Efficiency
of Seminal Fructose Estimation As A Marker of
Seminal Fluid Colonization with Bacteria
.........................................................................................................................
Zakeria A.Yaseen AL-KHAYAT*
Kawther I. AL-HARMINI**
Sardar Nouri AHMED***
*PH.D.Clinical microbiology, Dept.of microbiology
-College of medicine-Hawler medical university
**MS.c. Medical parasitolog,y, -Dept.of anatomy
and histology, -College of medicine- Hawler medical
university
***MS.c. Medical biochemistry, Dept.of clinical
biochemistry, College of medicine- Hawler medical
university
Correspondence:
Sardar Nouri
Email: sardarna2007@yahoo.com
|
ABSTRACT
Background and
objectives: Infertility is generally
defined as the inability of a couple to
conceive after 1 year of unprotected intercourse.
This study included semen fructose estimation
in two groups of men; fertile controls
without significant bacteriospermia and
nonazoospermic infertile men with significant
bacteriospermia.
Materials and
methods: A prospective study was carried
out from March 2003 to January 2004 by
collaboration between clinical biochemistry
and clinical microbiology departments
in the College of Medicine, Hawler Medical
University on eighty five infertile men,
and the results were compared with twenty
six fertile men.
Results:
The results obtained revealed the isolation
of different species of microorganisms
from asymptomatic infertile men. Fructose
level was unaltered in the two groups
of males. Thus the measurement of the
concentration of fructose in the semen
fluid was found to be non-discriminatory.
.
Conclusion: Estimation of seminal
fluid fructose is not an efficient marker
for the presence of bacterial colonization
in the semen.
Key words:
Infertility, Fructose, Bacteriospermia
|
Infertility is generally defined as the inability
of a couple to conceive after 1 year of unprotected
intercourse. Approximately 10 to 15 percent
of couples in the United States are affected(1).
One of every four Polish married couples is
infertile(2). In approximately 30%
of cases, pathology is found in man alone and
in another 20% both the man and woman are abnormal,
therefore, the male factor is at least partly
responsible in about 50% of infertile couples(3).
It is certainly appropriate for semen analysis
to be the first step on evaluation of the male,
besides a detailed history and physical examination(4).
Other suggested procedures include chemical
analysis of the seminal fluid, i.e: determination
of fructose level, hormonal assays, immunological
investigations of testicular biopsy(3).
According to the guidelines of (WHO, 1999)(5),
the parameters of seminal fluid can be classified
into four classes(5):
- Azoospermia: i.e: The seminal fluid with
no sperm and the condition may be caused either
by obstruction or by defects in spermatogenesis
or due to congenital azoospermia.
- Marginal seminal fluid: the defect is in
one of the principle parameters like morphology,
motility or number. It may or may not cause
infertility.
- Abnormal seminal fluid: Most or all parameters
are abnormal. It causes infertility.
- Normal seminal fluid.
Fructose is the principle source of the motility
of the sperm under anaerobic conditions. It
is secreted by the seminal vesicles under the
effect of androgen hormone(6).
Microorganisms can be isolated from most seminal
samples, but the significance of bacteriospermia
is uncertain because many males lack symptoms
associated with the bacterial infection of the
reproduction tract(7).
In many cases, opportunistic microorganisms
cause such classical infections of the urogenital
tract such as epididymitis and prostatitis,
as well as sub-clinical reproductive tract infection(8).
Thus such silent sub-clinical infection of the
reproductive tract could be one of the causes
of male infertility(9).
Asymptomatic inflammation is difficult to diagnose,
therefore investigations for different biochemical
markers e.g.; zinc, citric acid, acid phosphatase
and fructose are achieved to facilitate the
diagnosis of asymptomatic inflammation(10).
A-SUBJECTS
The studied group included eighty-five selected
infertile men who attended the laboratory department
at the infertility Care and IVF center in Erbil
city. This group were selected after exclusion
of other variables like genitourinary infection,
medical, surgical, and anatomical causes. Also
taking into consideration, they were not heavy
smokers and were non -lcoholics. Azoospermic
semen was extracted from this study. The control
group consists of twenty-six fertile men. This
work was carried out between March and January
2004.
B-COLLECTION OF SPECIMENS
A semen specimen was collected after 3 days
of sexual abstinence, in a wide-mouthed clean
and sterile container. Then after liquifection,
one drop of the semen was taken to determine
the number of the sperm and then after that,
the semen sample was divided into two parts;
one for fructose estimation and the other for
culture.
C- DETERMINATION OF SEMINAL FLUID FRUCTOSE
The method is adopted from that of Seliwanoff(11).
The principle depends on the presence of fructose
(ketoses), which produce hydroxyl methyl furfural
by the action of concentrated HCl. The intensity
of the red complex is proportional to the fructose
concentration and absorbed at 490 nm.
D-METHOD OF SEMEN CULTURE
Because seminal plasma has a rather strong bacteriostatic
capacity, it is recommended to be diluted before
innoculation. This was done by adding 0.2 ml
of sterile physiological saline to 0.2 ml of
semen. After mixing 50ul of the mixture was
homogenously spread on blood MacCoukeg, chocolate
agars, and incubated for 24 - 48 hours at 37c.
The number of colonies is counted and multiplied
by 40 to give the number of colony forming unit
CFUs per ml. Growth of 1000-3000 CFU/ml is considered
borderline. While CFUs fewer than 1000 suggest
contamination(12).
E-STATISTICAL ANALYSIS
Statistical analysis was carried out by using:
- Mean standard deviation and standard error
of the mean SEM.
- t-test to find out the significant difference
of the semen parameter in infertile as
compared to fertile men.
From the eighty five infertile semen samples
cultured, sixty five yielded positive growth
of bacteria, while the remaining twenty five
yielded no significant growth. Regarding the
control group, the results of all culturing
the twenty six semen samples showed non significant
growth.
Table (1) revealed the mean levels of fructose
in these four groups i.e.: control, infertile
group as a whole, infertile group with significant
growth and infertile group with non-significant
growth. The mean levels were: (10.2 ±
1.3), (13.3 ± 0.55), (14.05 ±o.6),
(15.02± o.998) respectively.
Tables (2, 3) demonstrate the results of the
sixty-five seminal fluid cultures. Six different
species of bacteria were isolated, with one
or two different bacteria from some samples.
The most frequently isolated bacteria was Diphtheroid
32 (49.23%), and this bacteria was isolated
alone and in combination. The other positive
isolated growths were: Staphylococcus epidermidis
28 alone, and in combination 43.07%.
Escherichia coli 16 (24.6%) alone and in combination.
Staphylococcus awreus 7 (10.8%) alone and in
combination, and one (1.5%) for each of Proteus
mirabilis and Pseudomonas aeuroginosa.
Table (4) demonstrates the frequency distribution
of fructose level in the two groups (infertile
and the control).
|
Table 1. The mean
level of fructose in both the control and
the infertile groups. |
| |
Control
26 |
Infertile
group 85 |
Infertile significant growth
65 |
Infertile no significant
growth 20 |
|
Fructose
level mmol/L |
10.2mmol/L±1.3 |
13.3±0.55 |
14.05±0.6 |
15.02 ± 0.998 |
|
Statistical
analysis |
N.S |
N.S |
N.S |
N.S |
|
Table 2. Results
of the seminal fluid culture of 65 infertile
men. |
| One bacteria |
N |
% |
Two bacteria |
N |
% |
| E-Coli |
7 |
10.8 |
E.Coli
& st.epid |
6 |
9.2 |
|
Diphtheriod |
21 |
32.3 |
E.Coli
& st.anurus |
3 |
4.6 |
| S.epidermides |
11 |
16.9 |
Diphtheriod
& st.epid |
11 |
16.9 |
| S.anurus |
4 |
6.2 |
|
|
|
|
P.valgaris |
1 |
1.5 |
|
|
|
|
Pseud.aeurogenism |
1 |
1.5 |
|
|
|
| Sum |
45 |
69.2 |
|
20 |
22.5 |
|
Table 3. Bacteria
isolated from the 65 infertile semen samples
alone or in combination. |
|
Bacteria isolated |
N |
% |
|
Diphtheriod |
32 |
49.2 |
| S.epidermides |
28 |
43.07 |
| E-Coli |
16 |
24.6 |
| S.anurus |
170 |
10.8 |
| P.valgaris |
423 |
1.5 |
| Pseud.aeurogenism |
513 |
1.5 |
|
Table 4. Frequency
distribution of fructose in different groups. |
|
Fructose level |
Fertile |
Infertile |
|
Non
sign. growth |
Significant
growth |
|
One
bacteria |
Two
bacteria |
|
E-Coli |
St. epid |
St. merues |
Dipth |
Prot Valg. |
Pseudo Aruo. |
E.coli + St.
epid |
E.coli + St.
merues |
Dipth + St.epid |
| 3.5-8.4 |
8 |
2 |
2 |
1 |
_ |
3 |
_ |
_ |
1 |
_ |
1 |
| 8.4-13.3 |
2 |
5 |
3 |
4 |
3 |
6 |
_ |
_ |
1 |
1 |
5 |
| 13.3-18.2 |
10 |
8 |
1 |
2 |
1 |
8 |
1 |
1 |
2 |
1 |
2 |
| 18.2-23.1 |
6 |
5 |
1 |
3 |
_ |
4 |
_ |
_ |
2 |
1 |
3 |
| 23.1-28 |
_ |
_ |
_ |
1 |
_ |
_ |
_ |
_ |
_ |
_ |
_ |
| Sum |
26 |
20 |
7 |
11 |
4 |
21 |
1 |
1 |
6 |
3 |
11 |
It has been found that prostate and vesicle
infection, and sub-clinical reproductive tract
infection may lead to dysfunction of sperm and
changes in semen parameters, and the latter
may consequently lead to infertility. Some possible
pathophysiological mechanisms of the development
of infertility linked either to inhibition of
spermatogenesis resulting from testicular damage
or autoimmune process (Bukharin, 2000(7),
Khalili, 2000(13), Omer, 1985(14),
Huertra, 2002(15)).
In this study, it had been taken into account
only positive semen culture with 1000-3000 and
more CFU/ml (Combaire, 1996(12))
in which this amount of bacteria was postulated
to be effective for inducing genital tract infection
and affect semen parameters producing male infertility.
Gregoria (1989(16)) had taken the
count of 100-1000 and more into consideration,
thus our results depending on this basis of
numbering of bacteria is considered perfect.
Numbering of the bacteria in the semen is of
importance because as Keck (1998) concluded,
detection of bacteria in semen does not necessarily
signify infection. Since bacteriospermia may
represent contamination, colonization or infection.
Aaccording to Susan (1999(17)), colonization
of the host is also an infection, but it is
one in which the host and organism evolve a
commensual relationship without the development
of disease (asymptomatic).
Therefore these microorganisms isolated in
this study were not contaminant. They were in
colonizing state because all the patients were
asymptomatic. More than one kind of microorganism
had been isolated from asymptomatic patients
in different percentages. The result of this
study is in accordance with the studies below
which showed that mixed bacteria were isolated
in 98%, 59.1%, 64.7% and 47% of the cases respectively
(Khalili, 2000(13), Huertra, 2002(15),
Swenson 1980(18), Corradi, 1992(19)).
Regarding fructose estimation, it was noted
from this study that fructose concentration
did not differ significantly between the whole
infertile group and the control group, beside
that, there was no significance difference between
the positive growth and the non-significant
growth groups within the infertile group.
This result is in accordance with the studies
below that indicate the measurement of seminal
fluid fructose does not contribute to the diagnosis
of infection, because its discriminating power
is lower than that of the ejaculate volume which
is equally dependent on seminal vesicle function
(Andrade,1999(6), Grizard,E;etal,1985(20))
The result of this study is in accordance with
the study of (Vicari,E;etal, 2006(21))
which pointed out that seminal fructose levels
did not reflect the extension of prostate-vesiculo-epididymitis.
Therefore, in conclusion, estimation of seminal
fluid fructose is not an efficient marker for
the presence of bacterial colonization in the
semen.
- Issacs,D,Jhon, Evaluation of the infertile
couple. In manual of clinical problems in
Obstetrics and gynecology.4th edition. Little
brown company's.(1994), pp335-341.
- Kamienczna, M, .Domagafa,A,.Kurpizs,M,.The
frequency of antisperm antibodies in infertile
couples-a polish pilot study.Med Sci Moint,
Apr, (2003).9(4): 142-149.
- McConnel, John, D, .Diagnosis and treatment
of male infertility. In text book of reproductive
medicine. 2nd edition. Appleton and Lange's.
(1998).PP:549-561
- Fisch, H, Lipshultz, L, I. Diagnosis of
male factors of infertility. Arch patho lab
med. (1992), 326, 611-618
- WHO.WHO Laboratory manual for the examination
of human semen and sperm cervical mucus interaction.4th
edition Word Health Organization. (1999),
pp.14-16.
- Andrade, Rocha, Ft, .Seminal fructose level
in male infertility: relationship with sperm
characteristics Int-Urol-Nephrol. (1999),
31(1):107-111.
- Bukharin,O,V, .Kuzmin,M,D, . The role of
microbial factor in the pathogenesis of male
infertility. Th Microbial Epidemol Immunobiol
(2), (2000): 106-110.
- Esfandiari, N, .Saleh,R,R, .Positive bacterial
culture of semen from fertile men with asymptomatic
leukocytospermia.Int.J.Fert.women Med.Nov-dec;(2002);47(6)
pp
265-270.
- Rajesh B,Pratap,K,Siya,S,S,. Role of semen
culture in infertility Obst. Gyn. India (1992)
5(1), 42-48.
- Burnazian RA,Oganisian WN, Burshtein GE.
The biochemical indices of the sperm in chronic
inflammatory disease of the accessory genital
glands., Urol Nefrol (Mosk). (1992) Jul-Dec
;( 4-6):36-38.
- Mant, t. "Fructose content and fructolysis
in semen" practical application in the
evaluation of semen quality.J.Agric.Sci, (1948),
38:323.
- Comhaire, F, H., Zalata,A. Some aspects
of male accessory gland infection and its
possible role in male infertility.Inmale infertility:causes
and management.Champan and Hall Medical. 1rst
edition. Belgium (1996), pp 143-153.
- Khalili, A, M,. Pourshaftie,R,M, Khalili,
B, M,. Bacterial infection of the reproductive
tract of infertile men in Iran. Middle East
Fertility Society Journal, (2000),vol (5),
No(2);126-130.
- Omer, E, F. Inflammatory conditions and
semen quality among infertile Sudanese males-Trop
Doct, (1985), 15(1):27-28.
- Huerta, M, Cervera,R,.Hernandez,I,.Anala,A,
R,. Frequency and etiology of seminal infection
in the study of infertile couples.Gynicol.Obtet
Mex. (2003). 70, 90-94
- Gregoria,O,.Botsis,d,. Papadias,K,.Kassanos,D,.Liapius,A,.Zourlas,P,.
Culture of seminal fluid in infertilic men
and relatioinship to semen evaluation. Int.
J. Gynaecolo Obstet, (1989).28(2);149-153.
- Susan, E, Sharp, Commensal andpathogenic
microorganisms of human. In manualof clinical
microbiology.7th edition. ASM press. USA.
(1999).pp:23-32
- Swenson, Ch.E, Toth, A., Wolfgruber, L.,
Oleary, W.M. Asymptomatic bacteriospermic
in infertile men. Andrologia. (1980). 12,
(1):7-11.
- Corradi, M.G., Panovies, J. Signifiant bacterriospermia
value and limits of sperm in andrology. Orv.Heitl.
(1992).133(43), p259-262.
- Grizard, G, Janny L, Hermabessiere,J,.Sirot,J,B,.
Seminal biochemistry and sperm characteristic
in infertile men with bacteria in ejaculate.Arch
Androl. (1985).15
(2- 3); p181-191.
- Vicari,E,Lavignera.S,Castiglione,R,Calogero,AE.
Sperm parameter abnormalities, low seminal
fructose and reactive oxygen species overproduction
do not determinate patients with unilateral
or bilateral post-infections inflammatory
prostato-visiculo-epididymitis.J Endocrinal
Imvest. (2006). Jan; 29(1) 18-25
|
