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OJHAS Vol. 10, Issue 3:
(Jul-Sep 2011) |
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| AZF Microdeletions
in Human Semen Infected with Bacteria |
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Hayfa H Hassani, Department of Biology,
College of Science, University of Baghdad, Iraq,
Zainab SH Khalaf, Ministry of Health, Kamal Al-Samarie/IVF
and Fertility Center, Baghdad, Iraq,
Ehaim J Samarie, Ministry of Health, Kamal Al-Samarie/IVF and Fertility Center, Baghdad, Iraq,
Mohammed A Ibrahim, Royal Scientific Society, Environmental
Research Center, Amman, Jordan. |
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Address for Correspondence |
Department of Biology,
College of Science, University of Baghdad,
Baghdad, Iraq.
E-mail:
hayfahassani@ymail.com |
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Hassani
HH, Khalaf ZSH, Samarie EJ, Ibrahim MA. AZF Microdeletions
in Human Semen Infected with Bacteria. Online J Health Allied Scs.
2011;10(3):8 |
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Submitted: Aug 8,
2011; Accepted: Oct 18, 2011; Published: Nov 15, 2011 |
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| Abstract: |
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Bacterial infections
are associated with infertility in men. This study was aimed to investigate
microdeletions on Yq chromosome in semen infected with bacteria by using
bacteriological, biochemical, and serological assays. The investigation
showed that 107 of 300 (84.80%) semen samples collected from infertile
men with primary or secondary infertility were infected with different
species of bacteria. Chlamydia trachomatis and Neisseria gonorrheae were the most frequently diagnosed bacteria in the infected
semen samples. The percentages of infections of semen samples with
C. trachomatis and N. gonorrhea were 42.31% and 35.28% respectively.
Genomic DNA from each semen sample infected with predominant bacteria
was analyzed for AZF deletions by using multiplex PCR. Different patterns
of AZF microdeletions were obtained. It can be concluded that sexually
transmitted bacteria may contribute in microdeletions of Yq chromosome
by indirectly producing reactive oxygen species and causing gene defect
in AZF regions.
Key Words:
Male infertility; Yq microdeletion, Chlamydia trachomatis,
Neisseria gonorrheae
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Male infertility has been associated with a number of non-genetic and
genetic factors.1 The non-genetic factors include hypogonadotrophic
hypogonadism, previous inguinal and scrotal surgery, and environmental
factors such as genital infections. In respect to male urogenital tract
infection, it was found that asymptomatic bacteriospermia had an important role in male infertility through affecting different
sites of male reproductive tract, such as the testis, the epididymus
and male accessory gland.2
The contribution of
genetic factors in male infertility has been reported. In this respect,
it was observed that Yq chromosome, and in particular Yq11.23 was involved
in the sexual development and spermatogenesis.3 Yq contains
three AZF regions designated AZFa, AZFb, and AZFc from proximal to distal
Yq.4 A more recent study reported a new region designated AZFd and it was mapped between STS and sY145-sY221.5 Many microdeletions on Yq have been implicated as significant causes of infertility.
These microdeletions are often observed at Azoospermia Factor (AZF)
locus.6 AZF locus harbor genes, RBMY
(RNA Binding Motif on Y) and DAZ
(Deleted in Azoospermia), that involved in spermatogenic failure.7
Many researchers have
linked positive bacterial semen cultures with poor semen quality8-10
but to best of our knowledge no study has correlated between the existence
of bacteriospermia and gene’s defect on Yq chromosome. Thus, this
study was aimed to investigate the occurrence of microdeletions on Yq
in semen infected with bacteria.
The study was conducted
during January 2010 to December 2010 on 300 infertile men attending
Kamal Al-Samaraie Hospital/IVF Center in Baghdad, Iraq. The patients
had either primary infertility (n=242) or secondary infertility (n=58).
Those patients were interviewed about their medical history, family
backgrounds, reproductive problems and possible consanguinities. In
addition, a physical examination was conducted in all cases, in order
to identify anatomical problems. In addition, 30 fertile men were used
as control. Semen samples were obtained after a 7 days period of sex-inhibition.
The semen samples were
collected from patients following the World health organization (WHO)
guidelines11, and were subjected to microbiological tests
for identification bacteria. Each sample was cultured on different selective
media (Blood agar, Chocolate agar, MacConkey agar, Mannitol salt agar,
Chromogenic UTI agar) and incubated at 37ºC for 24-48 hrs. After incubation
period, the colonies were identified by using standard bacteriological
and biochemical assays.12,13 Furthermore, several
specific tests were used to detect the suspected colonies, for example BactiCard Neisseria test was used to detect Neisseria
gonorrheae,
H.V.D.R.L test was used to identify Treponema
pallidum, and Furazolidone disk test was used to differentiate Staphylococci
from Micrococci.14,15 ELISA assay was also used to detect
anti-Chlamydia trachomatis antibodies IgG by using a specific
kit (Euroimmune, Germany).16
Genomic DNA was
isolated from each seminal fluid samples infected with predominant bacteria
by QIAamp®DNA Mini Kit (Qiagen, Valencia, CA). The screening for AZF
microdeletions was performed by multiplex PCR. A series of 16 of specific
STSs, mapped in four AZF regions, human zinc-finger protein-encoding
genes (ZFX/ZFY) located on the X and Y chromosomes, acting as an internal
control primers, and a unique region of SRY on Yp chromosome
for detection XX male arising from Y to X translocation were selected
17,18. The specific STSs included sY86 and sY84 for AZFa,
sY127,sY134,sY121,sY124,sY128,sY130 for AZFb, sY254,sY157,sY255,sY242,sY208
for AZFc, sY133,sY152,sY145 for AZFd. Multiplex PCR amplification was
performed in a 10µl reaction system, containing 200 ng of genomic DNA,
1.5 mmol/L Mg2+, 800 µmol/L deoxynucleotide triphosphates
(dNTPs), 10 pmol/L of each primer and 2 U Taq polymerase. Amplification
was carried out in thermocycler (Appendorf, Germany). The conditions
for PCR amplifications consisted of an initial denaturation of 6 min
at 94ºC, followed by 35 cycles of 40 s at 94ºC, 45s at 55ºC and 60s
at 72ºC, with final extension step at 72ºC for 6 min.
Thirty fertile men
were used as positive control to ensure the performance of the amplification
reaction. The PCR products were separated on 1.5%agarose gel for
2hrs at 5V/cm. The results were visualized using UV transilluminator
system.
Out of 300 cultured
semen, 107 (84.80%) showed positive bacterial contamination (Table 1).
Chlamydia trachomatis antibodies IgG were diagnosed in 58 (42.31%)
samples. This bacterium markedly formed higher prevalence in secondary
infertile men than primary infertility cases; the percentages were 24.13%
and 18.18%, respectively. The second dominant bacteria was Neisseria
gonorrheae; it was identified in 27 (11.15%) semen samples obtained
from patients had primary infertility and in 14 (24.13%) cases had secondary
infertility. In addition, Treponema
pallidum was successfully isolated from 3 (1.23%) samples obtained
from patients with primary infertility and 2 (3.44%) patients with secondary
infertility. Whereas Staphyloccocus
aureus was existed in lower incidence; it was found at 0.41% and
1.72% in primary and secondary infertile men respectively. Trailing
behind these was Escherichia coli which isolated only from one
patient (0.41%) with primary infertility. It is worth mentioning, no
culture yielded a mixed growth of bacteria; on the other hand no bacterial
isolates were recovered from samples of fertile men’s semen.
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Table 1:
Bacteria isolated from semen samples of infertile men. (Control showed no
bacterial infection)
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Type
of bacteria |
Type of infertility |
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Primary infertility
(n=242) |
Secondary infertility
(n=58) |
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No. (%) |
No.(%) |
| C.
trachomatis |
44 (18.18) |
14 (24.13) |
| N.
gonorrhea |
27 (11.15) |
14 (24.13) |
| T.
pallidum |
3 (1.23) |
2 (3.44) |
| S.
aureus |
1 (0.41) |
1 (1.72) |
| E.
coli |
1 ( 0.41) |
0 (0) |
| Total |
76 (31.38) |
31 (53.42) |
Subsequently semen
samples infected with prevalent bacteria, C.
tarchomatis and N. gonorrheae, were subjected to molecular
analysis of Yq microdeletion. Table 2 showed the analytical results
of multiplex PCR reactions concerning patients who had either primary
or secondary infertility infected by C. trachomatis. Semen samples from primary infertile men revealed
3 (6.81%) samples with complete deletion in AZFa+b+c+d, 11(25%) samples
observed with partial deletion in AZF b+d and complete deletion in AZFc,
8 (18.18%) harbored partial deletion in AZFb+c+d, and 22 (50%) showed
complete deletion in AZFc. While secondary infertile men who harbored
the same bacterium showed 10 (64.28%) samples had complete deletion
in AZFc, 3 (21.42%) samples indicated complete deletion in AZFc
and partial deletions in AZF b+d, and one (7.14%) sample partial deletion
in AZFb+c+d.
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Table 2:AZF deletions
detected in semen infected with C.
trachomatis |
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Type
of bacteria |
Type of infertility |
Semen samples No. (%) |
Deletion in AZF loci |
Type of deletion |
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C. trachomatis |
Primary infertility |
3 (6.81) |
a+b+c+d |
complete |
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11 (25) |
a |
* |
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c |
complete |
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b+d |
partial |
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8 (18.18) |
a |
* |
|
b+c+d |
partial |
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22 (50) |
a |
* |
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b |
* |
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c |
complete |
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d |
* |
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Secondary infertility |
10 (64.28) |
a |
* |
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b |
* |
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c |
complete |
|
d |
* |
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3 (21.42) |
a |
* |
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c |
complete |
|
b+d |
partial |
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1 (7.14) |
a |
* |
|
b+c+d |
partial |
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*Skipping band |
On the other hand,
DNA of semen from infertile patients infected by N. gonorrheae was analyzed for screening AZF microdeletions (Table 3).
In primary infertile men, 14 (63.63%) semen samples exhibited partial
deletion in AZFb+c+d, 3 (13.36%) revealed complete deletion in
AZFb+c+d, 3 (13.36%) showed partial deletion in AZFb and complete deletion
in AZFc, and 2 (9.09%) cases had complete deletion in AZFd. While
semen specimens of secondary infertile men showed 3 (42.85%) cases had
partial deletion in AZFc, 2 (28.57%) cases had partial deletion in AZFb+d,
one (14.28%) sample showed complete deletion in AZFb, and one (14.28%)
sample appeared with complete deletion in AZFa. Regarding semen
of fertile men, no AZF microdeletions were noticed.
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Table 3: AZF deletions
detected in semen infected with N.
gonorrhea |
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Type
of bacteria |
Type of infertility |
Semen samples No. (%) |
Deletion in AZF loci |
Type of deletion |
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N. gonorrhea |
Primary infertility |
14 (63.63) |
a |
* |
|
b+c+d |
partial |
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3 (13.63) |
a |
* |
|
b+c+d |
complete |
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3 (13.63) |
a |
* |
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b |
partial |
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c |
complete |
|
d |
* |
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2 (9.09) |
a+b+c |
*
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d |
complete |
Secondary infertility
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3 (42.85) |
a |
* |
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b |
* |
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c |
partial |
|
d |
* |
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2 (28.57) |
a |
* |
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b |
partial |
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c |
* |
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d |
partial |
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1 (14.28) |
a |
* |
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b |
complete |
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c |
* |
|
d |
* |
|
1 (14.28) |
a |
complete |
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b |
* |
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c |
* |
|
d |
* |
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*Skipping band |
The occurrence of male
genital tract and/or accessory gland infections has been considered
as a potential hazard to male infertility. In this study, 107 (84.80%)
of examined seminal fluid samples, collected from 300 patients with
primary and secondary infertility, were infected with different species
of bacteria: C. trachomatis, N. gonorrheae, T. pallidum, S.
aureus, and E. coli. The obtained results showed that
C. trachomatis and N. gonorrheae were the most prevalent bacteria isolated from semen;
the percentage was 42.31% and 35.28%, respectively. It is worth mentioning,
other investigators reported that the genital tract infections and inflammation
were associated with 8-35% of male’s infertility cases8,19
and these bacteria were considered as asymptomatic bacteriospermia.
Furthermore, there are indications that sexually transmitted infections,
especially N. gonorrheae and C. trachomatis, account for
a significant proportion of cases causing occlusion of the vas deferens
and subsequent oligospermia and azoospermia.20
Hosseinzadeh and his
group21 reported that the active component of C.
trachomatis, lipopolysaccharide, was responsible for sperm death
due its spermicidal properties. Research work conducted by Gomez and
coworkers showed that the presence of N. gonorrheae in semen may result in azoospermia or oligospermia, then
lead to infertility by binding to human sperms, and result in agglutation
of sperms and decreasing the sperm’s motility.22 Kauer and his research team23 were able to isolate S.
aureus (2.13%) and E. coli (0.14%) from infertile semen’s
cultures. These bacteria can be considered as commonsalty bacteria.
Whereas T. pallidum, a bacterium that cause syphilis, was reported
to be associated with male infertility by lowering the sperm count and
volume of ejaculate.24
In this study, the
possible connection between bacterial infection and potential gene defects
in human semen, which infected with predominant bacteriospermia,
C. trachomatis and N. gonorrheae, was investigated. Regarding
semen infected with C. trchomatis (Table 2), the most frequently
microdeletions were seen in AZFc (85.70%). It was reported that the
complete deletion in AZFc region (absence of sY254, sY157, sY255, sY242,
sY208) means defect in DAZ gene family that had been regarded
as the most likely candidate genes for spermatogenesis deficiency.17
In addition, other study reported deletion in AZFa+b+c+d regions (3
of 44 cases, 6.81%), they suggested that this region probably involves
the heterochromatic region on Y chromosome and causes severoligozoospermia.5
Whereas other study conducted by Kin (2004)5 reported that
7.14% of cases had deletion in AZFd, he suggested that deletion in this
region may not be associated with extent of defective spermatogenesis.
On the other hand,
our results (Table 3) showed that the semen infected with N. gonorrheae, revealed 3 (13.63%) cases with primary infertility and
the genome harbored deletion in AZFc. The obtained results showed that
14.28% of examined cases gave complete deletion in AZFa and AZFb. It had been reported that absence or deletion of AZFa, which harbors
two genes, USP9Y and DBY, will cause spermatogenic failure,
while deletion of AZFb, which contains RBMY1; will cause spermatogenic
arrest.5 Further studies are required to explain the mechanism
which causes the deletion.
In general, when microbes
invade the human body, it produces polymorphonuclear leukocytes and
macrophages, which are the major sources of reactive oxygen species
(ROS) production.27 Although there is no direct evidence
that N. gonorrheae or C. trachomatis increases ROS production,
the associated leukocytospermia is well known to produce ROS.28
Most importantly, leukocytospermia has been associated with occult sperm
DNA damage, this may occur directly in the form of leukocytospermia,
a manifestation of inflammation that is associated with cytokines, which
can potentially alter spermatogenesis and cause DNA aberrations, or
indirectly as a result of pathological ROS levels, which are frequent
in leukocytospermia patients.29
We are grateful to
the staff of Kamal AL-Samariae Hospital/IVF Center for their assistance.
We also acknowledge the technical assistance provided by the staff of
Molecular Biology Laboratory in the College of Science, Baghdad University.
- Dohle GR, Halley DJ, Van
Hemel JO et al. Genetic risk factors in infertile men with sever oligozoospermia
and azoospermia. Hum Reprod. 2002;17:13-16
- Golshani M, Taheri S, Eslami
G et al. Genital tract infection in asymptomatic infertile men and its
effect on semen quality. Iranian J Publ Health. 2006;35(3):81-84
- Tiepolo L, Zuffardi O.
Localization of factors control lingospermatogenesis in the nonfluorescent
portion of the human Y chromosome long arm. Hum Gene. 1976;34:119-124
- Vogt PH, Edelmann A, Kirsch
S et al. Human Y chromosome azoospermia factors (AZF) mapped to different
subregions in Yq11. Hum Mol Genet. 1996;5:933-943
- Kin CM. A study on the prevalence
of AZFd Y-chromosome microdeletion in Hong Kong Chinese men with severe
male factor infertility. Med Sc Dissertation submitted to the University
of Hong Kong. 2004;21-22
- Kilic S, Yuksel B, Yilmaz N et al. Results of ICSI in severe oligozoospermic and azoospermic patients
with AZF microdeletions. Iranian J Reprod Med. 2009;7 (2):79-84
- Ferlin A, Arredi B, Speltra
E et al. Molecular and clinical characterization of Y chromosome microdeletions
in infertile men: A 10-year experience in Italy. J Clin Endocrinol Metab.
2007;92(3):762-770
- Ibadin OK and Ibeh IN. Bacteriospermia
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- Marconi M, Pilatz A, Wagenlehner et al. Impact of infection on the secretory capacity of the male accessory
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- Onemu SO, Ogbimi AO, Ophori EA. Microbiology and semen incidices of sexually-active males
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- Wang RX, Fu C, Yang YP et al. Male infertility in China: Laboratory finding for AZF microdeletions
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- Okonofua FE. Female and
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- Hosseinzadeh S, Pacey AA, Eley A. Chlamydia trachomatis-induced death of human spermatozoa
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- Gomez CI, Stenback WA, James
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- Adejuwon AO, Bisi-Johnson
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