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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