Download File - medical laboratory technologist

CHAPTER ONE
INTRODUCTION
Semen is a viscous whitish secretion of the male reproductive organ
containing spermatozoa and consisting of secretion of the seminiferous tubules.
Semen can also be called seminal fluid. Semen analysis is the study of semen to
investigate mans infertility, where the numbers of sperms are counted and the
morphology and other parameters like motility, volume, and pH are all accessed.
The pathogenic bacteria which brings about low sperm count in infertility
in whole include E. coli, Candida albicans , Neisseria gonorrhea, Chlamydia
trachomatis, Treponema palidium, Fusobacterium nuletum, Bacteriodes fragilis,
Streptococcus faecalis, Staphylococcus species, Proteus vulgaris, Pseudomonas
pyocyanea, β- haemolytic streptococci, just to name a few. These pathogens are
usually isolated from seminal fluid after analysis from males presenting with
infertility (Mongra, et al.,2010).
Bacteria are not the only causes of male infertility other causes are
klinefelter’s syndrome which is based on hormonal disorder, surgery like
vasectomy , deficiency of zinc, selenium and vitamin c, environmental toxins
and radiation with heavy metal, auto immunity where antibodies of a mans
immune system may cause sperms to stick together stopping the penetration of
the cervix, heavy use of alcohol, tobacco, drug addicts, tight fitting underwear
which raises the crotch temperature and reducing sperm count.(Michael, 2003).
Seminal fluid is collected after a period of abstinence of 3-7 days. It is collected
by coitus interruptus, masturbation or using condoms free of spermicide. While
transporting to the laboratory it should be preserved at body temperature by
holding close to the abdomen or under the armpit to maintain the temperature
close to that of the body. Normal sperm count is about 20x106 million sperm
/ml and is termed Normozoospermia
less than this value is called
Oligozoospermia, no spermatozoa in the ejaculates is termed Azoospermia and
Aspermia is when no ejaculates is produced. A normal semen sample has a
1
grey opalescent appearance and liquefies within 60minutes in the presence of
fibrinolysin. If sample is too clear it indicates low sperm concentration. If
brownish or bloody, it indicates physical obstruction in the reproductive tract.
Normal semen pH is between 7.2 -8.0. pH less than 6.4 is indicative of digenesis
and pH > 8.0 indicates infections. 70% of sperm cells should be motile within
the first 60 minutes and should be graded as follows; rapid progressive motility,
sluggish or immotile. Total WBC is counted and should be less than
1x106WBC/ml. Morphology is checked together with viability. Culture is done
and Gram staining done from the colony with biochemical tests also done to
isolate particular pathogens (Cheesbrough, 2000).
1.1 Background Information
Spermatozoa were first described by Leeuwenhoek in 17 th century but it was
not until 1928 that the sperm count was found to be associated with infertility.
Bacterial infections are the main cause of low sperm count. A Hungarian
researcher isolated two types of bacteria from 42 men presenting with infertility,
these bacteria were Fusobacterium nuclentum, and Bacteriodes fragilis. They
affected sperms cells in many ways that is, it concentration, motility,
morphology just to name a few. They compete with sperms for the supplies of
fructose needed for their energy; and also interfere physically with the sperm
lashing of its tail. (Molnar, et al., 2010).
70 infertile males in a study of non specific seminal tract infection and male
infertility with epididymal tenderness, pus cells in semen; and with a history
of urinary tract infection where studied by semen culture , examination and
significant
growth of Streptococcus
faecalis, E. coli, Coagulase
staphylococcus, pseudomonas, and β-haemolytic streptococci were found in
42% of cases . Most of them were sensitive to Ampicilin, Nitrofurantion,
Erythromycin and Chloramphenicol after sensitivity was done (Mogra, et al.,
2010).
2
Pathogenic bacteria remain the main causes of infertility in males. A study
carried out on sperm analysis where samples were taken from 193 men seeking
fertility treatment with their partners in Mexico, 143 were infected with bacteria.
It was observed that these bacteria cause death of DNA of the spermatozoa in a
process called DNA fragmentation. Some recover fertility after treatment (José
and Michael, 2003).
15% of cases of male infertility are caused by bacteria, parasites, viruses,
vaginal infections and other external factors. Yeast cells use the same molecular
structure in reproduction making sperms cells to be fragile, immotile and
deformed, a key to infertility (Michael, 2003).Microorganism affect the
development , maturation and motility of sperm cells E. coli and other common
bacteria such as C. trachomatis, Ureaplasma urealyticum, Mycoplasma hominis
affect sperm morphology (Michael, 2003)
Semen analysis is not the only test for infertility determination. There are couple
related phenomena that require the initiation of a pregnancy. The patient cannot
be considered infertile based only on abnormal semen analysis.
(www.sunysh.edu/urology/male -infertility /semen analysis htmc).
1.2 Statement of Problem
An estimated 30.000 seminal fluid infections which subsequently lead to
infertility or sub fertility problems occur annually on worldwide bases of which
Cameroon and Shisong is included (WHO,1992). This high Prevalence of
infertility is brought about by high sexual activity of the subjects concerned
which subsequently exposed some to sexually transmissible diseases like
Chlamydia, Gonorrhea, Syphilis and HIV which could lead to infertility or sub
infertility.
Other factors such as poor development of male reproductive system, Hormonal
imbalance, social activities (occupation) where most subjects engage in black
collar jobs such as continuous ridding of motorcycles and driving cars are other
3
predisposing factors to infertility and sub infertility in which clients in this
study population would likely be part of these activities.Hence the need for the
study.
1.3 RESEARCH QUESTION AND HYPOTHESES
1.3.1 Research Question
Are bacteria the main cause of infertility in males consulting with fertility
problems in Saint Elisabeth Catholic General Hospital and Cardiac Center
Shisong?
HYPOTHESES
Ho; Bacteria are not the main causes of infertility in males presenting with
fertility problems in Shisong Hospital.
H1: Bacteria are the main cause of infertility in males presenting with fertility
problems in Shisong Hospital.
1.4 GOALS AND OBJECTIVES
1.4.1 Goals
The main goal of this study is to create awareness on infertility in males,
sensitize and educate the community on modes and prevention of male
infertility.
1.4.2 Objectives
 To know the different bacteria that causes infertility in males
 To study and know the measures that can be taken to prevent infertility.
 To investigate other causes of infertility other than bacteria.
 To identify better quality control measure that can be employed to ensure
proper semen collection and analysis.
4
1.5 RELEVANCE OF THE STUDY
The project will be beneficial as follows
 It will help the researcher to know the different bacteria that cause male
infertility.
 It will help to educate the study population on the preventive measure to take
in order to avoid bacteria infections which exposes them to infertility and
subinfertility.
 Help the St Elizabeth General Hospital and Cardiac Center Laboratory staff
to know the prevalence of azoospermia and preventive measures that can be
taken to avoid further bacteria infection so that they can better educate their
clients.
 It will enlighten the staff of the Saint Elizabeth General Hospital and Cardiac
Center laboratory on the importance of quality assurance and quality control
measures in semen collection, preservation, transportation and analysis.
5
CHAPTER TWO
LITERATURE REVIEW
2.0 INTRODUCTION
Infertility is the inability of a woman to get pregnant after a period of one
year despite regular and normal sexual intercourse with her male partner during
ovulation or without applying any family planning method. It can also be defined
as the inability of a woman to carry pregnancy due to miscarriages caused by
bacteria infections or other factors. Infertility is also a common case amongst men.
In their case it is defined as the inability to impregnate a woman after year of
regular sexual intercourse during a woman’s ovulation period (Author, et al.,
1986). As the incidence of infertility is gradually increasing, that is getting to
epidemic levels, health care providers have figured out sexually transmitted
infection as the major cause (Litwin, et al., 1999). Prominent amongst these
infections are gonorrhea, syphilis, and staphylococcal infection just to name few.
According to recent research, infertility is known to be caused mainly by bacterial
infection which are; sexually transmitted infections and urinary tract infections
(STI and UTI’s respectively).
There are two main groups of infertility these are; primary and secondary
infertility.
 Primary infertility occurs when the woman had never had a child before or
man had never impregnated a woman before. It can also be called sterility or
barrenness.
 Secondary infertility occurs when one partner or both had child or children
before and difficulties to get subsequent children.
Infertility can therefore be classified as infertility proper, when there is no child or
sub infertility with one child and great difficulty to get another.
6
In males a series of types of infertility occurs; Azoospermia, Oligozoospermia,
Necrozoospermia, Asthenozoospermia, Terathoszoospermia.
Bacteria
that cases
STI’s and UTI’s like staphylococcus specie , coliforms, Grams negative diplococci are
consistently being isolated from the semen cultures of men with low or no sperm
count.(Werner,2010)
A great number of medical conditions, physical, biological as well as
environmental factors, causes either infertility or sub infertility in both males and
females. for examples Hormonal disorders, (such as luteinizing hormone, follicle
stimulating hormone and testosterone)Genetic abnormalities like klinefelter’s
syndrome, Testicular or fallopian tubes injuries
or blockages with infections,
Malnutrition and deficiencies, Surgery, over heating of scrotum , cigarettes , drugs
alcohol and environmental factors like radiations, chemical toxins, varicocele which
can cause male infertility. Frequent criminal abortions in female and bacterial
infections (Werner, 2010).
2.1 THE MALE REPRODUCTIVE SYSTEM
The male reproductive tract consists of external genital and internal organs.
These organs are located in the pelvic cavity. The male’s reproductive system begins
to develop in early fetal life. Essentially, no testosterone is produced during childhood.
It starts at puberty and stimulates growth and maturation of the reproductive structures
and secondary sex characteristics. Testosterone is the male sex hormone secreted by
the intestinal cells of the testes.
The primary function of the male’s reproductive system is to produce male sex
cells which are called sperm cells. The primary organs of the male’s reproductive
system are the two testes in which the sperm cells are formed. The other structures are
the duct system and the accessory glandular structures. The testes are two almonds –
shaped glands whose function are for the production of spermatozoa and testosterone.
The testes are suspended in the scrotal sac outside the abdominal pelvic cavity. This is
because they are very sensitive to heat and high temperatures (Cliff, et al., 2010). The
7
high temperatures within the body are unfavorable for the production of spermatozoa.
The spermatozoa mature at temperatures of 36.2-36.6°. Each testis is enclosed by a
tough white fibrous capsule called the tunica albuginea. Capsules are divided into
lobes and each lobe contains tightly coiled seminiferous tubules where sperm cells are
produced. They are emptied into the epididymis where they stay and mature. When the
male is sexually stimulated the walls of the epididymis contract to expel sperm into the
next part of the duct which is the vas deferens. This part also enclosed with vessels
and nerves in the connective sheath called spermatic cord.
The vas deferens empties the sperms cells into the urethra which passes through
the accessory glands seminal vesicle, prostate glands, Cowper’s gland and the penis
into the external environment.
Seminal vesicles are 2 pouches that store seminal fluid. 60% of the fluid volume
of semen is produce here. The secretion is rich in sugar (fructose) which nourishes and
activates the sperm passing through the tract. Prostate gland is a single gland about the
size and shape of a chest nut. It encircles the upper area of the urethra just below the
bladder. It secretes a milky alkaline fluid which plays the role of protecting the sperm
cells against the acidic condition of the vagina (Cliff et al., 2010).
The Cowper’s glands are tiny pea size gland inferior to the prostate. They form
thick clear mucus, which drains into the urethra. The secretions are believed to serve
primarily as a lubricant during sexual intercourse. The penis is a cylinder shaped up
erectile tissues with the smooth cap of the penis called the glands penis and it is
covered by a fold of loose skin. Surgical removal of the foreskin is called
circumcision. It is performed mainly to beautify the outlook of the penis and to
facilitate easy penetration into the vagina. The penis apart from serving as the pathway
of exit of spermatozoa also functions as path in the urinary tract (Cliff, et al., 2010)
8
Figure 1: The male reproductive system
2.2 Sperm cell
The term sperm is derived from the Greek word sperma meaning seed and
refers to the male reproductive cell. A mature sperm contains 3 regions. The head,
the mid piece and the tail; the head of the sperm cell contains the haploid nucleus
with 23 chromosomes. At the tip of the spermatozoa head is the acrosome, a
lysosome containing enzymes which are used to penetrate the egg. The acrosome
originates from Golgi body vesicles that fuse to form a single lysosome. The mid
piece is the first part of the tail. Mitochondria spiral around the mid piece produces
energy (ATP) used to generate the whip-like movement of the tail that propel the
sperm. The tail is a flagellum consisting microtubule array (Wales, et al., 2010).
9
Figure 2: Structure of a Spermatozoon
2.3 Spermatogenesis
It is the process that creates sperm from initially undifferentiated germ cells.
Spermatogenesis is the process of sperm cell development. Rounded immature
sperm cells undergo successive mitotic and meiotic division (spermatogenesis) and
a
metamorphic
change
(spermatogenesis)
to
produce
spermatozoa.
Spermatogenesis begins during puberty and continues through out life. Millions of
sperms are produced in a 24 hour period. This occurs in the seminiferous tubules.
The process is begun by primitive stem cells called spermatogonia. Follicle
stimulating hormone is secreted by anterior pituitary gland at puberty FSH
influence division of spermatogonia into spermatocytes. Each spermatocyte
undergoes meiosis and produces spermatids. The male body contains 23 pairs of
chromosomes of each of the 23 pairs. The same occurs in the ovum. When the
sperm and the egg unite the total number of chromosome is 46 chromosomes
(L’Hernaults, et al; 2005).
10
Figure 3: Spermatogenesis
2.3.1 Effects of Temperature on Spermatogenesis
Sperm cells will not mature at body temperatures in most mammals. Spermatogenic
DNA polymerase beta and recombinase activates and exhibit unique temperature
[36.2-36.6°]. Testicular descent from the abdomen normally transpires during
neonatal life. If the testes fail to descend into the scrotum, a condition called
11
cryptorchidism occurs. The male will be sterile. If gone uncorrected, spermatogonia
will eventually be degenerated (L’hernaults, et al; 2005).
2.3.2 Hormonal regulation of spermatogenesis
The production of sperms is regulated by hormones. The hypothalamus begins
secreting gonadotropin releasing hormone (GnRH) at puberty which then
stimulates the anterior pituitary gland to secrete FSH and LH. LH activates
interstitial cells in the testes to produce testosterone and other male sex hormone
(androgen). In male LH is interstitial cell stimulating hormone (ICSH).
Testosterone stimulates the final stage of sperm development in the nearby
seminiferous tubules. It accumulates here because testosterone and FSH act
together to release androgen binding protein which holds testosterone here to
perform its function. The figure below shows the hormonal regulation in the male
reproductive system.
Hypothalamus
GnRH
Anterior Pituitary
FSH
LH (ICSH)
Interstitial Cells
Testosterone
Sustencular Cells
ABP
Seminiferous tubules
Sperm cells
Secondary Sex Characteristics
Figure 4: Hormonal Regulation in the Male Reproductive System
12
2.4 TYPES OF INFERTILITY
There are different types of infertility. These types are due to different causes
which affect the sperm cell in different way either morphologically, motility,
concentration, composition, these different types are; aspermia, azoospermia,
oligozoospermia,
hematozooseprmia,
Terathoszoospermia,
leucocytospermia,
bacteriospermia , necrospermia just to name a few.
2.4.1 Aspermia
This means no ejaculates at all. The semen volume is completely zero. This
can be due to psychological problem, since the man cannot achieve orgasm or
cannot get an erection. The commonest reason for this condition can be retrograde
ejaculation that is semen is ejaculated in the bladder instead of through the urethra
(Fertile, 2008)
2.4.2 Azoospermia
It is complete absence of sperm cells in the ejaculates (Werner, 2010) it is the
most severe form of male infertility. There are 2 main types of azoospermia that is
obstructive and non obstructive.
 Obstructive Azoospermia: It occurs when there is a blockage in the vas
deferens or epididymis. It accounts for 40% of cases of azoospermia.
 Non Obstructive Azoospermia: Here there is no problem with the passage
of sperms within the body. It is often the results of hormonal imbalance.
Hormonal changes account for 60% of cases of azoospermia for example
klinefelters syndrome azoospermia accounts for 10-20% of males seeking
fertility (Werner, 2010).
Signs and Symptom
Unfortunately it is very difficult to recognize azoospermia without
undergoing fertility testing, since there are no signs and symptoms with the
condition only sperm count can diagnose the condition.
13
Causes
There are typically 2 main causes of azoospermia either there is a problem
with sperm production or there is a problem with sperm transportation.
Sperm production problems sometimes azoospermia is as the result of a
dysfunction within the testes themselves making it impossible for the body to
produce enough viable sperm. Other causes of no sperm production are hormonal
abnormalities (caused by anabolic steroids) cryptorchisim (undescended testis)
vascular trauma varicocele (enlarge veins in the testes) (Fertile, 2008)
Sperm Transport Problems
Sperm leave the testes and travel through a series of ducts before mixing
with the ejaculates and exit out of the body. Transportation problems can be due to
vasectomy that is cut or blockage in the vas deferens, congenital absence of vas
deferens, infections with coliforms,or staphylococci. also cause blockage in the vas
deferens preventing sperm from mixing with the ejaculates (Fertile, 2008)
Diagnosis of Azoospermia
Spun Specimen
Even if no sperm cell is seen on the test slide, the sperm count may still not
be zero that is they may be very low number of sperm cells in the ejaculate. The
sample is spun so that all the sperm cells are concentrated in a pellet on the bottom
of the tube and then collecting the pellet from the bottom of the tube and then
examining it using the microscope. If no sperm cell is seen then azoospermia can
then be concluded (Chama, et al., 1994)
Post Ejaculatory Urinalysis (PEU)
Some men ejaculate all or part of the sperm back into the bladder. This can
ne detected by collecting man’s urine immediately after the ejaculates into a
separate cup. The post ejaculatory urine is then centrifuge to see it any sperm cell
are present. A FSH level test in the blood is also helpful to differentiate between
14
obstruction and testicular failure. If the FSH level is high, it means the problem is
testicular. If the FSH is normal then a testis biopsy is needed in order to arrive at
diagnosis. (Chama, et al., 1994).
CASA (Computer Assisted Semen Analysis)
Mostly for assessment of sperm concentration and specification and specific
pattern of sperm motility i.e. spermolyser.
2.4.3 Oligozoospermia
It means low sperm count. Here there are fewer sperm cells in the ejaculate
than normal. That is less than 20 x 106 spermatozoa/ml of ejaculate.
Oligozoospermia is the cause of male subinferitlity that is reduced ability to
achieve pregnancy.
Causes
Reduction in sperm production due to varicoceles (inflammation of vein of
the penis and testis) hormonal disorders, infections of the testicle like prostatitis,
epididymis, urethritis , also obesity , stress, smoking drug or alcohol use, toxins,
malnutrition, mumps, underweight
sexually transmitted diseases examples
Chlamydia , gonorrhea causes oligozoospermia. Complications are sub infertility
and infertility (Malpani, et al., 2004).
Diagnosis of Oligozoospermia
It includes sexual history, complete general and physical examination
including penis and testicle, semen analysis, FSH, LH and testosterone checking.
Treatment
Treatment depends in the type and severity of oligozoospermia examples
improving diet, gaining or losing weight, not smoking reducing or stopping alcohol
avoiding tied fitting underwear or treatment of infection or surgery.
15
2.4.4 Terathoszoospermia
These are s perm cells with abnormal morphology terathos mean monster. It could
also be term globozoospermia. These abnormalities greatly affect fertility (Guyton,
1986).
Causes
The cause of Terathoszoospermia are unknown in most cases, however
leukaemia and other conditions contributes to some instance.
Globozoospermia are sperm cells with round heads and is due to Golgi apparatus
which is not transformed into the acrosome which is needed for fertilization. The
presence of abnormal shape sperm can negatively affect fertility by reducing sperm
motility or preventing sperm form adhering to the ovum (Malpani., et al., 2004)
Diagnosis
In testing for Terathoszoospermia, sperm cells are collected, stained and
analyzed under the microscope to detect abnormalities. The abnormalities may
include head that are too large, too small, tapered double, pyriform, lack of tails or
tails that are abnormally formed. As seen in the figure below (Guyton, 1986).
16
Figure 5: Abnormal Sperm Morphology.
Treatment
Antiestrogens have been effective in treatment of Terathoszoospermia. Also
globozoospermia may be treated by Intra Cytoplamic Sperm Injection (ICSI) that is
injecting the sperm cell directly into the egg, once the egg is fertilized abnormal
sperm cell morphology does not appear to influence blastocyst development (Behre
and Niescllag , 2000)
2.4.5 Asthenozoospermia
It is the reduction in the strength or energy of sperm cell hence infertility. It
means weak sperms or reduced capacity of quality sperm cells function. This may
be due to genetic factors previous inflammation of the body. Surgery, injury, drugs,
smoking and used of addictive substances or radiations.
17
Causes
 Blockage of routes of sperm production
 Hormonal disorders e.g. Klinefelter’s syndrome.
 Testosterone, estrogen disorders
 Chromosomal/genetic disorders for example cystic fibrosis
 Anti-germinal antibodies (Johnson, 2009)
Treatment
Antiestrogens have been effective in treatment of asthenozoopsermia.
Treatment could also be through Intra Cytoplamic Sperm Injections (ICSI)
2.4.6 Haematospermia
It is defined as blood (RBC) in seminal fluid. It is perceived as symptom of
less significance. It is due to long period of sexual abstinence; and is also indicated
inflammations of prostate and seminal fluids (Litwin, et al., 1999). The prevalence
is unknown since many episodes go unnoticed. It affects males of all age at
maturity.
Causes
Infection of the prostate gland (prostatitis) strictures, unusual frequent sex,
blood clotting problems cancer, tumors.
Symptoms
Blood in semen, pains with urination pains with bowel movement,
tenderness in the scrotum, swelling scrotum, fever or chills, pain with ejaculation.
Diagnosis
Medical history and physical examination
Urine test with urinalysis and culture
Semen analysis and culture
Imaging studies or x-ray test to look for urethral stricture (Bhawna and Jonathan,
2010)
18
2.4.7 Leucocytospermia
This is the presence of White Blood Cells in seminal fluid. High WBC in
semen can cause infertility problems. That is more than 1 million WBC/ml, which
is high and can have detrimental effect on male fertility. This is because leucocytes
cause the oxidation of cells. If high WBC is present in the seminal fluid, this could
result in the oxidation of sperm cells, damaging their ability to fertilize and egg
(Lacker, et al., 2008)
Reactive Oxygen Specie s (ROS) Leucocytes triggers oxidation by releasing
reactive oxygen species. These molecules cause cellular damage by charging the
make up of individual cells. In particular, reactive oxygen species change the make
up of sperm cells affecting motility and morphology making pregnancy
achievement very difficult. The more WBC in seminal fluid the more the sperm
cells are being affected by reactive oxygen species.
A prospective cohort study carried out by Lacker Jakob and his colleagues
on spontaneous variation of leucocytospermia in asymptomatic infertile males.
Where 99 men were evaluated and 21% of the men had leukocytospermia;
concluded that WBC should be taken into consideration when treating infertility.
Diagnosis
Semen analysis can detect levels of WBC in the ejaculate. Also urethral swab
should be analyzed to check if active infections are present.
Treatment
It involves medication on any active infection with antibiotics. Also advised
is given to urinate frequently to eliminate the WBC out of the seminal tract.
(Lacker, et al., 2008)
2.4.7 Necrospermia
Also called watery seminal fluid that occurs when the seminal fluid is very
watery. This is due to bacterial infection of the seminal vesicles and prostate gland.
19
Diagnosis
Semen analysis by physical examination that is, aspirating the fluid with
syringe and allowing it to drop.
Treatment
Treatment of the various bacteria isolated from the semen culture causing
these infections.
Table I: COMMONLY USED NORMAL SEMEN PARAMETERS
ACCORDING TO WHO 1992
Standard test
Normal ranges
Volume
2.0ml or more
PH
7.2 – 8.0
Sperm concentration
20 x 106 spermatozoa/ml or more
Total sperm count
40 x106 spermatozoa per ejaculate or more
Motility
50% or more with forward progression or
25% or more with rapid progression within
60min
Morphology
30% or more with normal forms
Vitality
75% or more alive
WBC
Fewer than 1 x 106/ml
Immunobead test
Fewer than 20% spermatozoa with adherent
particles
MAR Test
Fewer than 10% spermatozoa with adherent
particles
20
2.5.0 Other
Causes of Male Infertility
2.5.1 Klinefelter’s Syndrome
A genetic condition in which each cell in the human body has additional X
chromosomes. Men with Klinefelter’s syndrome have one Y and two X
chromosome. Physical symptoms include peanut sized testicles and enlarged breast.
A chromosome analysis is used to confirm the analysis. If this condition is treated
in its early stage with the drug HCG (Human Chronic Gonadotrophine) sperm
production may commence or improved. However Klinefelter’s syndrome
eventually causes all active testicular structure to atrophy. Once testicular failure
has occurred, improving fertility is impossible (Jonathan, et al., 2010)
2.5.2 Varicoceles
These are dilated veins in the scrotum (similar to varicose veins in the legs)
these veins are dilated because the blood does not drain properly from them. These
dilated veins allow extra blood to pool in the scrotum which has a negative effect
on sperm production. This condition is the most common reversible cause of male
infertility and may be corrected by minor out patient surgery. Most experts perform
this surgery microscopically to preserve the arterial supply and lymphatic’s. a subinguinal incision (about 1 inch )above the penis and (1 inch) from the midline is
usually used as this avoids incising the abdominal muscles and creates less post
operative pain .(Werner, 2010)
2.5.3 Seminal Fluid Abnormalities
If the seminal fluid is very thick it may be difficult for sperm cells to move
through it and into the reproductive tract. Often the semen can be processed to
separate the moving sperm cells and seminal fluid. The processed sperm cell is
usually placed directly inside the uterus with a small tube. This is called
Intrauterine Insemination (IUI). Abnormalities of volume are not very common.
They usually reflect a problem with the accessory gland. For viscosity seminal fluid
21
should be liquefied after 30-60 minutes. Too viscous can be due to lack of fructose
which is provides energy for sperm motility and contributes to the seminal fluid.
(Rrumbullaku, 1992)
2.5.4 Ductal System Problem
Sperm carrying ducts may be blocked. A patient may have bilateral
congenital absence of vas deferens. He may have obstruction either at the level of
the epididymis or higher up in the more muscular vas deferens. The ducts may have
became mechanically blocked during hernia or hydrocele repairs or blocked by
scarred tissues as a result of bacterial infections. Sperm are stored in sacs called
seminal vesicles and are then deposited in the urethra, which is the tube through
which men urinates and ejaculates. The sperm cells must pass through the
ejaculatory duct to get out. In some situation the ducts may be repaired or blocked
hence sperm cells cannot flow through the track.
2.5.5 Immunological Infertility
Men can develop an immunological response (anti bodies) to their own
sperm cells. The causes for this may include testicular trauma, testicular infection.
Large varicoceles or testicular surgery. sometimes there are unexplained reasons
for this occurrence. These anti bodies have a negative effect on infertility although
the exact reason why this is the case is unclear. Most likely, these anti bodies act
negatively at several points along the path way to fertilization. They make it more
difficult for the sperm to penetrate the partner’s cervical mucus and its way into
uterus. They make it more difficult for the sperm to bind with the zona pellucida
(the external membrane or shell of egg)also the antibodies make it more difficult
for the sperm to fuse with the membrane of the oocytes themselves.(Rrumbullaku,
1992)
The treatment for anti sperm antibodies is some what controversial. Men may be
treated with corticosteroids. However, this can lead to significant morbidity in the
22
man. The most significant is aseptic necrosis of the hip. (Non infectious destruction
of the joint), requiring hip replacement. Most of the time the first level of include
intrauterine insemination. If the couple is planning In-Vitro Fertilization (IVF), the
presence of anti sperm anti bodies is usually and indication to inject the sperm
directly into the egg (ICSI) (WHO, 1992)
2.5.6 Difficulties with Erections and Ejaculations
About five percent of couples with infertility have factors relating to
intercourse. This includes the inability to obtain or maintain erection, premature
ejaculation, lack of appropriate timing of intercourse and excessive masturbation.
Interestingly, the most common problem is infrequency of intercourse. Many men
will have difficulty with erections under the pressure of trying to achieve
conception. These couples can easily learn the technique of self insemination.
Studies show that 5 out of 6 previously fertile couples having intercourse 4 times
per week will conceive over six moths. While 1 out of 6 with intercourse once per
week during the same period will conceive. (Werner, 2006)
2.5.7 Testicular Failure
This refers to the inability of the sperm- producing part of the testicles (the
seminiferous epithelium) to make adequate numbers of mature sperm cell. This
failure may occur at any stage in sperm production for a number of reasons. The
testicle may completely lack the cells that divide to become sperm cells (sertoli
cell only syndrome ) there may be an inability of the sperm cells to complete their
development (maturation arrest). Sperm may be made in such low numbers that
few, if any, successfully travel through the ducts and into the ejaculated fluid.
(Hypo spermatogenesis). This situation may be caused by genetic abnormalities.
Hormonal factors or varicoceles, even in the case where the testes are only
producing low numbers of sperm cells. They may be harvested and used in
23
conjunction with advanced reproductive techniques to attempt a pregnancy.
(Werner,2006).
2.5.8 Cryptorchidism
It is a condition when a baby boy is born with un-descended testis or
partially descended into the scrotum. It may be the cause of testicular failure. Since
the testes are very sensitive to the temperature. If they do not descend into the
scrotum before the adolescence, they will stop producing sperm altogether. In fact
they have a higher rate of malignancy. The current recommendation is that
approximately one year, of age. If the testes have not descended by themselves they
may be brought down surgically. Cryptorchidism is often associated with male
infertility. 81% of men who have one single testis which is cryptorchid have
normal fertility. However Cryptorchidism brings about infertility. This may be due
to something inherent in the testes, surgery or damage done by not having the testes
brought down in time. (Rrumbullaku, 1992)
2.5.9 Hormonal Abnormalities
The testes need pituitary hormones to be stimulated to make sperm cells. If
these are absent or severely decrease, the testes will not maximally produce sperm.
Importantly men who take androgens (steroids) for body building either by mouth
or injection shut down the production of hormones for sperm production. A
hormonal profile must be performed on all men with male infertility. This will help
out serious medical condition, given more information on the sperm producing
ability of the testes and my reveal situation where hormonal treatment is indicated
(Werner, 2006)
2.5.10 Retrograde Ejaculation
It is condition in which semen is ejaculated in to the bladder rather than out
through the urethra because the bladder sphincters does not close during
ejaculation. If this disorder is present ejaculate volume is mall and urine may be
24
cloudy after ejaculation. This affects 1.5% of infertile men and may be controlled
by medication like decongestants which contract the bladder sphincters or surgical
reconstruction of the bladder neck can restore normal ejaculations (Jonathan, et al.,
2010).
2.5.11 Infections
Infection of the reproductive tract include; epididymis, orchitis, prostatitis or post
pubertal viral infections of the testis and my cause absolute and irreversible
infertility. Bacterial infections or sexually transmitted diseases may cause blockage
of the sperm duct. The patient may have normal production of sperms, but ducts
carrying them are obstructed. Active bacterial or viral production or sperm
function. WBC which is the body’s response to infection may also have a negative
effect on the sperm membrane, making them less healthy. If excessive WBC is seen
in a semen specimen, culture should be done. This usually includes cultures of
commonly asymptomatic, sexually-transmitted diseases including Mycoplasma,
urealyticum and Chlamydia. Also a general genital culture is usually taken. If the
infection and the WBC are persistent antibiotics may be considered (Werner, 2006)
2.5.12 Drugs
There are a number of common drugs that may have negative effect on sperm
production and or function they include;
 Ketonazol (antifungal)
 Sulfasalazine (for inflammatory bowel disease)
 Spironolactone (an anti hypertensive)
 Calcium channel blockers (anti hypertensive)
 Allopurinol, colchicines(for gout)
 Antibiotics e.g. nitrofurantin, erythromycin
 Methotrexate (cancer, arthritis)
 Cimentidine (for ulcer or reflux)
25
The following drug can cause ejaculatory dysfunction
Antipsychotics, chlorpromazine, haloperidol, thioridozine
Antidepressants, amitriphyline, imipramine, fluoxetine (prazol) paraxethin (paxi)
sertraline(zolft) (WHO, 1992)
Recreational Drugs
Marijuana and cocaine often causes a decrease average sperm count, motility and
normal morphology. It affects the hormonal axis (HPG) causing decreased plasma
testosterone; it may also have a direct negative effect on the leydig cells. The effect
found in men who have been taking cocaine for 2 years preceding their initial
semen analysis. Cocaine also decreases the ability of sperm to penetrate the
cervical mucus, making it difficult for them to enter the uterus (Jonathan, et al.,
2010)
Anabolic steroid male hormones
The use of anabolic androgenic steroid has reached almost epidemic proportions.
Nearly 7% of 12 grade males use them to build muscles mass and improve athletic
performance. These males hormone suppress the testes ability to make testosterone.
This decrease the intra-testicular testosterone level. This may cause several
diminished spermatogenesis or complete absence of sperm (azoospermia) when
taken, these steroid cause a persistent depression of the hypothalamus and pituitary
gland which may be irreversible even when the steroid are stopped (Werner, 2006).
2.5.13 Lifestyle Factors
Cigarette Smoking: This has been shown to significantly affect semen quality.
Regular smoking causes a 23% decrease in sperm density or concentration and a
13% in motility and to a lesser extent smoking causes toxicity to the seminal
plasma or seminal fluid. Smoking affects the hypothalamic-pituitary gonadotropin
axis most commonly affecting levels of estradiol and estrone. (Estrogens which are
hormones found in higher concentration in women.) The leydig cells which are in
26
the testes that produce testosterone may have secretory dysfunction. Smoking leads
to congenital abnormalities in child hood; with cancer. (Rrumbullaku, 1992)
Alcohol: Excessive alcohol affects the hormonal axis and is a direct gonadotoxin. It
may cause associated liver dysfunction and nutritional deficiencies which are
detrimental to sperm cell production. (Rrumbullaku, 1992)
Lubricant: Most vaginal lubricants including K-Y jelly, surgilube and lubifax, are
toxic to sperm cells. Couples should avoid their use during the fertile times of a
woman’ cycle (Jonathan, et al., 2010)
Exercise: Men who ride more than 50miles/ week should be reduced during
attempting conception (Jonathan, et al, 2010)
Conclusively treatment of males with infertility 50% will regain their fertility with
the above mentioned factors and treatments. (Guyton,1986).
2.6.0 Bacterial Infections in Seminal Fluids
Bacterial infections of the seminal tract have been postulated to play a
potential role in male infertility. Pathogens include Neisseria gonorrhea,
Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis other
pathogens
include Enterococcus
species, Staphylococcus aureus Klebsiella
species, Escherichia coli and other Gram negative bacilli, possible pathogens in
seminal fluid
at concentrations >106CFU/L has been defined as significant
bacteriospermia which may be associated with infertility. However bacteria in this
fluid may also be in urethral track orifices (Keck, et al., 1998)
A study of 166 males who underwent fertility investigation using routine semen
analysis according to the guidelines of the WHO and bacteria analysis by culture
and using PCR assay showed that the prevalence of bacteriospermia in semen
samples was 56.91% independent of the presence
of leukocytes. The most
common bacteria isolated was C. trachomatis (41.4%), U. urealyticm(15.5%) and
27
M. homonis (10.3%). This was carried in Tunisia in SFAX hospital in department
of microbiology and research laboratory (Gdoura, et al., 2008).
Another study in prevalence of sexually transmissible pathogens in semen from
asymptomatic male infertility patients with and without leucocytospermia where
241 infertile male under went a prospective controlled studies and was found out
that DNA of STI pathogens were detected. Semen from a high percentage of
asymptomatic male suffering from infertility were associated with poor semen
quality. This was also associated with decrease in sperm concentration, motility and
total sperm count where 18.7% was the prevalence of CMV 8.7%, HPV 4.5%,
HHV 3.7%, EBV 0.4%, C. trachomatis 2.5%, It shows that efforts to diagnose and
treat subclinical genital tract infection should be intensified (Guntrum, 2007).
Another interesting study carried out in Benin City and Nigeria to determine the
presence of pathogenic microorganism and semen parameter is seminal fluid.
Where 229 volunteer were collected between 19-33 ages. This was diagnosed by
culture and using mycoplasma agar enrich with ceftrazidine to isolate Mycoplasma
species. The determination of spermatozoa, concentration motility test was carried
out using standard procedure and antimicrobial sensitivity test as carried out by the
disc diffusion method. Results show that microorganism percentage was 34.9%.
The isolated bacteria where Staphylococcus aureus (58.8%), E .coli (12.5%),
Klebsiella species(7.5%), Candida albican(6.3%) and mycoplasma species(15%).
Sensitivity to antimicrobial agents was highest among isolates to ciprofloxacin and
pefloxacin and was resistant mostly to Amoycilline and Tetracycline. Azoospermia
was 10.4% and oligozoospermia and semen with pathogenic micro-organism had
significantly P<0.001. Hence indicating that micro-organism semen is a marker of
deterioration is semen parameter and development of male infertility (Onemu, et
al., 2010).
28
The association of genital tract infection to spermatozoa concentration and
infertility has been seen as an important parameter that affects male reproduction.
Previous studies has shown that there is a close correction between prostatitis,
epididymis , sexual transmitted micro-organism and male infertility (Hawkins, et
al., 1996) clinical and experimental studies have linked the presence of bacteria in
semen and the immunological function of spermatozoa to decrease fertility (Ireton
and Berger, 1984) which ultimately lead to deterioration in spermatogenesis and
spermatozoa function (Keck, et al., 1998).
Yeast cells have the same molecular structure in reproduction like sperm cells
hence yeast infection makes sperm to be fragile, weak and deformed them since
they compete(Jose L.F et al;) and other pathogenic bacteria e.g. N. gonorrhea
causes death of DNA of spermatozoa in a process called DNA fragmentation,
which makes fertility treatment their to be difficult. Although some males recover
their fertility after treatment (Werner, 2010). Fusobacterium nuclentum, Bacteriode
fragilis affect sperm concentration and motility by competing with the sperm for
the supplies of fructose needed for their energy for movement. Also these bacteria
also produce toxins that interfers with physical structure of sperm cell especially its
tail. Other bacteria like S. faeclis , E coli, Coagulase positive, other Staphs species,
Proteus vulgaris, Pseudomonas pyocyanea, and B-haemolytic streptococcus was
found to reduce sperm concentration, motility and morphology. (Mogra, et al.,
2010).
Signs and Symptoms
Sub Infertility and Infertility
Infertility is the inability of a man to impregnate a woman after a year of regular
sexual intercourse during the woman’s ovulation periods. Incidence of fertility is
gradually getting to epidemics.
29
Sexual transmitted infections are the major causes. Prominent amongst these
infection are bacteria like N. gonorrhea, Staphylococcus, C. tranchomatis just to
name a few. The main sign and symptom are oligozoospermia, (low sperm count)
bacteriospermia, Azoospermia (zero sperm count), Necrospermia (watery sperm,
aspermia (no ejaculates), leucocytospermia (WBC in sperm) which is the main
indicator. Urinary tract infection indications like;
Discharge of yellowish or greenish pus from the penis
Semen ejaculated with urine
Prostatitis and epididymitis with mouth disease
Silent gonorrhea
Urinary tract infection by Streptococcus and Coliforms bacteria i.e. constant pain
in urethra and painful ejaculation (Nutcrakcer, 2010).
Pathologies and Pathogenesis
There are many pathologies of bacteria in seminal fluid which are urinary tract
infection, like epididymitis, prostatitis
Epididymitis
This is the inflammation of the epididymis where sperm cells are being stored. This
occurs with N.gonorrhea bacteria, when untreated travels it way through the
urethra or cervix in case of female to pelvic organs and settles in the epididymitis
causing inflammations . It is contracted through sexual intercourse. The main cause
of epididymitis in males is by bacterial infection and frequently between 20-39
years. In children is due to trauma, in general individual always have discomfort
and pain in the area of the testicle or groin at times fever, penile discharge and
blood in urine. The main function of the epididymis is to store sperm cells for
maturation.
30
Prostatitis
This is the inflammation of the prostate glands. There are two types
Acute bacterial prostatitis
Chronic prostatitis
Acute bacterial prostatitis is generalized infection of the prostate gland and is
associated with both the lower urinary tract infection (IUT) and generalized sepsis.
Chronic bacterial prostatitis is associated with recurrent lower UTI’s (cystitis)
secondary to areas of focal uropathogenic bacteria residing in the prostate gland
(Litwin, et al., 1999)
Causes
The Enterobacteria family E. coli,(65-80% pseudomonas aeruginosa Serratia
species, Klebsiella species and Enterobacter aerogenes
Gram
positive
bacteria
examples
Enterococci,
S.saprophytic
haemolytic
streptococcus, curves just to name a few .
These bacteria get into the system through many ways like indwelling urethral
catheters’, unprotected penetrative anal rectal intercourse, urethral pathways, and
transurethral surgery.
Dysfunctional voiding or urine
Chemical induced inflammation of the prostate
Psychological causes (Litwin, et al., 1999)
Diagnosis
 Urine culture before and after prostatic massage since bacteria count will
increase after massage
 Semen analysis and culture
 Urethral swab for culture
 Prostate-specific antigens test
 Pressure flow studies of urine.
31
Signs and symptoms
Dysuria
Urethritis
Urogenital cancer
Bacteriospermia
Bacteruria
Pyuria
Haematospermia
It is also absorbs fluid and add substances to help nourish the maturation of
sperm. Since the epididymis is directly attached to the testicle, if epididymis is
infected or inflamed then same the testicles called epididymo-orchitis that is
infection inflammation if both epididymis and testicles. This leads to retrograde
ejaculation (Nickel, et al., 2007).
Causes
Bacterial infection which gets in through the urethra prostate, vas deferens
into the epididymis. The responsible bacteria are usually indentified in about 80%
of cases sexually transmitted diseases example C. trachomatis, N. gonorrhea
Coliforms example E.coli which is also cause bladder infection
Other mycobacterium species
Medication example Amiodarone
Laboratory Diagnosis
 Physical examination shows a red tendered swollen lump of scrotum
 Complete blood count
 Testicular scan
 Urinalysis and culture
 Seminal fluid and culture
 Test for Chlamydia and gonorrhea
32
Treatment
o Antibiotics for treatment of infections
o If causes by medication do not stop drugs
o Bed rest while elevating the scrotum and applying ice packs to the area is
recommended
Urethritis in men
This is inflammation of the urethral in men due to bacterial or non bacterial
infections such as staphylococcus aureus, streptococcus species, gonococci
Signs and Symptoms
Men present with urethral discharge and dysuria. It is an infection acquired
by inoculation of organism into the urethra during sexual intercourse. It is classified
as gonococcal or non gonococcal urethritis based on the causative pathogens.
Gonococcal urethritis
It is caused by Gram negative diplococcus, Neisseria gonorrhea. It is a
short incubation period of 1-5 days and produced a purulent, yellowish discharge
with dysuria. (Macfarlane, 1995).
Diagnosis
Diagnosis is based on history of sexual contact, purulent discharge with dysuria and
positive Gram stain and culture. The specimen for culture must be carefully taken
from within at least 1 hour after the patient last voided. A Modified Thayer-Martin
culture medium is used as the selective medium for inoculation.
Treatment
Ceftriaxone 250mg in single dose followed by doxycycline 100mg bid x 7days.
Non-gonococcal urethritis (NGU)
It is believed to be the most common cause of urethritis in men with C. trachmatis
being the most important pathogen. It has prolong period of incubation of 5-21days
and procedure a mucoid whitish discharge with or without dysuria. The diagnosis
of NGU require the exclusion of gonorrhea and the demonstration of arthritis.
33
Treatment
Doxycycline 100mg bid x 7days with tetracycline or erythromycin.
2.7 LABORATORY DIAGNOSIS
Semen analysis is the starting point for the investigation of the man in the infertile
couple; the usefulness of semen analysis is to differentiate between prognostic
capacity and correlation between analysis parameters and various fertility success
parameters. (Douglas, et al., 2003) for accurate evaluation, at least 2 specimens
should be obtained with specific collection and transportation guidelines. The
semen analysis is typically divided into many parameters, volume, sperm
concentration morphology, motility , forward progression, PH, Bacterial
determination just to name a few.
Macroscopy
Appearance
The semen sample is first evaluated by simple inspection. A normal sample has a
grey opalescent appearance, homogenous and liquefies within 60min at room
temperature. Under the influence of enzymes of the prostatic origin (fibrinolysin) if
liquefaction does not occur within normal time period it suggest functional
disturbance of the prostate if it appears clear signifies low sperm or no sperm cell
concentration. If may appear brown when red of mucous streets or clots suggest
inflammation in one of the accessory glands (Douglas, 2003)
Consistency
Very viscous specimen’s impaired movements of fertile sperm at the fertilization
site.
Volume
It is important to determine the total same volume accurately since it is used to
calculate the total sperm number. The major component of the ejaculate volume is
made up of fluid from the accessory glands, Seminal vesicles and prostate glands
34
A low ejaculate volume can reflect abnormalities in accessory glands. It can also be
indicative of a physical obstruction in the reproductive tract. Or retrograde
ejaculation large volumes are associated with varicoceles or after long periods of
sexual abstinence. (Rrumbullaku, 2008)
pH
It is determine by an acidic secretions of the prostate and alkaline secretions of the
seminal vesicles it should normally be in the range 7.2-8.0. If it exceeds 8.0
infection should be suspected with decreased secretions of acidic products by the
prostate such as citric acid of the seminal vesicles (Douglas, 2003)
2.7.2
Microscopic Examination
Sperm Motility
A basic sperm function that is very apparent in basic semen analysis is the motility
(Cheesbrough, 2000) it is quite easy to understand that with no motility or poor
motility the chance for a man to cause a spontaneous pregnancy is very small. In
recent years, a number of techniques for assessment of movement characteristics of
human spermatozoa have analysis (CASA) systems. It has a simple classification of
sperm motility. That is;
Rapid progressive motility: when sperm cell move along a linear track covering a
distance of at least 20mm/sec
This is the one related to fertilization (Douglas, 2003)
Slow or sluggish progressive motility: when a sperm cell does not move in a linear
track and it is slow. Cannot cover 20mm/sec
Non- progressive motility: the sperm cell is quite alive but cannot move. This is
shown by viability testing using eosin 0.5 (Cheesbrough, 2000)
Immobility
Here sperm cells are completely death and are immotile hence cannot cause
fertilization (Cheesbrough, 2000)
35
Hypo osmotic Swelling Test
This is done in cases of low motility to determine live sperm cells where tail of live
spermatozoa swells and cuts in hypo osmotic solution. Reverse with viability
testing when only death sperm cells absorb the eosin test. (Douglas, 2003)
Sperm Count
This is done with a dilution of
1 in 20 using Sodium Bicarbonate Formalin as the
diluting fluid, which kills and maintain the position of the sperm cells on the
Improved Neubauer chamber for counting. Normal sperm count is 20×106
spermatozoa/ml and above. Below this value is term oligozoospermia or sub –
infertility. Men under this category are not infertile but it takes then substantially
longer period of time to achieve pregnancies. (Rrumbullaku, 2008). Zero sperm
count is term azoospermia and it signifies disgenesis. Males here are completely
infertile or sterile.
Cells other than spermatozoa could be seen these include polymorph nuclear cells
from the urethral tract spermatogenic cells and WBC which are referred to as
“round cells’’ and are present in almost every semen sample wet mount
preparations can not differentiate between WBC and spermatogenic cells.
Cytochemical peroxides method reliable identifies granulocytes the most prevalent
WBC in semen. The gold standard for the detection of all WBC population in
semen is immunocytology .using monoclonal antibodies which is expensive
Cytochemical Peroxidase Method
The working solution is a combination of NH4CL, sodium EDTA solution, or
tholuidine and H2O2. A dilution of 1 in 10 with well mixed semen sample, which is
then transferred to a counting chamber. The number of peroxidase – positive cells
stains brown are counted which are the granulocytes and peroxidase negative
(unstained) are the spermatogenic cells which suggest pathology at the level of the
36
seminiferous tubules with inadequate spermatogenesis and premature release of
spermatids ( Rrumbullaku, 2008)
Morphology
Sperm cells represent a unique population in which up to 50%
(up to 70%
according to WHO criteria 1992 and up to 86% according to strict criteria ) of the
cells can have morphological defects in normal fertile individual. Staining for
morphology using Sodium bicarbonate formalin which kills and fixes the sperm
cells, loefflers methylene blue stains the tail. This staining shows any defect in any
part of the spermatozoa. Papanicolaou staining of spermatozoa is the method
widely used. In andrology laboratory. Simple methods with Meyer’s haematoxilin
and Giemsa could equally be used.
Agglutination Testing
Agglutination of sperm means that motile spermatozoa stick to each other,
head to head, mid piece to mid-piece tail to tail or mixed. The adherence of either
immotile or motile spermatozoa to mucus threads, or to debris is not agglutination,
the presence of agglutination is suggestive but not sufficient evidence to prove the
existence of an immunological factor of infertility. In case of agglutination sperm
culture must be perform in order to exclude infection with E.coli, sperm
agglutination could be used also as indication for antisperm antibody testing of
infertile men( Rrumbullaku, 2008)
Hypo osmotic swelling test: this is done in cases of low motility to
determine live sperm cells where only tails of the lives spermatozoa swells and
curls in hypo osmotic solution. Reverse with viability testing where only death
sperm cells absorb the eosin test (Douglas, 2003).
37
Hypereactivation
Sperms that have undergone capacitation that is changes before fertilization
can occurs, usually exhibits a change in pattern of motility , high frequency , high
amplitude tail movement with poor progressive motility. This best studies with
CASA.
Fructose testing
Fructose should be 13mmol or more per ejaculate. Fructose is androgen
dependent and is produced in the seminal vesicle. Fructose level should be
determine in any patient with azoospermia and thus with low ejaculate volume or
less than 1ml suggesting seminal vesicle obstruction. Absence of fructose, low
semen volume and failure of the semen to coagulate indicates either congenital
absence of the vas deferens and seminal vesicle or obstruction of the ejaculatory
duct. (Anjali,2010)
CASA (Computer Assisted Semen Analysis)
Mostly for the assessment of sperm concentration and specific pattern of
sperm motility (velocity, linearity etc). The available medical data obtain here is
correlated with conception in vivo and fertilization in vitro but Quality Control
and quality assurance programs are necessary to ensure accuracy(Anjali, 2010).
Other tests like;
 Acrosome reaction test (to check presence of acrosome )
 Hamster egg penetration test(to check sperm fusion ability)
 Hemizona test (to evaluate sperm zona-binding capacity)
 PCR-based detection (to detect pathogens)
 These are other test which are hardly performed due to expenses and time
consuming (Anjali, 2010).
38
2.7.3 SEMEN BIOCHEMISTRY
Analyses of biochemical markers give important information about the
composition of the mixture of secretion the sperm encounters in the laboratory. The
secretory functions of the epididymis (α-glucosidase), prostate (Zinc) and seminal
vesicle (fructose) are important for normal sperm function and can give clues to the
reasons for abnormal results in basic semen analysis. Among men with abnormal
semen analysis, some can have abnormal sequence of ejaculation, leading to a
decreased protection of the nuclear chromatin. The majorities of their sperm are
expelled together with mainly the zinc binding seminal vesicular fluid and have
lower zinc content in the nuclear chromatin. The only way to discovered this
abnormally is to investigate a spilt ejaculate; assess sperm number, motility and
biochemistry in the different ejaculate fractions. Poor sperm motility combined
with decreased prostatic secretion should awaken the suspicion of sub acute
prostatitis and result in a full physical examination to detect any signs of prostatic
infection.(Douglas, 2004)
A semen sample without sperm, low volume, with only normal Zinc level
indicates disgenesis or lack of epididymis, vas deferens, and seminal vesicle or
could also be related to mutation in genes. These markers can be assessed with
spectrophotometric assays.
Antisperm Antibodies Testing
The presence if antisperm antibodies in semen can alter the fertilizing ability of the
spermatozoa. By causing sperm cell death or decrease motility or perhaps hinder
proper binding
of sperm to different receptors essential
for sperm-oocyte
interactions and fertilization ( Rrumbullaku, 1998) being haploid, sperm cells are
immunogenic and display different surface antigens form their diploid counterparts.
Under normal circumstance, they are protected from the man’s immune system by
a basal membrane constituting the blood-testis barrier. When this barrier is
39
ruptured, sperm cells induced the synthesis of anti-sperm antibodies. The presence
of sperm anti bodies coating the spermatozoa are typical of immunologic infertility.
These sperm antibodies belong to the immunoglobin classes IgG, IgA or rarely IgM
and IgE anti bodies have greater clinical importance than IgG antibodies. The
screening test for antibodies is performed on the fresh semen sample making use of
either immunobead method or the mixed anti globulin reaction test (MAR test).
Immunobead test
Immunubead are polyarcrylamide spheres with covalent bound rabbit antihuman immunoglobulin the presence of IgG, IgA, and IgM antibodies can be
assessed simultaneously with this test. Spermatozoa are washed by repeated
centrifugation and resuspended in buffer .This sperm suspension is mixed with a
suspension of immunobeads. The test is considered positive when 25% or more of
motile spermatozoa have immunobead bending (Rrumbullaka, 1992)
MAR test
The MAR test is performed by mixing fresh, untreated semen with latex
particle or sheep blood cells coated with human IgG. A monospecific antihuman –
IgG antiserum is added to this mixture. The formation of mixed agglutinates
between particles and motile spermatozoa proves the diagnosis of IgG antibodies
on the spermatozoa. The diagnosis of immunologic infertility is probable when
50% of motile spermatozoa have particles adherent which is typical of
immunologic infertility (Bijrndal, et al., 2010).
40
2.7.4 Semen Culture
Semen culture was performed on samples with leucocytospermia and in case
of agglutination. This was to isolate pathogen present which was causing the
infection the various bacteria isolated were. Staphylococcus aureus, Micrococci
species, Neisseria gonorrhea, Corynebacteria diphtheriae, E. coli, Streptococcus
species, Listeria species, Candida, species.
These bacteria mentioned are known for their capability of causing infections
of the UTI’s and reproductive organs of the male system which hence leads to
affecting seminal fluid parameters leading to infertility. Most of these bacteria get
into the system through sexually transmitted routs, dirty habits and nursing
procedures like catheterization (Awingnjia, 2010)
Aetiological Agents
Neisseria gonorrhea
It is a Gram- negative Diplococcic intracellular fastidious organism, and a
facultative anaerobe. Biochemically it ferments glucose (William, et al., 1955)
Pathogenicity
It is sexually transmitted. Causes gonorrhea when it attaches to the epithelial
cells of the urethra and cervix. Infection occurs when the pili that project from the
surface of the cocci attaches to the receptors on host cells. It has an enzyme which
during infection causes scar tissue formation which obstructs the urethra,
predisposing the man to urinary tract infections. This may spread to prostate gland,
testis epididymis. Sterility results when scar tissue blocks the tubes that carry sperm
cells and also when testes tissues are destroyed (Eugene, et al., 2004). Acute
conjunctivitis occurs in infants born of mothers with urogenital gonorrhea. The
eyes become infected at the time of delivery, known as ophthalmic neonatorum and
if untreated can lead to blindness
41
Vulvovaginitis in pre-pubertal girls.
Gonococci arthritis as a complication of gonorrhea bacteria.
Laboratory features:
Specimen includes seminal fluid and post ejaculated urine. Others specimens
are urethral and cervical exudates eyes ear swabs. N. gonorrhea is a non-capsulate
gram negative intracellular Diplococci, aerobe or facultative anaerobe. Modified
New York City medium or Thayer martin medium is the selective agar. It also
grows on BA or CA with small, raised grayish or translucent colonies after
overnight CO2 incubation. (Cheesbrough, 2000)
Biochemical test:
 N. gonorrhea is oxidase positive
 It ferments glucose not maltose, sucrose or lactose
 DNase negative
 Beta (B) –galactositase negative ( Cheesbrough, 2000)
Staphylococcus aureus
Pathogenicity; S. aureus causes boils, urethritis styles, pustules, epidydimitis,
orchitis, infections of ulcers and burns. In general urinary tract infections (UTI’S)
Laboratory features
Specimen include seminal fluid, urine .US and VS, pus, wound swabs,
C.S.F, and blood. Staphylococci species are Gram positive cocci of uniform size
occurring characteristically in groups, singly or pairs, non motile and non
capsulated. They grow well aerobically in 10% co2 enriched atmosphere.
They produce yellow to cream white 1-2mm in diameter colonies after an
over night incubation on blood and CA.Some strains are B-haemolytic most strains
are late lactose fermenting on MCA. Manitol salt agar is the selective media.
(Cheesbrough, 2000).
42
Biochemical test; Staphylococcus aureus is:
Catalase positive
Coagulase positive
DNase positive. (Cheesbrough, 2002)
Mode of Infection
Ascending through the urethra
Dirty toilets and dirty habits
Through the penis orifice
Through the blood
Staphylococcus aureus express many potential virulent factors
Surface proteins that promote colonization of host tissue
Invasion that promote bacterial spread in tissues (leukocidin protein A)
Biochemical properties that enhances their survival in phagocytes
(carotinoid, catalase production)
Immunologic disguises(protein A, Coagulase, clotting factor)
Membrane damaging
toxins that lyses eukaryotic
cell membrane
chemolysis , leukcocidin)
Acquired resistance to antimicrobial drugs.
Streptococcus Species
They are gram positive cocci occurring characteristically in short chains but
also in pairs and singly. The organism
are non motile and some strains are
capsulated streptococcus pyogenes produce β Haemolytic colonies on BA and
Streptococcus agalactiae produces grey mucoid colonies about 2mm in diameter
surrounded by small zones of β-haemolysis. Streptococcus pneumoniae are αhaemolytic . They are all associated with UTI’s ( Sunjo, 2010)
Biochemical Test
All are catalase negative
43
PYR positive (pyrrolidonyl test) using strip (for S. Pyogenes)
.S. pneumonia is sensitive to optochin (Cheesbrough, 2000)
S.pyogenes is sensitive to Bacitracin
Escherichia coli:
This pathogens belongs to the large group of Gram negative rods referred to as
Enterobacteria. They are naturally found in H2O, soil, and intestinal tract. Some are
aerobes and some facultative anaerobes with their optimum growth temperature at
36-37°C (Cheesbrough, 2000).
Laboratory features: specimen depends on the site of infection which includes
seminal fluid, urine, pus, faeces, Blood for culture. They are motile rods with some
strain being non motile with few being capsulated. They produce 1-4mm diameter
colonies after over night incubation on BA in which colonies appear mucoid and
some strains are haemolytic (Cheesbrough, 2000).
On MCA and cystine lysine electrolyte deficient (CLED) agar, they ferment
lactose, producing smooth pink colonies on KIA (kligler Iron Agar|). Most strains
of E.coli produce yellow slope and yellow butt with gas production.
Pathogenicity
 UTI in which it is the commonest pathogen isolated from patients with
cystitis, urethritis, prostatitis which block the accessory glands leading to
blockage of passage of sperm cells or no production at all in case of
epididymitis . Recurring infections are common e.g. uropathogenic E.coli
 Infections of wound, peritonitis, sepsis and endotoxin including shock.
 Meningitis and bacteriaemia in neonates.
 Diarrhea
disease,
travelers
diarrhea,
diarrhea(Cheesbrough, 2000).
44
dysentery
and
haemorrhagic
Biochemical Reactions: Most strains are ;
 Indole positive
 lysine decarbxylase positive
 Beta(β)-glucoronidase positive
 Citrate and H2S negative(Cheesbrough, 2000)
Listeria monocytogenes
It is the causative agent of listerosis.
Pathogenicity
It causes genital tract infection in the gravid female with infection of the
offspring. It causes abortion of both man and animals; premature delivery and still
birth or even death within a short period after birth. It also cases a generalized
blood infection or inflammation of the coverings of the brain (meningitis) (Joklik,
1980)
Morphology and Laboratory Diagnosis
Listeria species are small, Gram positive coccobacilli that have a tendency to
occur in short chains of 3-5 organisms. In stained preparation they assume a typical
diptheroid palisade arrangement. It is motile, non sporulating aerobic bacillus
(Wilson, 1979)
Specimens include spinal fluids, exudates, for culture, blood for culture,
vaginal swabs form post partum mothers with infected babies, cultures of
meconium seminal fluid and lesions.
Prevention and treatments
Control of reservoir sources and transmission routes
Education of pregnant women exposed to domestic animals
Pasteurization of milk
Treatment of infected pregnant women.
45
Diphtheroids (Corynebacterum Pseudodiphtheriticum and C. Xerosis)
Corynebacterium include 3 groups
Human and animal parasites and pathogens
Plant pathogenic Corynebacteria
Nonpathogenic Corynebacteria.
C.pseudodiphtheriticum and C. xerosis are the non pathogenic species and are
found in the nasopharynx of man skin and mucous membrane of man, especially
the conjunctiva.
Laboratory Diagnosis
Gram positive short rods that stain unevenly biochemically they are non
haemolytic, nitrate positive, urease negative for C. xerosis, and sucrose fermenter
for C. xerosis (Joklik, 1980)
Klebsiella
Pathogenicity
Klebsiella aerogenes is associated with hospital –acquired infection of
urinary tract and wounds it is also found in the respiratory tract where it may cause
infections , particularly in immune suppressed patients.
Laboratory Features
Specimens depend on the site of infection and include seminal fluid, urine,
pus, and infected tissue. They are Gram negative, non motile usually capsulated
rods. They are aerobes and facultative anaerobes and produce large-grey-white
usually mucoid colonies on blood agar. On MacConkey agar is lactose fermenting
producing mucoid pink colonies (Cheesbrough, 2000)
46
Biochemical Test
Vagos –proskauer positive,
Lactose fermentation positive
Urease positive
Citrate positive
Malonate utilization positive and
Lysine decarboxylase positive (Cheesbrough,2000)
Candida Albicans
It causes candidiasis
A yeast-like fungus and infection of the UT and normal inhabitants of the
GIT, It occurs in a unicellular form and as long thread like pseudomycelia. The
normal bacteria flora usually suppresses its level of growth. Candida’s thread like
pseudomycelia tend to cluster forming small bezoars or fungus balls which can
cause urethral obstruction ( Macfarlane, 1995)
There exist vagina candidiasis and cuteneous candidiasis.
Laboratory Diagnosis
Specimens include vaginal swab, skin, seminal fluids, urine and blood
cultures. Candida is Gram positive large cocci joint in mass. The selective media
for Candida species is Sabourald Dextrose agar with Chloramphenicol to inhibit
bacteria growth. Colonies are raises, 1-3mm, cream white or yellow, and mucoid
with spirk –like appearance.
Biochemical Test
Germ tube positive (Macfarlane, 1995)
47
CHAPTER THREE
METHODOLOGY
3.0 INTRODUCTION
The study was a survey of different bacteria isolates in semen of males
presenting with fertility problems attending the Saint Elizabeth General Hospital
and Cardiac Center Shisong: The main goal of the research was to create awareness
to the population concerned on different bacteria pathogens associated with
infertility in males, and to educate them on modes of prevention of male infertility.
One of the objectives was to identify better quality control measures that could be
employed to ensure proper semen collection and analysis. The research question
pending clarification was “Are bacteria the main cause of infertility in males
presenting with fertility problems in Saint Elisabeth Catholic General Hospital and
Cardiac Center Shisong”?
3.1 Research type and Time line
The study was a cohort
prospective cross sectional laboratory based
research which ran from the 27th of October to the 28th of May 2011 with a sample
size of 70.
3.2 Study Area
The study was carried out in the St. Elizabeth Catholic General Hospital and
Cardiac Centre Shisong. Shisong is situated in the kumbo East Health District of
the North West Region. It is bounded to the north by kingomen, to the south by
kimbo, the west by Riba and to the East by Mbah. Two seasons exist in Shisong
throughout the year. The rainy and the dry seasons. During the dry season
temperatures fluctuates, with high temp of 37-40oC during the day and 20oc and
below during the nights and early morning hours. (Frost in the morning and heavy
48
sunshine during the day) During the rainy seasons, there are mild temperatures
changes with heavy rainfall observed in August, September and early October.
This area has a hilly topography and muddy roads with a population of
15.000 inhabitants. The indigenous population is made up of the Banso, with
Lamnso as their mother tongue and English Language as their main official
language. The main economic activity of the people is farming. The youths are into
education while others take part in black collar jobs like motorcycle riding, others
as hawkers. The traditional dish here is cornfufu and huckleberry known in the
lamnso language as "kiban” and “Nyoseji” while their main drinks are palm wine
and “shar” or “Nkang”. They have coffee as their main cash crop. Christainity is
the most dominant religion in the area with few Muslims. There are few secondary
schools and many primary schools in this area.
3.2.1 Research Population
Samples were collected from males presenting with fertility problems
attending the St Elizabeth General Hospital and Cardiac Centre Shisong.
3.2.2 Ethical Consideration
The study was carried out in St Elizabeth general hospital laboratory and
Catholic school of health science practical laboratory. Authorizations were taken
from the Matron of the hospital, the director of the school and the head of the
laboratory. Consent was sought from the patients involved ahead of time before
collections of the samples.
3.3 MATERIALS
Equipment
Microscope
Autoclave
Incubator
Bunsen burner
49
Electric balance
Bijou bottles
Graduated Pasteur pipettes
Fridge
Sterile graduated syringe
Sterile bottles
Slides
Petri dishes
pH paper
Wire loops
Stain and dry racks
Reagents
Nutrient Ager (powder)
Mac Conkey Agar (powder)
Kligler Iron Ager (powder)
Oxidase Reagent
3% hydrogen peroxide
Kovac’s reagent
0.5%Eosin
Sodium Bicarbonate Formaline
Carbol fuchsin Methylene blue
Lugos Iodine
Crystal violet
Alcohol
Distill water
Hypochloride
Poor of serum \plasma
50
sterile normal saline
Antibiotic Discs
A pine of blood
Omo
3.4 Sample Collection and Examination
3.4.1 Sample Collection
Individuals were identified by their address, names, ages, occupations and
marital status. Seminal fluid was collected from males after instructing them on;

Sexual abstinence of between 2-7 days and not more than 7 days
Collection to be done by masturbation.

Proper washing of hands and Penis with savon and clean water before
masturbating.

The patient should urinate at least twice before collection of the sample.

Preservation of the sample at body temperature by holding it under the armpit
or holding it closed to the abdomen during transit to the laboratory.

It Should be transported to the laboratory between 30-60 minutes after
collection

The time of collection and period of abstinence to be noted before bringing the
sample to the laboratory.
3.4.2 Examination
Patients were duly instructed on proper collection of the specimen as
explained above. This helped in the reliable interpretation of the results particularly
sperm concentration. For, if abstinence is less than 2 days sperm concentration will
be reduced and more than 7 days progressive or active motility will be reduced.
The samples were obtained by masturbation after the patient had washed both
hands and penis and urinated at least twice.
51
The patient ejaculated in to a sterile wide neck bottle.
Coitus interruptus was not accepted as a means of collection since the first portion
of the ejaculate with the highest concentration could easily be lost and more over
to avoid cellular and bacteria contamination from the vagina. Improperly collected
samples were not analyzed, particularly if the first portion of the ejaculate was lost.
The samples were protected from extremes of temperature by holding them closed
to the abdomen or under the armpit to maintain them at body temperature.
The samples were then examined within 30-60 minutes after allowing them on the
bench or at room temperature for liquefaction to occur ( Rrumballuku,1992 )
3.4.3
Macroscopic Evaluation
Appearance
The sample was first evaluated by simple inspection. A normal sample had
a grey – opalescent appearance homogenous (without clots) and liquefies within 60
minutes at room temperature from ejaculation. Samples that did not liquefied
within this period or over liquefied (watery) were recorded as abnormal. Samples
appeared clear if sperm concentrations were too low, could also be brown if RBC
were present in the ejaculates (Haematospermia).
Consistency \Viscosity
This was evaluated by carefully aspirating the semen in to a syringe and
then allowing the semen to drop by gravity such that the sample leaves the needle
as a small discrete drop forming a thread of less than 2cm long. It was confirmed
by introducing a glass rod in the sample and observing the thread that it formed on
withdrawal of the rod which should not exceed 2cm (Cheesbrough, 2000).
52
Volume
The volume was measured using a sterile graduated syringe of 10ml, by
aspirating all the ejaculate in to the syringe and reading its volume. A graduated
cylinder was also used to measure the volume. Normal semen volumes are above
2ml. (Rrumballaku, 1992)
pH
The pH was measured using a pH paper. A drop of the ejaculate was placed
on the pH paper and the colour change observed was compared with the colours on
the pH meter. Where the value was then written against it .The normal pH values
were 7.2 – 8.0 (Cheesbrough, 2000)
3.4.4 Microscopic Investigations
During initial microscopic investigation of the sample estimations of
motility, concentration of spermatozoa, viability, morphology, Gram stain for
detection of bacteria were performed.
Motility
 One drop of well mixed liquefied semen was placed on a slide and covered
with a cover slip 22 x 22cm.
 The specimen was focused using the 10x objective while closing the
condenser iris sufficiently for good contrast.
 The 40x objective was used to better examine the preparation. 10 fields were
assessed and 100 spermatozoa were counted noting;
.Excellent, rapid or progressive motility.
.Slow or sluggish progressive motility
. Non – progressive motility
. Immotile spermatozoa.
53
Normal motility is over 50% active spermatozoa within 60minutes after
ejaculation. If more than 60% were non motile, within 60minutes then viability was
checked, from the 100spermatozoa counted a percentage of active motile, sluggish
and immotile sperm cells were calculated.
Viability
Viability was assayed using 0.5% eosin
 5µl of the well mixed liquefied sample was placed on a clean slide.
 5µl of 0.5% eosin was then pipette and added to it.
 It was then well mixed to get a homogenous mixture.
 It was then allowed to stand for 2-3 minutes for the spermatozoa to absorb
the stain
 It was covered with a cover slip and mounted on the microscope, observed
using 10 x objectives and 40 x objectives.
 10 fields were assessed and 100 spermatozoa were counted taking note of
those that absorbed the stain (colored) and those which did not absorb
(uncolored).
Results
Dead sperm cells were colored, that is absorbed the stain. They were stained
and appeared pink or orange red because the stain was absorbed in their membrane.
Non stained indicated that the cells were still alive so could not be absorbed the
stain. The percentage of dead to living cells was estimated from the number of dead
cells to living cells which were counted.
Total sperm count
The sperm count was done using the improved Neubauer chamber to count
this was to assessed the total number of spermatozoa concentration present per ml
then per total volume.
54
Procedure
 1ml of the well mixed liquefied semen was placed in a tube
 19ml of the diluting fluid (sodium bicarbonate formalin) was added to this
tube.
Note: sodium bicarbonate fixed the sperm cells and formalin killed the sperm cells
all these maintain the sperm cells on a fixed position for proper counting.
 An improved Neubauer chamber was wiped and using a Pasteur pipette it
was filled with the sample mixture.
 It was then mounted on the microscope and was counted using 10x objective
with condenser iris closed.
 The spermatozoa were counted in 2 large squares (2mm2 area)
 The number of spermatozoa counted was multiplied by 100.000.
Marker chamber was also used for counting. Normal sperm count was 20 x
106spermatozoa /ml (Cheesbrough, 2000).
Sperm Morphology
The various shapes of the spermatozoa were assessed .
Procedure
 A thin smear of the liquefied semen was made and while still wet, it was
fixed with 95% alcohol for 5-10 minutes and allowed to air dry.
 It was then washed with sodium bicarbonate formalin solution to remove
mucous , after which it was the rinsed several times with water.
 It was covered with dilute carbol fuchsin and allowed to stain for three
minutes which was again washed with tap water.
 It was now counter stained with loeffler’s methlyene blue for 2 minutes.
which was then washed with tap water, drained and allowed to dry.
 Using oil immersion the slide was then observed at 100x objective.
55
 10 fields were assessed and 100 spermatozoa were counted from different
fields noting the different abnormal forms for example large heads, double
heads, pyriform heads, double tails, no middle part, not tail or absent of
acrosomal head.
The percentage of normal to abnormal morphology was then estimated. 50% of the
100 spermatozoa should show normal morphology. (Anjali, 2009)
White Blood Cell Count
WBC in semen indicates bacterial infection and it was assessed as below
Procedure
Using a Pasteur pipette, 1 drop of the well mixed liquefied semen was
transferred in to a clean tube.
19 drops of the diluting fluid (sodium bicarbonate formalin) was also transferred to
the semen tube.
It was then mixed and allowed to stand for 5 minutes.
Neubauer chamber was cleaned and filled with the sample which was then mounted
and counted using the 10x objective
Large squares were counted and the number counted was multiplied by 50
Number of spermatozoa counted x dilution factor
in ml
Area X Depth
Or
Spermatozoa counted X 100,000 /ml(Anjali, 2009)
Gram Stain
This was done to identify bacteria present in the sample before and after culture.
Procedure
 A representative part of the sample was smeared on a clean grease
free slide and allowed to air dry.
56
 It was then fixed on a Bunsen flame by passing it 3 times over the
flame.
 It was placed on the staining rack and crystal violet was dropped on
it and allowed to stain for 30 -60 seconds.
 The smear was rapidly washed off with clean water
 The smear was then covered with lugol’s iodine for 30 seconds. This
is the mordant.
 It was again washed with tap water and decolorized with 70% alcohol
and washed .
 The smear was covered with dilute carbol fuchsin which was the
counter stain for 2 minutes. Which was then washed and the back of
the slide was wiped and dried and observed under the microscope
using 40x and 100x objectives and oil immersion .
 The slide was thoroughly scanned for the presence of bacteria which
described their gram reaction and morphology.(Cheesbrough, 2000)
3.4.5 Culture
Culture was done on the sterile semen sample before performing any other test on
the samples. This was to prevent contamination of the sample.
Inoculation on the first day, the various plates were removed from the fridge and
dried in the incubator (MA, BA, and CA)
The plates were then divided to contain a maximum of four samples each.
The codes on the samples were written on the Petri dishes on the space in which the
sample was to be inoculated.
A wire loop was flamed several times; when it was red hot, it was then cooled by
swaying in air.
Representative parts of the samples were picked up with several streaking lines so
as to have discrete colonies.
57
The sample was inoculated on MacConkey agar, blood and chocolate agar.
The BA and CA were incubated in a candle jar at 37°c so as to have a 10% CO2
atmosphere favourable for the growth of facultative bacteria. The MCA was
incubated at 37°c in the presence of O2. All the plates were incubated overnight and
readings of the plates were done the next day. It was extended to 48 hours in case
Neisseria species were suspected since it grows with difficulties.( Werner, 2009)
Reading of Plates
After 18-24 hours of incubation, the plates were read. The numbers of colonies on
the samples were noted. Their different sizes were measured especially on MCA
where pink colonies were lactose fermenting and pale yellow were non lactose
fermenters. Haemolysis was checked on the both BA and CA.
Swarming was also checked on BA and CA
The ordours and shapes of Colonies were also assessed.
Opacity was assessed since different bacteria had different opacity.
The texture of the colonies was checked by flaming the wire loop and touching the
colony to see if it had mucoid or soft. (Ochei, 2008)
Gram Stain
Gram stain was done on each of colony of the sample
A grease free clean slide was partition into six portions and each colony was
picked with a sterile wire loop and smear on the slide using normal saline, to better
spread the colonies on the slide.
The slide was then air dry
It was then fixed by passing the smear 3 times over the flame.
The slide was then stain by Gram technique as seen above.
The smear was then allowed to dry and was observed under the microscope using
100x objective (Cheesbrough, 2000)
58
3.4.6 Biochemical Tests
Biochemical test were done to better isolate the different bacteria present in the
samples.
Catalase Test
A drop of H2O2 was placed on a clean grease free clean slide.
The edge of another clean slide was flamed and was used to pick up a colony.
It was then emulsified on the H2O2
The production of air bubbles was observed which is a positive test and no air
bubble is negative.
Note ; A platinum wire loop was not used to pick up the colony because it reacts
with H2O2 to give a positive reaction. (Ochei, et al., 2008)
Coagulase Test
A drop of plasma was placed on a sterile slide
Using the edge of another sterile glass slide, a colony was picked and emulsify in
the plasma.
Clumping within 10 seconds was a positive test and no clumping within 10 seconds
was a negative test (Cheesbrough, 2000)
Oxidase Test
A filter paper was used,
A drop of the oxidase reagent was drop on the filter paper
A sterile edge slide was used to pick the colony and emulsified on the soaked part.
A deep red purple colour observed within 20 seconds indicated the test was
positive, No colour change was a negative test (Cheesbrough, 2000)
Urease Test
The colonies were inoculated in bijou bottles containing 3ml sterile Christensen’s
Urea broth
A colony was picked with a sterile wire loop and emulsify in the reagent.
59
It was incubated at 35-37°c for 18-24 hours
The next day, color change was observed where pink colour showed a positive test
and no pink colour showed a negative test ( Ochei, et al., 2008)
Kligler Iron Agar (KIA)
A straight wire loop was used to pick up the colony and a butt and stab was made
in the agar.
The mouth of the KIA tube was sterilized by passing over the flame before and
after inoculation.
It was then corked and incubated at 37°c for 18-24 hours.
Yellow butt and slope indicated that the organism was a lactose fermenter.
Blackening indicated that H2S was produced and bubbles of gas indicated that the
organism produces gas. From here different bacteria of the Enterofamily were
isolated. (Cheesbrough, 2000).
Indole Test
The colony was placed with a sterile wire loop and inoculated in a bijou bottle
containing 3ml of sterile Tryptone water. The mouth was flamed and corked.
It was then incubated at 35-37°c for 24-48 hours
The next day 0.5ml of kovac’s reagent was then added.
It was sharked gently and allowed for 10-15 minutes to observed colour change at
the surface layer.
Red surface or pink layer indicated positive indole test and no colour change was
negative.(Cheesbrough, 2000)
3.5 Reagent preparation
The reagent prepared and used for this study were Gram stains, eosin stain, sodium
bicarbonate formalin (the diluting fluid) normal saline, methylene blue, nutrient
agar from which blood agar and chocolate were prepared, Mcconkey agar, H2O2
60
oxidase reagent. All preparations were done according to (WHO and Cheesbrough,
2000.)
Preparations of Gram Stain.
Crystal violet (CV) crystals
20g
Ammonium oxalate
9g
Absolute ethanol or methanol
96ml
Distilled water
100ml
CV was weighed and put into a brown bottle; ethanol was then added and mixed
for the dye to completely dissolve. Ammonium oxalate was then weighed and
dissolved in distilled water the stain was then added and volume was made up to
the requires amount. It was the labeled, dated and stored at room temperature.
(Cheesbrough, 2000)
Lugol’s Iodine Solution
Potassium iodide
20g
Iodine crystals
10g
Distilled water
100ml
The KI2 was weighed and transferred to a brown bottle, distilled waster was added,
and iodine was also weighed and added. It was then mixed till the iodine was
dissolved. The volume was made up to 1000ml. the bottle was dated and was stored
in the dark at room temperature. (Cheesbrough, 2000).
70% Alcohol Decolorizer
Acetone
500ml
Absolute ethanol or methanol
475ml
Distilled water
25ml
61
The acetone was measured and diluted in the distilled water a, absolute ethanol was
added. It was mixed thoroughly and stored at room temperature after labeling
(Cheesbrough, 2000)
0.5% Eosin Preparation
Eosin powder
0.5g
Distilled water
100ml
The eosin powder was weighed and put in transparent container. Distilled water
was then added and make up to the mark. It was well mixed and stored in the dark
for future use. (Cheesbrough, 2000)
Sodium Bicarbonate Formalin (diluting fluid)
Sodium bicarbonate
5g
Formaldehyde solution concentrated
1ml
Distilled water
100ml
It was then weighed and put in a clean container. Distilled water was then added
and mixed well.
N.B formaldehyde solution is toxic, with harmful and irritating vapour. The bottle
was labeled toxic.
Leoffler’s Methylene Blue
Methylene blue
0.5ml
Ethanol (ethyl alcohol) absolute
30ml
Potassium hydroxide 200g/l
0.1ml
Distilled water
100ml
It was then weighed, mixed and transferred into a brown bottle and was labeled
corrosive. (Cheesbrough, 2000)
Sterile Normal Saline
Nacl salt
8.5g
62
Distilled water
100ml
It was then mixed in a clean container after the salt was completely dissolved, then
it was sterilized by autoclaving (Cheesbrough, 2000)
Strong Carbol Fuchsine
Basic fuchsine
10g
Ethanol or methanol absolute
100ml
Phenol
50g
Distilled water
100ml
It was weighed, transferred in to a brown bottle and mixed thoroughly.
N.B methanol is highly flammable and phenol is corrosive. It was then stored at
room temperature and it was always filtered before use (Cheesbrough, 2000)
Preparation of Culture Media
Culture media preparations were done according to the manufacturer’s instruction
written on the container of the various agars.
Preparation of 10 Plates of BA (10%)
One plate contained 20 ml of BA, 10 plates then contained 1l0 x 20 ml = 200ml.
from the nutrient agar (NA) container, 23g of agar were to be dissolved in 1 liter of
distilled water (1000ml)
To get the amount of nutrient agar to be dissolved in 200ml of distilled water.
23g x 200ml
4.6g of NA was then weighed out on the balance and dissolved in 200ml of
distilled water. The dissolved agar was then autoclaved at 121c for 15minutes. The
agar was removed from the autoclave and allowed to cool to 55-560C and 20ml of
blood was added and mixed. It was the poured in 20ml amount in to the sterile
Petrie dishes, and allowed to cooled and solidified. The plates were labeled with
name and date of preparation and stored in the refrigerator to be used when ever
needed. (Cheesbrough, 2000)
63
Preparation of 10 Plates of Chocolate Agar
One plate contained 20ml of chocolate agar, 10 plates then contained 10 x 20ml.
from the nutrient agar (NA) container 5.2g of the dehydrated powder were dissolve
in 100ml of distilled water, then 10 plates which is 200ml = 5.2g x 200ml
10.4g of NA was then weighed out on a balance and dissolved in 200ml distilled
water which was then autoclave for 15minutes at 121c.The agar was removed from
the autoclave and allowed to cool to temperature of 70c, which 20ml blood was
then added and stirred. The color of the medium was then changed in to chocolate
colour. It was then dispensed into 20ml amounts in to the sterile Petri dishes;
covered and allowed to cooled and solidified. The plates were labeled and dated
and stored in the refrigerator for future used. (Cheesbrough, 2000)
5% blood agar was prepared and used for sensitivity testing.
Analysis Plan
After collection and examination of the required number of samples, the statistics
shall be presented on tables and pie charts from which the me.The research
hypotheses shall be proven using Chi square independence test. The confidence
intervals shall be alpha = 0.5. Frequency distribution tables shall used to classify
data according to ages, occupation, and marital status.
Vaibility and Reliability
 Semen samples were collected by masturbation and in sterile containers
 Hands and penis were washed with soap and water and the patient urinated at
least twice before the collection was done.
 The samples were kept at body temperature because extreme temperature
altered motility of the cells.
 The time between collection and analyses was strictly recorded in order to
better interpret the results because sperm cell motility reduces greatly with
time.
64
 Semen samples were inoculated immediately before any other analysis was
done to prevent contamination of sample (Werner, et al., 2010)
 To ensure that growth observed after culture wasn’t as a result of
contamination, empty culture plates were incubated without inoculation to
ensure they were prepared with sterility.
 Results obtained from culture were often compared with those of the direct
Gram examination during analysis to ensure they are similar.
1.6 LIMITATION AND DELIMITATION
1.6.1 Limitations
 Majority of the clients preferred collection of samples at their homes before
transporting them to the hospital laboratory which lead to wrong or faulty
results as analysis is suppose to be done with one hour of collection.
 Most patients had difficulties preserving the samples close to the body to
maintain the temperature close to that of the body.
 Some clients expressed difficulty in producing the specimen by
masturbation. They only prefer coitus interuptus which could
bring faulty
results.
 Most clients had little knowledge about the test and so needed explanations
and education leading to time wastage.
 There was no room or convenient place in the laboratory for the collection of
this sample hence using the toilets which was not convenient for most clients.
1.6.2 Delimitations
 Proper quality control in analyzing the sample was done and samples were
analyzed 60 minutes after collection.
 Equipments were available for correct analysis and culture. The results were
compared
with
automated
results
65
hence
working
within
standard.
CHAPTER FOUR
RESULTS AND INTERPRETATION
4.0 RESULTS
The study analyzed 70 seminal fluids from males presenting with fertility problems, 48
of the seminal fluids had bacteria growth with an overall prevalence of 68.6%. 49
participants were married while 21 were celibates, married males presented with a
prevalence of 54.3% while celibates had 14.29%. According to age group, participants
of the age group 30-39 presented with the highest prevalence of 28.6%
With respect to occupation, Farmers and Drivers had the highest prevalence of 17.1%
each.
Clients were from primary, secondary and higher educational levels. With those with
primary education being a modal level presenting with the highest frequency and a
prevalence of 37.1%.
Of the 70 samples, 33 had Staphylococcus aureus (28.95%), 15 had Diphtheroids
(13.2%), 10 had Escherichia coli (8.8%), 13 had Streptococcus species (11.4%), 8 had
Micrococci species 7.02%, 4 had Corynediphtheriae(3.5%), 3 had Candida albicans
and Neisseria gonorrhea(2.63%). Bacillus species, Listeria species and Lactobacilli
with a prevalence of 0.9% each.
Of the 70 samples, 33 had Normozoospermia (34.3%), 5 had azoospermia (7.14%), 2
had necrozoospermia (2.9%) 1 had astheriozoospermia and Terathoszoospermia with a
prevalence of 1.4%.
66
4.1 Analyses and Interpretation.
4.1.1 Overall Prevalence of Bacteria Isolates in Seminal Fluid
Table II
Number Analyzed Number Positive
70
48
Number Negative
22
% Prevalence
68.6%
Figure 1: Overall Prevalence of Bacteria Isolates from Seminal Fluid.
Out of the 70 samples analyzed 48 showed evident growth and 22 presented with no
growth.
4.1.2 Prevalence of Bacteria Isolates According to Age
Table III: Prevalence of Bacteria Isolates According to Age
Age
Number
Number
Number
%
Group
Analyzed
Positive
Negative
Prevalence
20 – 29
22
8 (21.6%)
14
11.4%
30 – 39
34
20 (54.0%)
14
28.6%
49 – 49
12
8 (21.6%)
4
11.4%
≥50
2
1 (2.7%)
1
1.4%
TOTAL 70
37(100%)
33
2
χ Tabulated (df = 3 , 0.05) = 7.815, χ2 Calculated = 3.819
67
Figure 2: Prevalence of Bacteria Isolates According to Age.
From the date above the age group 30-39 had the highest prevalence giving a
percentage of 28.6%, being the modal class. This is the most active age group in life
socially, and is exposed to many of these infections. Many are involved in high
sexual activities like prostitution and promiscuity, and also in other occupations like
motorcycle/car driving which leads to destruction of sperm cells in the testis due to
the strenuous nature of these activities to the male genitals. Coupled with the fact,
that when a couple is facing infertility problems, that is married for more than 3
years without a child, the man likely goes in for another sexual partner to verify if
he is the one having the problem, he can possibly gets infected with Sexually
transmissible pathogens that will further helps to aggravate the disease condition.
Though from X2 test of independence, X2
calculated
< X2 tabulated satisfying the claim
that there is not enough evidence to prove that the prevalence of bacteria isolates is
independent on the age group, there is still an insignificant relationship between age
and the prevalence of these disease condition.
68
4.1.3 Bacteria Species versus Age
Age Group
S. aureus
Diphtheroids
Strep. Species
E. coli
Micrococci
C. diphtheriae
N. gonorrhoea
C. albicans
Bacillus species
Listeria species
Lactobaccili
NO Isolates
Total
% Prevalence
Table IV: Bacteria Species versus Age
20 – 29
30 – 39
49 – 49
≥50
TOTAL
8
16
8
1
33
1
9
5
0
15
2
8
2
1
13
2
4
2
2
10
1
5
1
1
8
3
0
0
1
4
1
1
1
0
3
2
0
1
0
3
0
0
1
0
1
0
1
0
0
1
0
0
1
0
1
14
14
4
1
33
22
34
12
2
70
11.4%
28.6%
11.4%
1.43%
52.9%
From the data above, the age group 30-39 had the highest prevalence with a
percentage of 66.7%. this age group is the most active socially, probably due to
high promiscuity with Staphylococcus aureus presenting as the most prevalent
isolate because it’s a normal flora of the skin and Diptheroids which are normal
flora of the male genital(urethra).The least isolated were Lactobacilli, Listeria and
Bacillus species which are mostly isolated in trivial disease conditions except for
lactobacilli which might be isolated from the male patient due to recent sexual
intercose with a female as the organism is a normal flora of the female genital
tract. Indicating that some of the participants might not have followed the
instructions given to them strictly.
69
4.1.4 Prevalence of Bacteria Isolates According to Marital Status
Table V: Prevalence of Bacteria Isolates According to Marital Status
Marital
Status
Married
Single
TOTAL
Number
Number
Number
% Prevalence
Analyzed
Positive
Negative
49
38 (79%)
11
54.28%
21
10 (21%)
11
14.3%
70
48
22
100
2
2
χ Tabulated (df = 1 , 0.05) = 3.841, χ Calculated = 6.1
P revalenc e B ac teria Is olates by Marital S tatus
21%
Married
S ingle
79%
Figure 3: Prevalence of Bacteria Isolates According to Marital Status
From the table v above, it can be seen that clients who were married were more
infected in which 38 clients out of 49 screened were infected presenting as the
modal class giving a percentage prevalence of 54.28%. This may be due to
sexual activity involving more than two partners (intersexual activity) of which
one of the partners may be infected. Males presenting with infertility always
have 2 or more partners to see if they can impregnate them and disprove the fact
that they have a problem. Also due to the fact that married participants will want
to procreate inorder children, infertility consultations from them becomes high
relative to frequency sample explaining in part the reason for that high
prevalence in them.
70
For singles, 10 of the 21 screened were infected giving a prevalence of 14.3%.
This may be due to premarital sex or, unhygienic habits such as dirty under
wear.
From X2 test of independence, there is enough evidence to support the claim that
bacteria infection in seminal fluid of males is dependent on marital statues. With
married participants presenting with the highest prevalence.
4.1.5 Prevalence of Bacteria Isolates According to Occupation
Table VI. Prevalence of Bacteria Isolates According to Occupation
Occupation
Number
Number
Number
% Prevalence
Analyzed
Positive
Negative
Business
23
12 (25%)
11
17,1%
Drivers
10
9 (18.8%)
1
12.9%
Farmers
14
12 (25%)
2
17.1%
Health Personnel 10
4 (8.3%)
6
5.7%
Herdsmen
5
5 (10.4%) 0
7.14%
Other
8
6 (12.5%) 2
8.6%
TOTAL
70
48
22
68.6
2
2
X Tabulated (df =5 , 0.05) = 11.071, X Calculated = 14.04
Figure 4: Prevalence of Bacteria Isolates According to Occupation
71
From the data above it is shown that those in the Business sector and farmers
followed by drivers had the highest prevalence of 17.1% each and 13 %
respectively. For drivers, 9 were infected out of 70 samples (13%), business
men, 12 were infected (25%), while farmers,12 were also infected (25%). This
high prevalence identified in Businessmen, farmers, and drivers may be due to
ignorance of sanitation procedures, lack of personal hygiene as most of them
cover long distances and stay in farm bushes for days without bathing. Some
drivers are highly promiscuous hence leading to infections. In addition to the
factors above, most of these affected individuals due to little education would
hesitate to consult immediately to a medical practitioner when notice symptoms
of genital infections until it becomes chronic which could possibly contribute to
infertility. The lowest infected occupation was recorded with health personnel
with a prevalence of 5.7%. This may be due to awareness on basic hygiene
procedures and health education talks on the general preventive methods of
infection
Since X2
calculated
> X2
tabulated
there is enough evidence to prove that the
prevalence of bacteria isolates is dependent on the occupation of the clients
involved; with Businessmen, farmers and drivers presenting with
high
prevalences.
4.1.6 Prevalence of Bacteria Isolates According to Educational Level
Table VII: Prevalence of Bacteria Isolates According to Educational Level
Educational
Level
Primary
Secondary
Higher
TOTAL
Number
Number
Number
% Prevalence
analyzed
Positive
Negative
34
26 (54.1%)
8
37.1%
17
13 (27.1%)
4
18.6%
19
9 (18.8%)
10
12.9%
70
48
22
100
2
2
χ Tabulated (df =2, 0.05) = 5.991, χ Calculated = 6.3
72
Figure 5: Prevalence of Bacteria Isolates According to Educational Level
Of the 70 samples analyzed 34 were from patients with the primary level of
education, 17 with the secondary level and 19 of those participants had gone
beyond secondary level as elaborated in the table above. Out of the 34
participants with primary level 26 were infected and 8 uninfected , it falls in the
modal class level with the highest frequency and a prevalence rate of 37.1%
which was an indication of ignorance of good sanitation rules and personal
hygiene as compared to the decreasing prevalence rate 12.9% in the higher level
of education. Since this level is well aware of proper sanitation methods
pertaining to infections.
X2 tabulated, Df 2 at 95% confidence interval level is less than X2 calculated
there is enough evidence to support the claim that the prevalence of bacteria
isolates is dependent on level of education.
73
4.1.7: Prevalence of Individual Bacteria Isolates
Table VIII: Prevalence of Individual Bacteria Isolates
Isolates
Number
%Prevalence
Staphylococcus aureus
33
28.95
Neisseria gonorrhea
03
2.63
Escherichia coli
10
8.77
Candida albicans
03
2.63
C . diphtheria
04
3.5
Streptococcus species
13
11.4
Bacillus species
01
0.88
Listeria species
01
0.9
Micrococci species
08
7.02
Diphtheroids
15
13.1
Lactobacilli
01
0.9
No Growth
22
19.29
Total
114
2
X Tabulated (df =11, 0.05) = 19.675, X2 Calculated = 105.23
Prevalence of individual bacterial isolates
staph
Neiseria
19.29
E.coli
28.9
Candida
0.9
C. Deph
Streph spp
Bacillus spp
13.1
Listeria spp
2.63
8.77
7.09
Micrococi
Dephteroids
Lacto
0.9
11.4
No growth
3.5
2.63
0.88
Figure 6: Prevalence of Individual Bacteria Isolates
74
From the table above, Staphylococcus aureus comes up with the highest
prevalence and also the modal pathogen infecting 33 participants making a
prevalence of 28.95%. This might probably be due to contamination of the
samples with the skin which contain Staphylococcus aureus as its normal flora.
Bacillus species, Listeria species and Lactobacilli have the lowest frequencies
infecting one client each giving a prevalence rate of 0.9%
From the X2 goodness of fit test, it is clear that clients show preference to some
pathogens like Escherichia coli and Staphylococcus aureus because of
colonization from intestinal flora and the surface of the skin.
4.1.8: Prevalence of the Different Types of Infertility
Table IX: Prevalence of the Different Types of Infertility
Types of Infertility
Number Positive
%prevalence
Normozoospermia
33
47.1%
Oligozoospermia
28
40%
Azoospermia
05
7.1%
Necrozoospermia
02
2.9%
Asthenozoospermia
01
1.4%
Terathoszoospermia
01
1.4%
TOTAL
70
X2 Tabulated (df =5, 0.05) = 11.071, X2 Calculated = 80.14
75
Figure 7: Prevalence according to types of Infertility
From the table above, 33 participants had Normozoospermia with a prevalence
of 47.1%, 28 had Oligozoospermia with 40%, 5 had Azoospermia with 7.1%, 2
had
Necrozoospermia
with
3%
while
Asthenozoospermia
and
Terathoszoospermia were each observed in 1 client with a prevalence of 1.4%.
From the X2 goodness of fit test there is enough prove to support the claim that
males presenting with fertility problems at Saint Elisabeth Catholic General
Hospital and Cardiac Center Shisong Show preference to specific types of
infertilities with Oligozoospermia with reasons as explained in the tables above.
76
CHAPTER FIVE
DISCUSSION, CONCLUSIONS AND
RECOMMENDATIONS
5.0 INTRODUCTION
This study was carried out on seminal fluids of males presenting with
fertility problems visiting the Shisong Hospital .The study had 70 samples
analyzed and cultured, with an overall prevalence of 52.9%.Married males had a
prevalence of 54.3% while celibates had 14.3%.According to age group ;the age
group 30-39 had the highest prevalence of 28.60%.Prevalence according to
occupation was 68.6% with businessmen and farmers having the highest
prevalence of 17.1%.According to the type of infertility ;Normozoospermia
had 47.14%;Oligozoospermia had 34.3% and Azoospermia had
7.14%.Staphylococcus aureus had the highest pathogenic Prevalence of
28.95%.
5 .1 DISCUSSION
The prevalence of seminal fluid infections according to age in a
similar work carried by Onemu in 2010 where 229 volunteers of active age
group (19-33)participants gave a prevalence of 34.9%.While with the results of
this study the highest prevalence were recorded in a similar age group (38.0%)
and the lowest prevalence at ages > 50years(2.7%).
The high prevalence of 52.9% in this research was similar to the results of
the study conducted by Agbakoba, et al in genital bacteria in semen samples of
males where 54married men were screened presenting with a prevalence of
57.2% in 2007 and Rodriquez 2001 in Genital Infection and infertility where
487 men where screen with a prevalence of 47.3%.
The Prevalence of seminal fluid infection according to Pathogenic species
in this study was also similar to the Works of Obiechina and Okoye in Seminal
fluid indices of men attending infertility clinic (1994) where 628 men screened
77
with Staphylococcus aureus had 46% as the highest prevalent and low
prevalence of 0.3% with Neisseria gonorrhoea. The prevalence according to
pathogens disagreed with the results of Mehta, et al, 2002 in males Partners in
infertile couple where Streptococcus faecalis had (53%);Micrococci species
(20%); alpha Haemolytic streptococcus (16%) with Oligozoospermia and
Terathoszoospermia being insignificant. This could be due to the fact that the
high prevalence of Staphylococcus aureus in this research was due to
contamination with skin normal flora.
According to the type of infertility ;Normozoospermia had the highest
prevalence of (47.14%);Oligozoospermia (40%) and Azoospermia(7.1%) in line
with the work of Obiechina and Okoye who had 53.8%; 44% and 6.2%
respectively
5.2 CONCLUSIONS
With respect to this study in which 70 seminal fluid samples were
analyzed within the period of October 28th 2010 to May 28th 2011 the
following conclusions were drawn.
 The prevalence was higher amongst the primary level of education than in
the other with a prevalence of 37.1%.
 Equally the prevalence were high in Businessmen, and farmers than all
the other occupations (17.1% each).
 Staphylococcus aureus was more prevalent (28.9%) than any other
pathogens within this study population.
 Most patients presenting with fertility problems had significant bacteria
growth after culture; even if it was not requested by the consulting doctor.
Therefore all semen specimens from males presenting with infertility
should be examined by analysis and culture and proper antibiogram carried out
to identify all cases of bacterial infections and treatments administered .Also
78
not only are bacteria alone incriminated in infertility,other factors like
immunologic problems,physical activities,and physiology of male reproductive
system can also be responsible for infertility in males.
5.3
RECOMMENDATIONS
 Sperm count should be done manually when ever a sample presents with
Oligozoospermia when using the sperm analyzer.
 Proper instructions of washing the penis; hands and urinating at least
twice before collection of the samples should be properly explain to the
patient.
 A comparative study should be done between seminal fluid having
leucocytes and non leucocytic seminal fluid to prove or disprove the fact
that leucocytes are not indicators of bacteriospermia.
 There should be health education to the general public on the predisposing
factors to male infertility.
 Semen cultures should always be done for any samples collected to roll
out bacteria as the probable cause of infertility
 There is urgent need to establish centers for assisted reproduction which
will help in solving the already overwhelming problem of infertility in our
country
79
REFERENCES
Alkarhatkar, J.V (2008). Medical Laboratory Science (Theory and Practice).
New Delhi McGraw-Hill Publishing Company Limited.
Bar-Chama, N and Lamb, D.J (1994). Modern Andrology Laboratory Urologic
Clinics. North America 433-446.
Berhre, N and Eberhard, N (2010). Andorlogy Male Reproductive Health and
Dysfunction. Page 155
Bezold, G (2007). M.D Laboratory Department of Obstetrics Gynaecology and
Reproductive Biology. Boston Massachusetts.
Carell, D.T (2003). Reproductive Endocrinology and Infertility. Editor C
Mathiew Peterson District
Cheesbrough , M. (2000). Laboratory practice in tropical countries. Cambridge
university low price edition (130-132)
Department of urology . retrived on 20th may 2011 from
[email protected]. New Presbyterian hospital
medical college of comell; University of New York.
Anderson,Nester&Pearsall,(2004).Microbiology a Human
Perspective,Newyork;McGrawhill (2003).
Guyton, AC (1986). Medical physiology WB Saunders 7th edition page 955-958
Jokeb,L Department of urology medical university of Vienna(N.D)
Kchau,GR (2008). Department of microbiology and research, laboratory
microganismes et pathologie humaine. Tunisia U.S national
library of medicines
Leon, S (1994). clinical gynaecology , endocrinology and inferitility
Litwin, MS(1999). Health in chronic prostatitis symptoms. National institute
Benin
Mbeacho, LA (2010). Prevalence of urinary tract infection in HIV/AIDS
patients visiting the St Elizabeth General Hospital Shisong
(40-52) unpublished
80
Mogra,N.N(1981). Non specific seminal tract infections and male infertility
postgrade.
Nester, E.W(2004). Microbiology a human prespective. Mc Grahill companies
by Martins J Lange
Onemu, S.O, Ogbimi, A.O Ophori, E.A(2010).Department of medical biology
Benin
Patrick, A (2010). Health communities anatomy and function of the urinary
tract male reproductive system.
Rrumbullaku,L.M (1992).Reproductive Medicine and Reproductive Biology
Geneva A. de Agostini
Schiff,JD, MD(2010). Urology erectile dysfunction premature ejaculation and
sexual dysfunction.
Speroff, L (1994). Clinical Gynaecology , Endocrinology and infertility.
Baltimore Maryland by Charles Mitchel 5th edition
Strome, S.W and L’Hernault S.W(2005). Department of biology and Emory
university Atlanta.
Wales,J(1984). .Assessment of Human Sperm function biology reproduction
888-894.
Werner,M.A(2006). MD Facs urologist and specialist in sexual dysfunction.
WHO, (1992). Laboratory manual for examination of human semen and sperm
cervical mucus interaction. Cambridge university 3rd edition.
81
APPENDICES
Appendix IV
S/N
Occupation
Residents
33
Marital
status
M
Herdsman
Bamti-Din
Education
al level
01
31
M
Farmer
Mbeh
01
28
M
Business
Tobin
01
35
M
Farmer
Kitiwum
01
40
M
Business
Tobin
02
33
M
Others
Dschang
03
37
M
Others
Batouri
03
25
S
Business
Ndu
02
36
M
Secretary
B’da
02
33
M
Herdsman
Bango
00
11. Normozospermi 36
a
12. Yaya Joussouffu 41
13. Normozospermi 33
a
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
M
Heath
personnel
Herdsman
Business
Nkavikim
03
Microcolli spp
Strep Pyogene
S. aureaus
Y’de
Y’de
01
02
N.gonorrhoea
No Pathogen
1.
2.
3.
4.
5.
6.
Type of
infertility
Terathozoosper
mia
Oligozoospermi
a
Normozospermi
a
Normozospermi
a
Oligozoospermi
a
Oligozoospermi
a
7. Oligozoospermi
a
8. Normozospermi
a
9. Normozospermi
a
10. Azoos
Age
M
S
82
Pathogens isolated
S. aureus,
Diphtheroids
S. aureus,
Diphtheroids
No Pathogen
S.aureus, S.
Pyogenes
S. aureus,
Diphtheroids,
Lactobacilli
S. aureus,
S.aureus, S.
Pyogenes
Candida spp S.
aureus, E. Coli
S. aureus
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
83
CHAPTER FIVE
DISCUSSION, CONCLUSIONS AND
RECOMMENDATIONS
5.0
INTRODUCTION
A research carried out in seminal fluid in males presenting with fertility
problems visiting the Shisong Hospital .The study had 70 samples analysed and
cultured ; with an overall prevalence of 52.9%.Married males had a prevalence
of 54.3% while celibates had 14.3.According to age group ;the age group 30-39
had the highest prevalence of 38.0%.Prevalence according to occupation was
68.6% with Herdmen having the highest prevalence of 100%.According to the
type of infertility ;Normozoospermia had 47.14%;Oligozoospermia had 34.3%
and Azoospermia had 7.14%.Staphylococcus aureus had the highest pathogenic
Prevalence of 28.95%.
5 .1
DISCUSSION
The prevalence of seminal fluid infections according to age in a
similar work carried by ONEMU.S.O in 2010 where 229 volunteers of active
age group (19-33)participants gave a prevalence of 34.9%.While with the
results of this study the highest prevalence were recorded between this age
group (38.0%) and the lowest prevalence at ages > 50years(2.7%).
The high prevalence of 52.9% in this research was similar to the results of
the study conducted by AGBAKOBA. N.R et al; In genital bacteria in semen
samples of males where 54married men were screened presenting with a
prevalence of 57.2% in 2007 and Rodriquez 2001 in Genital Infection and
infertility where 487 men where screen with a prevalence of 47.3%
The Prevalence of seminal fluid infection according to Pathogenic species
in this study was also similar to the Works of Obiechina and Okoye in Seminal
fluid indices of men attending infertility clinic (1994) where 628 men screened
84
with Staphylococcus aureus had 46% as the highest prevalent and low
prevalence of 0.3% with Neisseria gonorrhoea. The prevalence according to
pathogens disagreed with the results of Mehta. R. H et al ;in 2002 in males
Partners in infertile couple where Streptococcus faecalis had (53%);Micrococci
species (20%); alpha Haemolytic streptococcus (16%) with Oligozoospermia
and terazoospermia being insignificant.This could be due to the fact that the
high prevalence of S.aureus in this research was due to contamination with skin
normal flora.
According to the type of infertility ;Normozoospermia had the highest
prevalence of (47.14%);Oligizoospermia (34.3%) and Azoospermia(7.4%) in
line with the work of Obiechina and Okoye who had 53.8%; 44% and 6.2%
respectively
5.2 CONCLUSIONS
With respect to this study in which 70 seminal fluid samples were
analysed within the period of November 24th 2010 to may 28th 2011 the
following conclusions were drawn
 The prevalence was higher amongst the primary level of education than in
the other with a prevalence of 76.5%.
 Equally the prevalence was higher in Drivers and Herdsmen than all the
other occupations (90%and100%).
 Staphylococcus aureus was more prevalent (28.9%) than any other
pathogens within this study population.
 Most patients presenting with fertility problems had significant bacteria
growth after culture; even if it was not requested by the consulting doctor.
Therefore all semen specimens from males presenting with infertility
should be examined by analysis and culture ;and proper antibiograme carried out
to identify all cases of bacterial infections and treatments administered .Also
85
not only would bacteria alone be incriminated in infertility ;other factors like
immunologic problems ;physical activities ;and physiology of male reproductive
system can also be responsible for infertility in males
5.3
RECOMMENDATIONS
 Sperm count should be done manually when ever a sample presents with
Oligozoospermia when using the sperm analyser.
 Proper instructions of washing the penis; hands and urinating at least
twice before collection of the samples and should be properly explain to
the patient.
 A comparative study should be done between seminal fluid having
leucocytes and non leucocytic seminal fluid to prove or disprove the fact
that leucocytes are not indicators of bacteriospermia .
 There should be health education to the general public on the predisposing
factors to male infertility.
 Semen cultures should always be done for any samples collected to roll
out bacteria as the probable cause of infertility
 There is urgent need to establish centres for assisted reproduction which
will help in solving the already overwhelming problem of infertility in our
country
86