Home | Archive | AJA @ Nature | Online Submission | News & Events | Subscribe | APFA | Society | Links | Contact Us | 中文版 |
 |
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
Abstract
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.
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
| |
Sperm |
Total number |
Motility |
Sperm |
Number of peroxidase-positive
cells |
Number of round cells |
pH |
Elastase |
| R |
-0.443 |
-0.470 |
-0.323 |
-0.233 |
0.588 |
0.290 |
0.187 |
0.613 |
| P |
0.0001 |
0.0001 |
0.0001 |
0.002 |
0.0001 |
0.001 |
0.023 |
0.0001 |
| N |
176 |
176 |
176 |
176 |
123 |
126 |
176 |
128 |
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
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.References
[1]
Henkel R, Mller C, Miska W, Gips H, Schill W-B. Determination of the
acrosome reaction of human spermatozoa is predictive of fertilization
in vitro. Hum Reprod 1993; 8: 2128-32.
[2] Schill W-B, Henkel R, Snchez R, Khanaga O, Turley H, Haidl G,
et al. Sperm function tests: do they have any predictive value? Hum
Reprod 1994; 9 Suppl 4: 94-5.
[3] Töpfer-Petersen E, Henschen A. Acrosin shows zona and fucose
binding, novel properties for a serine proteinase. FEBS Lett 1987; 226:
38-42.
[4] Tesarik J, Drahord J, Peknicov J. Subcellular immunochemical localization
of acrosin in human spermatozoa during the acrosome reaction and zona
pellucida penetration. Fertil Steril 1988; 50: 133-41.
[5] De Jonge CJ, Mack SR, Zaneveld LJD. Inhibition of the human sperm acrosome reaction by proteinase
inhibitors. Gamete Res 1989; 23: 387-97.
[6] Nuzzo NA, Anderson RA, Jr Zaneveld LJD. Proacrosin activation and
acrosin release during the guinea pig acrosome reaction. Mol Reprod Dev
1990; 25: 52-60.
[7] Schill W-B. Determination of active, non-zymogen acrosin, proacrosin and total acrosin in
different andrological patients. Arch Dermatol Res 1990; 282: 335-42.
[8] Henkel R, Mller C, Miska W, Schill W-B, Kleinstein J, Gips H. Determination
of the acrosin activity of human spermatozoa by means of a gelatinolytic
technique. A simple, predictive method useful for IVF. J Androl 1995;
16: 272-7.
[9] Wolff HH, Schill W-B, Moritz P. Rundk-pfige Spermatozoen: Ein seltener andrologischer Befund
(Kugelkopfspermatozoen, Globozoospermie). Hautarzt 1976; 27: 111-6.
[10] Schill W-B. Acrosin and seminal plasma proteinase inhibitors in the
diagnostic workup of male infertility. In: Insler V, Bettendorf G, editors.
Advances in diagnosis and treatment of infertility. North Holland, New
York: Elsevier, 1981. p 321-37.
[11] Liu DY, Baker HWG. Relationships between human sperm acrosin, acrosomes,
morphology and fertilization in vitro. Hum Reprod 1990; 5: 298-303.
[12] Goodpasture JC, Zavos PM, Cohen NM, Zaneveld LJD. Relationship of
human sperm acrosin and proacrosin to semen parameters. I. Comparison
between symptomatic men of infertile couples and asymptomatic men, and
between different split ejaculate fractions. J Androl 1982; 3: 151-6.
[13] Tummon IS, Yuzpe AA, Daniel SAJ, Deutsch A. Total acrosin activity
correlates with fertility potential after fertilization in vitro.
Fertil Steril 1991; 56: 933-8.
[14] Schill W-B. Some disturbances of acrosomal development and function
in human spermatozoa. Hum Reprod 1991; 6: 969-78.
[15] Terquem A, Dadoune JP. Aniline blue staining of human spermatozoa
chromatin: evaluation of nuclear maturation. In: Andr J, editor. The
Sperm Cell. The Hague: Martinus Nijhoff, 1983. p 249-52.
[16] Auger J, Mesbah M, Huber C, Dadoune JP. Aniline blue staining as a
marker of sperm chromatin defects associated with different semen characteristics
discriminates between proven fertile and suspected infertile men. Int J Androl 1990; 13: 452-62.
[17] Hofmann N, Hilscher B. Use of aniline blue to assess chromatin condensation
in morphologically normal spermatozoa in normal and infertile men. Hum
Reprod 1991; 6: 979-82.
[18] Hammadeh ME, Stieber M, Haidl G, Schmidt W. Association between sperm
cell chromatin condensation, morphology based on strict criteria, and
fertilization, cleavage and pregnancy rates in an IVF program. Andrologia
1998; 30: 29-35.
[19] Hofmann N, Hilscher B, Bierling C. Quantitative Untersuchungen der
Korrelation von Störungen der Chromatinkondensierung mit der Spermatozoenmorphologie
(Quantitative studies on the correlation between disturbed chromatin condensation
and sperm morphology). Fertilität 1990; 6: 208-13.
[20] Dadoune JP, Mayaux MJ, Guilhard-Moscato ML. Correlation between defects
in chromatin condensation of human spermatozoa stained by aniline blue
and semen characteristics. Andrologia 1988; 20: 211-7.
[21] Van Ranst H, Bocken G, Desmet B, Joris H, Vankeleccm AS, Liu J, et
al. Chromatin condensation assessment
in spermatozoa used for intracytoplasmic sperm injection. Hum Reprod 1994;
9 Suppl 4: 24.
[22] Hammadeh ME, Al-Hasani S, Stieber M, Rosenbaum P, Kupker D, Diedrich
K, et al. The effect of chromatin condensation (aniline blue staining) and
morphology (strict criteria) of human spermatozoa on fertilization, cleavage
and pregnancy rates in an intracytoplasmic sperm injection programme.
Hum Reprod 1996; 11: 2468-71.
[23] Henkel R, Franken DR, Lombard CJ, Schill W-B. The selective capacity
of glass wool filtration for normal chromatin condensed human spermatozoa: A
possible therapeutic modality for male factor cases? J Assist Reprod Genet
1994; 11: 395-400.
[24] McLeod J. The role of oxygen in the metabolism and motility of human
spermatozoa. Am J Physiol 1943; 138: 512-8.
[25] Aitken RJ, Clarkson JS. Cellular basis of defective sperm function
and its association with the genesis of reactive oxygen species by human
spermatozoa. J Reprod Fertil 1987; 81: 459-69.
[26] Aitken RJ, Clarkson JS, Fishel S. Generation of reactive oxygen species,
lipid peroxidation, and human sperm function. Biol Reprod 1989; 40:
183-97.
[27] Mortimer D. Sperm preparation techniques and iatrogenic failures of in vitro fertilization.
Hum Reprod 1991; 6: 173-6.
[28] Aitken RJ, West KM. Analysis of the relationship between reactive
oxygen species production and leukocyte infiltration in fractions of human
semen separated on Percoll gradients. Int J Androl 1990; 13: 433-51.
[29] Gavella M, Lipovac V, Marotti T. Effect of pentoxifylline on superoxide
anion production by human sperm. Int J Androl 1991; 14: 320-07.
[30] Ford WCL. The role of oxygen free radicals in the pathology of human
spermatozoa: Implications of IVF. In: Matson PL, Lieberman BA, editors.
Clinical IVF Forum: Current Views in Assisted Reproduction. Manchester:
University Press, 1990. p 123-39.
[31] Henkel R, Schill W-B. Sperm separation in patients with urogenital
infections. Andrologia 1998; 30 Suppl 1: 91-7.
[32] Iwasaki A, Gagnon C. Formation of reactive oxygen species in spermatozoa
of infertile patients. Fertil Steril 1992; 31: 531-7.
[33] Agarwal A, Ikemoto J, Loughlin KR. Relationship of sperm parameters
with levels of reactive oxygen species in semen specimens. J Urol 1994;
152: 107-10.
[34] Henkel R, Ichikawa T, Snchez R, Miska W, Ohmori H, Schill W-B.
Differentiation of ejaculates in which reactive oxygen species are generated
by spermatozoa or leukocytes. Andrologia 1997; 29: 295-301.
[35] Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, WU FCW. Analysis
of the relationship between defective sperm function and the generation
of reactive oxygen species in cases of oligozoospermia. J Androl 1989;
10: 214-20.
[36] Snchez R, Concha M, Ichikawa T, Henkel R, Schill W-B. Glass wool
filtration reduces reactive oxygen species by elimination of leukocytes
in oligozoospermic patients with leukocytospermia. J Assist Reprod Genet
1996; 13: 489-94.
[37] Rana N, Jeyendran RS, Holmgren WJ, Rotman C, Zaneveld LJD. Glass wool-filtered spermatozoa and their
oocyte penetrating capacity. J In Vitro Fert Embryo Transf 1989; 6: 280-4.
[38] Schiessler H, Jochum M, Friesen A, Schill W-B, Hofstetter A. Granulozyten-Elastase
im Ejakulat als Entzndungsparameter bei Adnex-Affektionen. In: Schirren
C, Semm K, editors. Fortschritte der Fertilitätsforschung 12.
Berlin: Grosse Verlag, 1984. p 157-63.
[39] Jochum M, Pabst W, Schill W-B. Granulocyte elastase as a sensitive
diagnostic parameter of silent male genital tract inflammation. Andrologia
1986; 18: 413-9.
[40] Neumann S, Jochum M. Elastase-1-proteinase inhibitor
complex. In: Bergmeyer HU, Bergmeyer J, Grassl M, editors. Methods of enzymatic analysis, 3rd ed. Weinheim:
Verlag Chemie, 1984. p 184-95.
[41] Wolff H, Anderson DJ. Evaluation of granulocyte elastase as a seminal
plasma marker for leukocytospermia. Fertil Steril 1988; 50: 129-32.
[42] Wolff H, Bezold G, Zebhauser M, Meurer M. Impact of clinically silent
inflammation on male genital tract organs as reflected by biochemical
markers in semen. J Androl 1991; 12: 331-4.
[43] Reinhardt A, Haidl G, Schill W-B. Granulozytenelastase-Bedeutung
fr die andrologische Diagnostik (Granulocyte elastase-significance for
andrological diagnosis). Fertilität 1995; 11: 153-7.
[44] World Health Organization. Laboratory manual for the examination of human semen and sperm-cervical
mucus interaction, 3rd ed. Cambridge: Cambridge University Press, 1992.
[45] Kirby RS, Kirby MG, Feneley MR, McNicholas T, McLean A, Well JA.
Screening for carcinoma of the prostate: a GP based study. Br J Urol 1994;
74: 64-71.
[46] Lehmann K, Simmler F, Schmucki O, Hauri D. PSA in prostatic fluid.
Urologe (A) 1994; 33: 232-4.
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
