Departments of Medical Microbiology 1, Physiology 3, Pharmacology 6, College of Medicine and Department of Microbiology 4, Faculty of Natural Sciences, Ambrose Alli University, Ekpoma and Departments of Obstertrics & Gynaecology2 and Pathology5, Irrua Specialist Teaching Hospital, Irrua.
*Corresponding Author: mcsionelphilrazzy @yahoo.com
ABSTRACT A total of 52 semen samples were analysed, following collection through masturbation by males with history of infertility. Various bacterial strains were isolated with Staphylococcus aureus being the most common with a prevalent rate of 60%, Streptococcus pyogenes 10%, Proteus mirabilis 23%, Klebsiella spp 35% and Escherichia coli 23%. Chlamydia trachomatis was diagnosed in 88% of the subjects, using serological means. A statistical significance (x2= 10.83; P<0.001) revealed Chlamydia trachomatis as the most probable cause of male infertility compared to Escherichia coli. Mean spermatozoa motility in the studied subjects reduced with active motile 26.6+17.63, slow motile 23.1+3.01 and non-motile 42.6+4.11. Significant improvement was noted in sperm motility with accompanying decrease in Leukocytospermia and combined antibiotics therapy also reduced Leukocytospermia and enhanced spermatozoa motility. Key Words: Male infertility, Semen analysis, Leukocytospermia, Combined antibiotic therapy. INTRODUCTION
cells(RBC), no white blood cells (WBC) nor
Male infertility is an important issue, a common
problem occurring worldwide. It is a neglected
revealed that various microbial infections
reproductive health issue in Nigeria (Okonofua
reduce the viability of semen. Chlamydiaet al., 2005). Reports indicate that the male
trachomatis with chronic epididymitis and
factor accounts for 20-50% of the causes of
epidididymal granuloma formation could lead to
blockage of the epidididymis, leading to
(Chukwudebelu et al, 1979; Esimai et al.,
infertility due to azoospermia (Ochsendorf et al,
2002). Male infertility is actually the inability of a
1999). Ness et al (1999) reports an association
couple to achieve pregnancy, despite regular
between Chlamydia antibodies in semen and
unprotected intercourse, usually after a period
male infertility. Similarly the presence of
of 12 months, with detected factors attributed to
antibody to Chlamydiatrachomatis is correlated
with the presence of white blood cells in semen
(Ojengbede et al., 1992) as well as sexually
and development of autoimmune response to
transmitted diseases may be associated with
spermatozoa (Witkin et al., 1995)
2001).Studies also revealed high rates of
For Ureaplasma urealyticum infection, induction
hyperprolactinemia (Modede, 1994; Adejuwon
of leukocytospermia, leads to elevated levels of
et al., 1999), antisperm antibodies (Ekwere,
leukocyte-derived reactive oxygen species
1995) and genital infections (Onemu and Ibeh,
which damage sperms by causing peroxidation
2001; Momoh et el., 2007) in infertile Nigerian
of Lipids (Potts et al., 2000). Leukocytospermia
men than those in fertile control. Aflatoxin may
in infertile males is associated with decreased
equally contribute to male infertility (Ibeh et al.,
sperm numbers and impaired motility with
The minimal criteria for normal sperms include
a volume of 1.5ml; a minimal count of 20million
Another associated factor in male infertility in
sperms/ml; motility of 60%, with no red blood
Nigeria include sexually transmitted infection
www.nansjournal.com NIGERIAN ANNALS OF NATURAL SCIENCES, VOLUME 8(2) 2009 (pp 36 – 41) PRINTED IN NIGERIA
(Imade et al., 1993),while health care seeking
trachomatis was assessed and confirmed for all
behaviour for STI equally play a vital role in
patients using the Chlamydia monoclonal
male infertility(Okonofua et al.,2005).
antibody spot test kit (Immunocomb) and the
Though, pregnancy is the most verifiable
means of determining fertility, diagnosis of
infertility should not be hurriedly reached even
ANTIMICROBIAL SUSCEPTIBILITY TESTING
after1 year of regular unprotected intercourse
The disc diffusion method of Bauer et al.,(1996)
by a cohabiting couple as the age of the
was used. All isolates were subjected to
because fertility is optimal in a woman,
between ages 18 and 24 years and begin to
Staphylococcus aureus Oxford strain NCTC
6751 was used as control for the Gram positive
organisms, while Escherichia coli strain NCTC
effectiveness of combined antibiotic therapy on
10418 was used as control for Gram negative
sperm motility and viability in infertile males in
The result of the susceptibility test were
MATERIALS AND METHODS
interpreted as sensitive, intermediate or
Specimen
resistant following the criteria below as earlier
A total of 52 clinical specimens of semen were
collected randomly. All the semen samples
A zone’s radius equal to or not more than 3mm
Specimens were sent to the laboratory for
smaller than the control was taken as sensitive,
analysis within 15minutes of collection and all
a zone’s radius more than 3mm smaller than
specimens were from patients with primary
the control and not less than 3mm was reported
as intermediate and a zone’s radius 2mm or
criteria(WHO,1999),ie, collection of semen by
less (ie, no zone of inhibition) was reported as
specimen bottles, following abstinence from
intercourse 2-3 days prior to collection.
Following antimicrobial susceptibility testing,
Immediate transfer of sample to the laboratory
selected antibiotics were prescribed with a four
for analysis, a wet preparation made and quick
months follow-up period to assess for clinical
examination, followed by culture and sensitivity.
From a total of 52 samples of semen collected
ISOLATION AND IDENTIFICATION
and analysed by standard microbiological
All samples were inoculated onto Nutrient agar,
methods, different bacterial strains were
isolated from the individual sample. Serological
method was used to detect Chlamydia spp. The
incubated aerobically at 37OC for 24hours,
prevalence of Chlamydia spp was 88%,
thereafter, discrete colonies were picked from
Staphylococcus aureus 60%, Escherichia coli
the growth and Gram stained, while further
23%, Klebsiella spp 35%, Streptococcus
subculturing was done to obtain pure cultures
pyogenes 10% and Proteus mirabilis 23 %.(
for biochemical tests. Bacterial isolates were
identified using the method described by Bauer
et al (1996). The presence of Chlamydia
www.nansjournal.com NIGERIAN ANNALS OF NATURAL SCIENCES, VOLUME 8(2) 2009 (pp 36 – 41) PRINTED IN NIGERIA
Table 1: Prevalence of Microorganisms obtained from semen of males with History or Infertility. Microorganism diagnosed Isolated Bacterial strains
Via serology Chlamydia trachomatis Table 2: shows some selected antibiotics and the in-vitro antimicrobial properties. Augmentin showed similar activity as Ciprofloxacin when its efficacy against Proteusmirabilis, Klebsiella spp as well as Escherichia coli was assessed. Perfloxacin and Ofloxacin had a MIC of 0.125µg/ml against Proteus mirabilis, with the same concentration of Ofloxacin being bacteriocidal with respect to Streptococcus Pyogenes and Klebsiella spp. Table 2 In Vitro MICs/Antimicrobial Activity of selected Antibiotics Against Isolated Microorganisms CRP PEF OFX AUG Chlamydia trachomatis N\A N\A N\A N\A Proteus mirabilis 0.250 0.125 0.125 0.250 Streptococcus pyogenes 0.250 0.250 0.125 0.500 Klebsiella spp 0.250 0.250 0.125 0.250 Escherichia coli 0.250 0.125 0.250 0.250 Staphylococcus aureus 0.250 0.250 0.250 0.500 Key: CRP = Ciprofloxacin PEF = Perfloxacin OFX= Ofloxacin AUG= Augmentin N/A = Not Applicable. Table 3 show actively motile sperms, slow motile sperms and non- motile sperms of male with infertility, have associated white blood cells. While there are numerous leukocytes associated with non- motile sperms and slowly motile sperms, about 0-1 white blood cells were seen among the active motile sperms. Table 3: Viability of spermatozoa using mean motility and Leukocytospermia. Motility Mean +sd Leukocytospermia
Active motile 26.6+_17.63 + Slow motile 23.1 +_ 3.0 +++ Non-motile 42.6 _+4.11 +++ Table 4 shows various degree of improvement in sperm viability when patients are placed on antibiotics. However, viability is greatly enhanced when combined therapy (Ofloxacin and doxycycline) are used and leukocytospermia was eliminated among active motile sperms.
www.nansjournal.com NIGERIAN ANNALS OF NATURAL SCIENCES, VOLUME 8(2) 2009 (pp 36 – 41) PRINTED IN NIGERIA
Table 4 Leukocytospermia in combined and mono-Antibiotic therapy Sperm motility Leukocytospermia in Leukocytospermia in Ofloxacin Ofloxacin therapy + doxycycline combined therapy Treatment Duration in months Treatment Duration in months 1 2 3 4 1 2 3 4 Active motile + _ _ _ _ _ _ _ Slow motile +++ +++ ++ + + + _ _ Non motile +++ +++ ++ + +++ + + + Key: + = 0-5 WBC \HPF ++ = 6-9 WBC \HPF +++ = > 10 (Numerous) WBC \HPF DISCUSSION
phosphate, fructose). Leukocytes are the main
Chlamydia trachomatis and Staphylococcus
radicals) which can decrease sperm function
organisms associated with male infertility in this
(acrosome reaction and zona-binding).MAGI is
work, with percentage prevalence of 88% and
also associated with an increased prevalence
31% respectively. Other isolated organisms
of sperm antibodies (Witkin,1995). The role of
were Streptococcus spp (10%), Escherichia coli
different micro-organism such as Mycoplasma
(23%), Proteus mirabilis (23%) and Klebsiella
or Chlamydia in prostatitis and infertility and the
role of leukocytes in seminal fluid are also
responsible for male accessory gland infection
(MAGI) occurs twice as often in the male
partner of infertile couples than in infertile men.
However, the role of infection and antibiotic
spontaneously, however, they tend to reoccur
treatment in male infertility is still controversial
and only frequent ejaculation together with
antibiotics treatment have a long-lasting effect
Escherichia coli, an organism isolated in this
study has previously been reported as the most
implicated microorganism in urinary tract
The protocol in some cases, is to treat with
infection (Momoh et al, 2007) MAGI, can be
prostatitis, if it is associated with positive
diagnosed when 2 or more criteria are fulfilled:
urethral Chlamydia, Mycoplasma or bacterial
(1.) history of recurrent UTI or prostatitis, (2.)
culture in semen, with positive Mar test or
expressed prostatic fluid with more than 40
associated genital tract infection in the female
leukocytes or urinary sediment with more than
15 leukocytes after prostatic massage and\or
positive bacterial culture, (3.) more than 1
In this study, the quinolones, particularly
million\ml leukocytes in the ejaculate , (4.)
Ofloxacin is effective in the treatment of
growth of 1,000 or more pathogens (E coli,
bacterial associated with infertility, as this study
Streptococcus faecalis, Proteus spp) in seminal
0.125µg/ml -0.25µg\ml, Ofloxacin has inhibitory
(Staphylococcus epidermis, Corynebacterium
effects on all the bacterial isolates. The
spp, Acinetobacter)(Comhaire,1986).
implication is that Ofloxacin can be relatively
relied upon to decrease the bacterial load in
MAGI could affect male infertility by decreasing
these condition. Augmentin at concentrations of
sperm count or motility and accessory gland
0.250µg\ml-0.050µg\ml, also has inhibitory
function (decreased levels of zinc, acid
effects on the isolated bacterial strains.
www.nansjournal.com NIGERIAN ANNALS OF NATURAL SCIENCES, VOLUME 8(2) 2009 (pp 36 – 41) PRINTED IN NIGERIA
Yanushpolsky(1996),reports that if no germ is
Sperm motility is an index of sperm viability and
isolated, suspected cases are treated with
male infertility while leukocytospermia is an
important critical index for assessing male
metronidazole (2x400mg) and AINS (diclofenac
100mg) in the male partner in case of severe
The use of a combined therapy of doxycyline
and Ofloxacin proved to be better in the
management of these patients, leading to
improved sperm motility and reduction and
elimination of white blood cells from semen.
associated with the semen samples. While
active motile sperms has between 0-1 white
Finally, this study has shown that the role of
blood cells with a mean value of 26.6+17.63,
Chlamydia in prostatitis and infertility as well as
the role of leukocytes in seminal fluids are not
numerous white blood cells (>10) associated
with their mean value being 23.1+3.01 and 42.6
REFERENCES
Adejuwon, C.A.,Ilesanmi,A.O.,Ode,E.O. (1999)
However, the inability to culture Chlamydia trachomatis, did not help much. It was however
infertility in Ibadan. West Afr J. Med. 18:17-
observed that serological test remained positive
for Chlamydiatrachomatis among patients who
Agboola, A. (2004) Infertility and Sub-infertility
commencement of Ofloxacin therapy. Hence,
In: Akin A., ED. Textbook of Obstetrics and
an attempt was made to empirically treat for
Chlamydia by adding doxycycline to the
Bauer,A.W., Kirby,W.M., Sheris,J.C., Turk, M.
Branigan(1994), reports that when Mycoplasma
(1996) Antibiotic susceptibility testing by a
or Chlamydia is implicated, either doxycycline
standardized single disc method. AMJ Clin
(2x150mg\d for 2 weeks) could be used for
Branigan,F. (1994) Treatment of Male Infertility.
Similarly, leukocytospermia was completely
eliminated in combined therapy among the
Comhaire, T.(1986) Role of Infection in Male
active motile spermatozoa, with active motility
of 60%-70%,on the average. While among the
slow motile sperm population, there was a
Chukwudebelu, W.O., Esege, N., Megafu, U.
drastic reduction of leukocytospermia. There
(1979) Etiological factors in infertility in
was a statistical significance (x2 = 10.83; P<
0.001) indicating Chlamydiatrachomatis is
strongly associated as a causative factor in
Ekwere, P.D. (1995) Immunological infertility
male infertility when compared to Escherichia
circulating antisperm autoantibodies and
some clinical observation: a preliminary
CONCLUSION
This study has shown various microorganisms
Esimai, O.A., Orji, E.O., Lasisi, A.R.(2002) Male
environment. The study also highlights a high
contribution to infertility in IIe-Ife, Nigeria.
rate of leukocytospermia as strongly associated
with a drastic reduction in sperm motility, as
numerous leukocytospermia was associated
Ibeh,N.,Uraih,N.,Ogonor, J.I. (1994) Dietary
exposure to aflatoxin in human infertility in
www.nansjournal.com NIGERIAN ANNALS OF NATURAL SCIENCES, VOLUME 8(2) 2009 (pp 36 – 41) PRINTED IN NIGERIA
Benin city, Nigeria. Int. J. Fertile Menopausal
Okonofua, F., Menakaya,U., Onemu, S.,Omo-
Imade,G.E.,Towobo,O.A.,Sagay,A.S.,Otubu,J.A.
control study of risk factors for male infertility
(1993) Sexually transmitted diseases and
in Nigeria. Asian Journal Andrology 7(4)
Medico- social factors associated with male
infertility in Nigeria. Arch AIDS Res. 7:245-
Onemu, S.O., Ibeh, N. (2001) Immunological
Infertility among Nigerian men: incidence of
Keck, P. (1998) Chlamydia in prostatitis and
circulating antisperm autoantibodies and
some clinical observation: a preliminary
report. British Journal of Urology. 76: 366-
Potts,J.M., Sharma, R., Pasqualotto, F., Nelson,
D., Hall, G., Agarwal,A., (2000) Association
of Ureaplasmaurealyticum with abnormal
Momoh, A.R.M.,Odike M.A.C., Samuel, S.O.,
reactive oxygen species levels and absence
Momoh, A.A., Okolo P.O. (2007) Resistant
of Leukocytospermia. Journal of Urology.
pattern of Urinary tract infection bacterial
journal of post graduate medicine. 9(1) 22-
Stoke,E.J.(1975) Clinical bacteriology 4th ed.
Ness,R.B.,Markovic,N.,Carlson,C.L.,Coughlin,M.
Witkin,S.S.,Kligman,I.,Bongiovanni,A.M.(1995)
Relationship between an asymptomatic male
having sexually transmitted Urethritis? An
epidemiologic examination. Fertility and
trachomatis and an autoimmune response to
spermatozoa. Human reproduction 10: 2952-
among sexually transmitted diseases clinic
Wolf,H. (1995) The biologic significance of white
Ochsendorf, F.R., Ozdemir,K., Raabenau, H.,
Fenner, T., Doer, H.W. (1999) Chlamydia
Laboratory Manual for the examination of
trachomatis and male infertility: Chlamydia-
interaction. 4th ed. Cambridge University
associated with an inflammatory response.
Yanushpolsky, M.(1995) Treatment of Male
Infertility. Fertility and Sterility 63:142-7
Ojengbede, O.A., Omonira, W.E., Ladipo, O.A.,
Yanushpolsky, M.(1996) Role of Mycoplasma
(1992) Screening for obstruction of the vas
and Chlamydia in male Infertility. Fertility and
deferens in Nigerian men with azoospermia
using the alpha-alpha-glucosidase reaction in semen. Afri J Med Sci: 21:79-81.
www.nansjournal.com
DOSSIER N'16: Reflux gastroœsophagien chez une asthmatique Madame V. B., 40 ans obèse, souffre d'un asthme sévère depuis l'adolescence et qui est actuellementcortico-dépendant. Elle prend 15 mg de prednisone chaque jour depuis six mois, en raison de l'échec detoutes les autres thérapeutiques. L'enquête allergologique a retrouvé des IgE spécifiques dirigées contredes acariens et de
AUDRA GOACH SOSTARECZ EDUCATION Ph.D. , Chemistry, December 2004 The Pennsylvania State University, University Park, PA Dissertation: “Structure Analysis of Langmuir-Blodgett Films with Time-of-Flight Secondary Ion Mass Spectrometry” Advisor: Dr. Nicholas Winograd B. S. , Chemistry (summa cum laude), Phi Beta Kappa, May 1998 Muhlenberg College, Allentown, PA PROFESSIONA