory processes need antibiotic-antiphlogistic therapy, whereas complaints of the autonomic nervous system should be treated quite differently, either with tranquilizers or by different psychosomatic techniques. To prove an inflammatory semen pattern, >1106 peroxidase-positive round cells (neutral granulocytes) per mL, known as leukocyto-spermia, indicate a reproductive tract infection. However, the absence of leukocytes does not exclude the possibility of an accessory sex gland infection. Therefore, biochemical markers have been suggested to be used as sensitive indicators of an inflammatory reaction[38].

An enzyme immunoassay for determination of elastase in seminal plasma as a specific inflammatory parameter of polymorphonuclear granulocytes (PMN) enables the diagnosis of a silent genital tract inflammation[39]. In addition, sequential determinations allow control of the course of the disease during and after therapy.

Apart from elastase measurements, a permeable blood-seminal plasma barrier, indicating adnexal impairment, can provide valuable information about an accessory sex gland infection. Particularly helpful is the quantitative determination of the complement component C'3 and coeruloplasmin. Normally, C'3 complement component is not or only in traces detectable in seminal plasma. During an inflammatory reaction, transudation from the blood is increased, and both C'3 complement component and coeruloplasmin are found in significantly elevated amounts in semen samples[38]. Also, determination of ROS might be helpful, which have been found to be highly significantly correlated with elastase. ROC curve analysis for ROS production in the ejaculate using PMN elastase as a decisive parameter revealed a cut-off value of 49 489.9 counts/107viable spermatozoa for 1000 ng/mL PMN elastase with the following statistical parameters of the assay: sensitivity 63.2%, specificity 100%, positive predictive value 100%, negative predictive value 36.1%. With 500 ng/mL PMN elastase as a decisive parameter, a cut-off value of 35 405.3 counts/107 viable spermatozoa was calculated. The statistical parameters were as follows: sensitivity 66.3%, specificity 87.2%, positive predictive value 92.2%, negative predictive value 53.1%[31].

2.2.1 Determination of granulocyte elastase

Granulocyte elastase is determined in cell-free seminal plasma according to the method by Neumann & Jochum[40], using an enzyme-linked immunoabsorbent assay provided by E Merck, Darmstadt, Germany. Due to the relatively rapid reaction of extracellularly liberated elastase with its major inhibitor, (1-proteinase inhibitor (1 PI), the enzyme can be detected in body fluids only in an inactive, complexed form (E-1 PI). PMN elastase levels above 1000 ng/mL are diagnostic of leukocytospermia[41]. Thus, clinically silent inflammations can be measured by PMN elastase in semen[42]. Recent investigations with an exact quantification of granulocyte elastase in 305 andrological patients confirmed its high specificity and sensitivity to distinguish inflammatory from non-inflammatory male adnexal affections[43].

2.2.2 Quantitative determination of C'3 complement component and coeruloplasmin

C'3 complement component and coeruloplasmin can be quantified in human seminal plasma by radial immunodiffusion (Partigen®, Behringwerke, Marburg, Germany). An increase in coeruloplasmin in seminal plasma is only seen during a significant inflammatory semen reaction, whereas C'3 complement determinations seem to be much more sensitive[38]. An increase in seminal plasma IgA and IgG has also been found to give relative information about genital tract inflammation, but is less sensitive and specific than determination of granulocyte elastase in seminal plasma.

2.3 Determination of accessory sex gland secretory function

The secretory capacity of seminal vesicles, prostate and epididymis can be determined by means of various biochemical markers, eg, fructose, PSA and neutral -glucosidase[44]. Low secretory function or lack of it due to an occlusion is reflected by low total output of the specific markers; therefore, they may be used for the assessment of both accessory sex gland secretory function and location of an obstruction.

2.3.1 Fructose

Fructose in semen is determined according to the WHO laboratory manual by means of a colorimetric reaction with indole[44]. Also, an enzymatic assay using spectrophotometry may be performed. In case of azoospermia caused by congenital absence of the vasa deferentia, low fructose levels indicate an associated dysgenesis of the seminal vesicles. Fructose levels are also reduced in case of postinflammatory atrophy of the seminal vesicle epithelium or relative androgen deficiency. Oral androgen medication (eg, 120 mg testosterone undecanoate or 75 mg mesterolone) allows differentiation between androgen-sensitive and androgen-resistant forms of seminal vesicle insufficiency.

In case of ejaculatory duct obstruction or agenesis of the vasa deferentia and seminal vesicles, semen samples are characterized by low volume, low pH and absence of fructose; this indicates that the ejaculate consists exclusively of prostatic fluid.

2.3.2 -Glucosidase

Determination of L-carnitin as a common epididymal marker has been abandoned. Assessment of neutral -glucosidase, which originates from the corpus and cauda epididymidis, has been found to give more reliable and reproducible results, is simpler and cheaper and less time-consuming[44]. Distal ductal obstruction shows significantly decreased -glucosidase values.

Unfortunately, functional disturbances at the level of the epididymis cannot be characterized by the -glucosidase assay. In case of an occlusion at the level of the rete testis, -glucosidase activity in seminal plasma is found within the normal range. Increased amounts of macrophages in semen have been shown to be associated with chronic epididymitis. Future research will hopefully provide more specific and sensitive markers of epididymal function.

2.3.3 Prostatic gland secretion

To give a reliable measure of the prostatic gland secretion, several marker substances can be used, including zinc, citric acid and prostatic acid phosphatase[44]. For clinical purposes, however, determination of prostatic gland secretions is of no great diagnostic value to localize obstructive disease. On the other hand, assessment of the specific prostatic marker PSA, a kallikrein-like protease, may give information about inflammatory reactions and tumor cells in the prostate[45,46]. PSA can be determined in semen and serum by means of a commercial radioimmunoassay. This determination is used as a screening test to exclude prostatic cancer or chronic inflammatory processes within the prostatic tissue. However, despite considerably decreased prostatic secretory function by infectious agents, the total amount of markers in semen may be still within normal range.

3 Conclusions

In conclusion, biochemical markers of seminal plasma allow to assess the functional state of the accessory sex glands and will help to localize obstructive azoospermia. Thus, absence of fructose in combination with a low pH and a low ejaculate volume allows to identify men with obstructions in the periphery, because pure prostatic fluid with an acid pH around 6.4 and a low semen volume with or without traces of fructose are observed in these cases. Future investigations will also clarify whether other biochemical markers (eg, the testis-specific marker transferrin, acridine orange as marker of the stability of sperm DNA, the cholesterol/phospholipid ratio as marker of human sperm capacitation) have any diagnostic relevance for clinical andrology.


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Correspondence to Prof W-B Schill, Zentrum fr Dermatologie und Andrologie, Gaffkystrasse 14, D 35385 Giessen, Germany.
Tel: +49-641-994 3200   Fax: +49-641-994 3209
e-mail: Wolf-Bernhard.Schill@derma.med.uni-giessen.de
Received 1999-05-13     Accepted 1999-05-18