INFECTION AND SEMEN QUALITY IN INFERTILITY
IMPROVEMENT OF SEMEN QUALITY IN INFECTED ASYMPTOMATIC INFERTILE
MALE AFTER BACTERIOLOGICAL CURE
ESTELA M. CARDOSO1, JORGE
E. SANTOIANNI2, ADRIANA N. DE PAULIS2, JUAN A. ANDRADA1, SILVIA C.
PREDARI2, ALEJANDRO L. ARREGGER1
1 Departamento de
Endocrinología y 2 Departamento de Microbiología, Instituto de
Investigaciones Médicas Alfredo Lanari, Facultad de Medicina,
Universidad de Buenos Aires
Key words: male genital tract infection, sperm parameters,
antibiotic therapy, sperm agglutinating antibodies, leukocytospermia
etiology was investigated in 29 infected asymptomatic infertile males.
The localization of the infection and the effect of a long term
antibiotic therapy on semen parameters were evaluated. The most
frequent etiological agent isolated was Enterococcus faecalis.
Positive bacteriological culture was obtained in prostatic fluid in 16
patients and in semen in 13. Bacteriological cure was achieved in 24
cases and it was associated with improved seminal parameters: sperm
concentration, motility, viability and total motile sperm per
ejaculate. In 5 patients without bacteriological cure there was no
change in semen analysis after antibiotic therapy. In 45% of the
infected patients there were less than 0.5 x 106/ml seminal
polymorphonuclear leukocytes. In view of these findings granulocyte
concentration seems to be a poor marker to predict infection.
de la calidad del semen en pacientes infértiles infectados
asintomáticos después de una cura bacteriológica. En 29 pacientes
infértiles infectados asintomáticos fue estudiada la etiología de
la infección, su localización y el efecto de la terapia antibiótica
prolongada sobre los parámetros espermáticos. El agente etiológico
más frecuente fue Enterococcus faecalis: 16 pacientes presentaron
cultivo positivo en líquido prostático y 13 en semen. La cura
bacteriológica se alcanzó en 24 pacientes y se asoció a mejoría de
los parámetros seminales: concentración espermática, motilidad,
viabilidad y número de espermatozoides totales móviles en el
eyaculado. Luego de la terapia antibiótica, 5 pacientes no lograron
cura bacteriológica y no presentaron mejoría en los parámetros
seminales. El 45% de los pacientes infectados presentó una
concentración seminal de leucocitos polimorfonucleares menor de 0,5 x
106/ml, por lo cual concluimos que la concentración de granulocitos
no constituye un marcador confiable para predecir la presencia de
Postal address: Dra. Estela Cardoso, Departamento de
Endocrinología, Instituto de Investigaciones Médicas Alfredo Lanari,
Avenida Combatientes de Malvinas 3150, 1427 Buenos Aires, Argentina.
Received: 10-XII-1997 Accepted: 15-I-1998
It is well known that certain bacterial infections such as those
produced by Neisseria gonorrhoeae, Mycobac-terium tuberculosis and
Chlamydia trachomatis, are related to male infertility1. However, the
phenomenom is less clear for other microorganisms that often colonize
the anterior urethra and may become potential etiological agents of
prostatitis and seminal infections. Moreover, little is known about
how often microorganisms are the cause of leukocytospermia in
infertile patients. Most papers reviewed mention no significant
association between white blood cells (WBC) and microorganisms in
semen. WBC are present in most human ejaculates, but abnormally high
concentrations of seminal WBC may reflect an underlying pathological
The absence of leukocytospermia does not exclude the possibility of
infection. As infertile patients usually do not show symptoms of
chronic genital tract infection the search for microorganisms in semen
and genital tract secretions described by Stamey and Meares3 should
always be performed, regardless of the number of leukocytes in semen4.
In order to evaluate the impact of male genital tract infections on
semen quality in asymptomatic infertile patients we determined: 1) the
microbiological etiology of infection; 2) the concentration of
neutrophilic poly-morphonuclear leukocytes (PMN) in semen as a marker
of leukocytospermia; 3) the result of the administration of specific
antibiotic therapy during a long period of time, according to the
susceptibility pattern of microorganisms and the pharmacodynamics of
Materials and Methods
All couples included in this study had a history of infertility.
Patient evaluation: On the first visit patients were clinically
evaluated and two consecutive semen analysis were performed according
to WHO guidelines, with an interval of seven days. Four months later,
sperm analysis were repeated. We found that among the 3 samples from
each patient the coefficient of variation remained < 20% for sperm
concentration, < 10% for sperm motility, < 5 % for sperm
viability and < 10% for sperm morphology. After this, a new semen
sample was obtained for microbiological cultures, antisperm
antibodies, and evaluation of semen parameters. Those patients with
positive microbiological cultures (MC+) received specific antibiotic
therapy during 90 days. Two months after antibiotic therapy was
completed, another microbiological study and semen evaluation were
performed. A second semen sample was studied a week later, for sperm
Following this protocol, 70 asymptomatic male partners of infertile
couples were evaluated during the last two years. Of these, 29
patients between 20 and 38 years old with MC+ completed all the steps
described and their results are shown.
Microbiological study: patients were asked to produce the samples
in the laboratoy after three days abstinence and should have full
bladder and desire to void. All specimens were obtained by a
microbiologist and a physician for prostatic massage procedure.
Microbiological cultures were performed in all patients from urethral
swab (US), first voided urine (VB1), midstream urine (VB2), prostatic
fluid (PF), voided urine after prostatic massage (VB3), and semen (S),
following the four specimen technique described by Stamey and Meares3,
with the addition of urethral swab and semen5.
None of the subjects presented urethral discharge or urinary tract
infections at the time of cultures. All samples were also assessed for
normal genital tract microorganisms and sexually transmitted
pathogens, including aerobic and anaerobic bacteria, fungus, yeasts
and Ureaplasma urealyticum using standard procedures6, 7, 8, 9.
The investigation of Chlamydia trachomatis was performed only in semen
samples by direct immunofluorescence method (Syva trak Chlamydia
direct IF kit).
Prostatic fluid or semen were considered infected if: 1) their colony
counts were > 1 log10 with regard to VB1, or 2) PF was not
obtained, but VB3 colony counts were > 1 log10 with regard to VB1,
or 3) a microorganism was not present in VB1 or US, but was recovered
from PF,S or VB3 at any count.
Antibiotic therapy administered during 90 days consisted of:
trimethoprim/sulfamethoxazole, ciprofloxacin, doxycyclin or minocyclin
at usually doses according to the susceptibility pattern of the
Semen evaluation: semen samples were collected by masturbation into
sterile containers after 3 days of sexual abstinence, and examined
within 1 hour after ejaculation. After liquefaction, semen analysis
included the determination of sperm concentration, motility and
viability performed by two different experimented observers following
WHO recommendations4. Sperm morphological assessment was performed on
ethanol-ether fixed smears of fresh ejaculates stained by the
Papanicolaou procedure recommended by WHO4.
The concentration of PMN cells was measured by peroxidase stain method
according to WHO4 and adapted by Wolff10.
The presence of sperm agglutinating antibodies in seminal plasma was
investigated by the tray agglutination test. This method (TAT) is
routinely applied in our laboratory using an antigen suspension
composed of only motile spermatozoa obtained by swim up from semen
with normal parameters, and was performed following WHO standard
Friedman test, Wilcoxon signed rank test, and Spearman rank order
correlation were used for statistical analysis of data. A probability
value p < 0.05 was considered significant.
Microorganisms isolated from prostatic fluid and semen are shown in
Table 1. All patients were infected with only one microorganism, 16
(55%) had prostatic localization and the other 13 (45%) presented
The median concentration of PMN in semen of these infected males was 1
x 106/ml (0.05 x 106 to 3.5 x 106). There was no evident difference in
PMN concentration between those who presented prostatic localization
(median 0.61 x 106/ml, range 0.05 x 106 to 3.5 x 106), and those with
seminal infection (median 1 x 106/ml, range 0.05 x 106 to 3.5 x 106),
Bacteriological cure was achieved in 24 patients (83%). Four patients
relapsed and one had a reinfection.
Sperm parameters before and after antibiotic therapy are shown in
Table 2. For the patients who achieved bacteriological cure as there
was not a significant variation on different samples during the 4
months preceding microbiological diagnosis, we considered each patient
as his own control (A versus B, p: NS). Total sperm concentration,
motility, viability, total motile sperm per ejaculate and PMN
concentration improved after antibiotic therapy. Sperm morphology did
not show significant differences.
There was a low correlation between PMN concentration and total motile
sperm per ejaculate pre and post therapy or the percentage of normal
forms after bacteriological cure (Spearman rank correlation
coefficient, p: 0.387, p: 0.481 and p: 0.734 respectively).
The 5 patients without bacteriological cure (2 asthenozoospermic and 3
oligoasthenozoospermic) did not show changes in semen analysis before
and after antibiotic therapy (Table 3).
Two patients showed antisperm antibodies in seminal plasma, with
titers ranging from 1/64 to 1/128. They had a median of 1.5 x 106
PMN/ml at the moment of the infection (one patient with
Arcanobacterium haemolyticum in PF, and one with Ureaplasma
urealyticum in S). After antibiotic therapy both patients had negative
antisperm antibody titers, and PMN concentration diminished to 0.25 x
We consider the infection of the male genital tract as an important
morbidity factor. It is known that it may affect seminal quality
through a direct action on spermatozoa or their environment, including
local inflammatory reaction and composition of seminal plasma13-16.
We have been involved in the diagnosis of seminal infection in
infertile men since 197917, 18. We followed the technique described by
Stamey and Meares that allows for the localization of the infection in
prostatic gland or in other sites of the male genital tract different
from the prostate (excluding urethra). The culture of semen alone may
lead to controversial results when compared to those obtained by the
Stamey and Meares protocol that we have used in the present study. We
have previously shown a prevalence of infection of about 46% in two
large populations of infertile men. Enterococcus faecalis was the most
frequent etiological agent followed by Ureaplasma urealyticum17, 18.
After specific antibiotic therapy we waited 2 months to control sperm
parameters since the entire process of spermatogenesis takes
approximately 70 days19 and requires between 4.3 to 4.7 cycles of the
seminiferous epithelium20. Until microbiological cure was achieved, we
advised the couples to use condom during sexual intercourse to prevent
reinfections and to avoid harmful effects of antibiotics on semen
Males have been treated during three months according to Meares22, 5,
who showed that in patients with long-term therapy the cure rates have
been 32% to 71%, more than twice those noted with short-term therapy
(two weeks or less). In spite of a long term antibiotic therapy, 17%
of patients in this study did not achieve bacteriological cure, and
did not modify their seminal parameters (including PMN concentration).
In this group of patients we have found Enterococcus faecalis as the
main etiological agent in PF and S. Most of the etiological agents
isolated such as Enterococcus faecalis, Enterobacteriaceae,
coagulase-negative staphy-lococci and Streptococcus agalactiae (Table
1) are constituents of the normal enteric flora that often colonize
the anterior urethra and that may also cause urinary tract
infections23-25. Other microorganisms such as Ureaplasma urealyticum,
Gardnerella vaginalis and Chlamydia tracho-matis, are well recognised
etiological agents of sexually transmitted diseases26-28. The
quantification of all of them is extremely important to determine
their clinical significance in each localization, and to avoid
confusion with the normal flora of skin and mucous membranes.
The finding of Chlamydia trachomatis in semen samples by
immunofluorescence does not allow differential counts, therefore
patients must be treated even if the infection site cannot be
The World Health Organization has defined leukocytospermia as the
finding of > 106 WBC/ml in semen. Granulocytes are the
predominating WBC in semen, and were reported to represent about 50 to
60% of all wBC in ejaculates from fertile and infertile men29. To
identify PMN granulocytes in semen routine analysis, we used the
peroxidase test recommended by WHO because it is fast, specific and
cost-effective. It is based on the visualization of peroxidase
activity to identify and quantify PMN. In agreement with previous
reports by Endtz30 and Yanushpolsky2 we defined the threshold for
leukocytospermia as a concentration of PMN equal to 0.5 x 106/ml.
Thirteen infected patients had a concentration of PMN in semen less
than 0.5 x 106/ml (45% of the total infected population). For this
reason we believe that leukocy-tospermia is a poor marker of
infection. On the other hand, most leukocytospermic patients (9 of 16,
56%) resolved it after antibiotic therapy.
In accordance with Yanushpolsky31, we consider that a single positive
finding of leukocytospermia does not require antibiotic therapy
without further evidence of a specific genital tract bacterial
infection. For this reason, it is important to conduct a complete
microbiological study such as the one described here.
Our results show that after microbiological cure, total sperm
concentration, viability, motility and total motile sperm clearly
The percentage of teratozoospermic patients did not change after
antibiotic therapy, and those with normal morphology values remained
In two patients with antisperm antibodies, the antibody titer became
negative after bacteriological cure. Genital tract infections in males
may trigger immunoresponse and elicit the formation of antisperm
antibodies32. A possible mechanism was reported by Kurpisz &
Alexander33 regarding common antigenic determinants between infectious
organisms and components of the reproductive system.
In conclusion, in this study we have shown that sperm parameters
improve after bacteriological cure in infected asymptomatic infertile
patients. These results stress the importance of proper
microbiological studies and specific antibiotic therapy in individuals
suspected of having genital tract infections.
Acknowledgements: This study was inspired on the work of Dr.
Franco E. von der Walde, who died in 1992, and his restless search for
the etiology of infertility.
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TABLE 1.- Microorganisms isolated in prostatic fluid and semen in
29 infected patients
Microorganisms Prostatic Fluid Semen
Enterococcus faecalis 6 7
Arcanobacterium haemolyticum 3 2
Escherichia coli - 1
Staphylococcus aureus 1 -
Staphylococcus hominis 1 -
Staphylococcus warneri - 1
Streptococcus pyogenes 2 -
Streptococcus pneumoniae 1 -
Streptococcus agalactiae 1 -
Gardnerella vaginalis 1 -
Ureaplasma urealyticum - 1
Chlamydia trachomatis - 1
Total 16 (55%) 13 (45%)
TABLE 2.- Semen parameters in 24 patients before and after 90 days
antibiotic therapy l
Before therapy After therapy
Parameter A B C
(106/ejaculate) 35.2 (0.017 to 415.4) 33.7 (0.016 to 378.4) 41.1 (0.47
Sperm motility (%) 18.5 (0 to 67.0) 18.5 (0 to 65.0) 41.0 (4.0 to
Total motile sperm
(106/ejaculate) 3.37 (0 to 278.3) 2.98 (0 to 283.8) 8.1 (0.017 to
Sperm viability (%) 69.0 (0 to 88.0) 70.0 (0 to 90.0) 72.0 (41.0 to
(% normal forms) 25.0 (5.0 to 67.0) 25.0 (3.0 to 70.0) 28.0 (5.0 to
PMN (106/ml) 1.0 (0.050 to 3.5) 1.0 (0.050 to 3.5) 0.25 (0.045 to
The initial andrological status of the 24 patients (A and B) was: 4
normospermic, 4 oligoasthenozoospermic, 9
oligoasthenoteratozoospermic, 3 asthenozoospermic, 3
asthenoterazoospermic, 1 teratozoospermic.
l Values are medians from two consecutive semen analysis in A, B and
C, with ranges in parentheses, for 24 patients
A: Five months before culture; B: At the time of bacteriological
culture; C: Two months after finishing antibiotic therapy
In all cases p express the significance between each parameter before
(A or B) versus C (after treatment) (Wilcoxon signed rank test)
C p < 0.05, Z p: 0.01, ? p: 0.0001, 9 p: NS
TABLE 3.- Semen parameters before and after antibiotic therapy in 5
patients not achieving bacteriological cure l
Before therapy After therapy Difference
Parameter A B C between
A, B, C
(106/ejaculate) 37.0 (32.4 to 136.5) 37.5 (33.0 to 126.5) 36.9 (30.4
to 145.0) NS;
Sperm motility (%) 14.0 (8.0 to 16.0) 15.0(8.0 to 15.0) 14.0 (8.0
to 17.0) NS;
Total motile sperm
(106/ejaculate) 5.1 (3.2 to 13.5) 5.0 (3.7 to 13.6) 5.8 (3.3 to 11.9)
Sperm viability (%) 56.0 (15.0 to 65.0) 54.0 (15.0 to 65.0) 54.0
(16.0 to 65.0) NS;
(%normal forms) 38.0 (30.0 to 50.0) 39.0 (31.0 to 50.0) 39.0 (30.0 to
PMN (106/ml) 0.72 (0.12 to 4.0) 0.43 (0.15 to 1.35) 0.4 (0.14 to 1.3)
l Values are medians from two consecutive semen analysis in A, B
and C, with ranges in parentheses, for 5 patients
A: Five months before culture; B: At the time of bacteriological
culture; C: Two months after finishing antibiotic therapy
; p: NS (Friedman test)