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17MEDICAL SCIENCES
INFECTIOUS FACTORS DETECTIN IN AZOOSPERMIA OF INFERTILE MEN
Melnyk O.,
Assistant Professor, PhD, Department of Microbiology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Kovalenko I.,
Assistant Professor, PhD, Department of Microbiology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Vorobets M.,
Assistant Professor, PhD, Department of Urology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Onufrovych O.,
Associated Professor, PhD, Department of Medical Biology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Borzhievsky A., Professor, D Sci, Department of Urology, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Fafula R.
, D Sci, Department of Biophisics, Danylo Halytsky Lviv National Medical University, Lviv, Ukraine
Abstract
One of the reasons for reduced male fertility is urogenital infections. In the urogenital tract of men and women, there are numerous microorganisms. Persisting for a long time in the urogenital tract, they may not cause any clinical manifestations, but cause sluggish inflammation that tends to spread and develop not only urethritis, prostatitis, epididymitis, orchitis, as well as a violation of the process of spermatogenesis. Microorganisms damaging the spermatogenic epithelium cause disruption of spermatogenesis and the appearance of abnormal and pathological forms of sperm. The study included 122 patients diagnosed with a non-obstructive (NOA) form of azoospermia and 68 diagnosed patients with obstructive azoospermia (OA). Patients with a mixed form of azoospermia were not included in the study. The number of men with a history of Trichomonas infection in NOA was 1.6%, and in OA - 1.5%. The frequency of detection of chlamydia by PCR in NOA was 3.3%, and in OA -2.9%. Mycoplasma in NOA was detected in 5.7%, and in OA - in 2.9%. Ureaplasma was diagnosed in 12.3% in NOA and in 11.7% in OA. In NOA, gonorrhea was detected in 6.6%, and in OA - in 13.2%. Herpes simplex virus type 2 was observed in 5.7% of men with NOA and in 7.3% of men with OA. Gardnerella was diagnosed in 9.0% of men with NOA and 13.3% of men with OA. The frequency of enterococci in men with azoospermia in OA is 32.3%, almost twice higher than in the PLA (17.2%). Infection of ejaculate/prostate secretion with Escherichia coli in NOA was detected in 13.1% of patients, and in OA - in 23.5%. Staphylococcus aureus was detected only in patients with OA (4.4%). p-hemolytic streptococcus was detected in both NOA (7.4%) and OA (13.2%). Epidermal streptococcus is found in men only in OA - 4.4%. The results obtained indicate that infections of the male urogenital tract and infection of seminal fluid or prostate juice are important factors, causing the development of NOA and OA.
Keywords: azoospermia, infections, seminal plasma, urogenital tract.
Introduction. One of the reasons for reduced male fertility is urogenital infections [5-9, 13, 15]. In the urogenital tract of men and women, there are numerous microorganisms [6, 7, 9, 13]. Chlamydia trachomatis and Mycoplasma genitalium are pathogenic microorganisms while Ureaplasma urealyticum and Mycoplasma hominis are considered conditionally pathogenic, that is, they only cause trouble if our immune systems are weakened. Persisting for a long time in the urogenital tract, they may not cause any clinical manifestations, but cause sluggish inflammation that tends to spread and develop not only urethritis, prostatitis, epididy-mitis, orchitis, as well as a violation of the process of spermatogenesis. Chronic inflammation of the urogenital tract and prostate can lead to impaired patency of the vas deferens, as a result, there is a lowered sperm
count as well as changes in the physicochemical properties of seminal plasma and decreased sperm activity [1-4]. Microorganisms damaging the spermatogenic epithelium cause disruption of spermatogenesis and the appearance of abnormal and pathological forms of sperm [2].
Getting into to ejaculate, infectious agents can disrupt sperm motility by producing reactive oxygen species, which leads to peroxidation of membrane lipids and mitochondrial damage [6, 10, 12]. Chlamydia, my-coplasma, ureaplasma can cause DNA fragmentation, damage chromatin and lead to sperm apoptosis [4]. In addition, interacting with components of the immune system, microorganisms are able to induce the activation of macrophages, production of proinflammatory cytokines and antisperm antibodies [6].
There is a large group of opportunistic pathogens such as enterococci, staphylococci, streptococci, gardnerella, etc., which may not cause clinical manifestations. Asymptomatic latent genital tract infection in men is often the cause of infertility. Infection often leads to chronic inflammation of the gonads, which causes damage to the spermatogenic epithelium. Bacteria also cause depletion of seminal plasma by fructose and zinc, which disrupts the metabolism of both sper-matogenic epithelium and sperm. Bacteria support chronic inflammation of the urogenital tract, which is often accompanied by an increase in the number of neutrophils, which produce free radicals and reactive oxygen species [6, 10, 12]. Reactive oxygen species are normally required for spermatogenesis, as they contribute to the compaction of chromatin and sperm maturation. However, if antioxidant protection is unsatisfactory, there is oxidative stress occurs, which is considered the main mechanism of cell damage [6, 11, 12]. ROS overproduction causes both the fragmentation of nuclear and mitochondrial DNA of sperm [4, 6]. Apop-tosis is more common in such sperm.
So, pathological effects of chlamydia, mycoplas-mas, ureaplasmas, as well as opportunistic pathogens on the male reproductive system are closely related to inflammatory and autoimmune processes which lead to impaired spermatogenesis.
Material and Methods. The study included 122 patients diagnosed with a non-obstructive form of azo-ospermia and 68 diagnosed patients with obstructive azoospermia. Patients with a mixed form of azoospermia were not included in the study. Infectious
screening included analysis of urethral secretions, bacteriological examination of sperm and prostate secretion, and assessment of the species and quantitative composition of the microflora of the male urogenital tract. PCR diagnosis of sexually transmitted infections and invasions was performed using a set of reagents "DNA technology" on an automatic fluorescent analyzer. The titer of opportunistic pathogens was quantified, the indicator > 104 CFU/ml was considered diag-nostically significant. In accordance with WHO recommendations (2010) [14] when the threshold values of the number of leukocytes in the seminal plasma exceeded 1.0 million/ml, microbiological studies of ejaculate were performed. Ejaculate was collected in sterile tubes. Diluted semen samples were cultured on 5% blood agar, MacConkey agar, Saburo agar for the isolation of microorganisms. Colonies of a single type of micro-organism (>103 CFU/ml) were picked for identification. The research was conducted on the basis of the urology department of the Lviv Regional Clinical Hospital and Synevo Laboratory.
Results. The number of men with a history of Trichomonas infection in NOA was 1.6%, and in OA -1.5% (Table 1). The frequency of detection of chlamydia by PCR in NOA was 3.3%, and in OA -2.9%. Mycoplasma in NOA was detected in 5.7%, and in OA - in 2.9%. Ureaplasma was diagnosed in 12.3% in NOA and in 11.7% in OA. In NOA, gonorrhea was detected in 6.6%, and in OA - in 13.2%. Herpes simplex virus type 2 was observed in 5.7% of men with NOA and in 7.3% of men with OA. Gardnerella was diagnosed in 9.0% of men with NOA and 13.3% of men with OA.
Table 1
Sexually transmitted infections in men with azoospermia
Transferred infections of men with azoospermia Non-obstructive azoospermia, Number (n = 122) Obstructive azoospermia, number (n = 68)
Trichomonas vaginalis 2 (1,6 %) 1 (1,5 %)
Chlamidia trachomatis 4 (3,3 %) 2 (2,9 %)
Micoplasma genitalium 7 (5,7 %) 2 (2,9 %)
Ureaplasma urealyticum 15 (12,3 %) 8 (11,7 %)
Neisseria gonorrhoeae 8 (6,6 %) 9 (13,2 %)
Herpes simplex virus 2-muny 7 (5,7 %) 5 (7,3 %)
Gardnerella vaginalis 9 (13,3 %)
Total 52 (44,2 %) 36 (52,8)
In accordance with the results of bacteriological men with azoospermia in diagnostically significant ti-culture revealed that the species spectrum of opportun- ters was various, however with low indicators (table 2). istic pathogens in the ejaculate or prostate secretion of
Table 2
Bacteriological examination of ejaculate/prostate secretion
Infectious factors Non-obstructive azoospermia, number, (%) (n = 122) Obstructive azoospermia, number, (%) (n = 68)
Enterococcus faecalis 21 (17,2 %) 22 (32,3 %)
E. coli 16 (13,1 %) 16 (23,5 %)
Staphylococcus aureus - 3 (4,4 %)
ß-hemolytic streptococcus 9 (7,4 %) 9 (13,2 %)
Epidermal streptococcus - 3 (4,4 %)
Bcboro 46 (37,7 %) 53 (77,8 %)
The frequency of enterococci in men with azoospermia in OA is 32.3%, almost twice higher than in the PLA (17.2%). Infection of ejaculate/prostate secretion with Escherichia coli in NOA was detected in 13.1% of patients, and in OA - in 23.5%. Staphylococ-cus aureus was detected only in patients with OA (4.4%). p-hemolytic streptococcus was detected in both NOA (7.4%) and OA (13.2%). Epidermal streptococcus is found in men only in OA - 4.4%.
Noteworthy, correspondence of bacteriospermia to the data of clinical examination of men with a history of chronic inflammatory diseases of the genital organs. As the data of table 2, microbial contamination of ejaculate and prostate secretion samples was predominant in men with OA (77.8%) compared with 37.7% in men with NOA. It should be noted, that the question of the role of subclinically and clinically pronounced infections of the male genital tract in the formation of infertility is still debated. Different mechanisms are involved in the disruption of the process of spermatogen-esis and sperm quality caused by infection of the gonads or inflammatory processes of the urogenital tract of men. Leukocytes are markers of infection, but there are controversial views on the effects on seminal plasma and male fertility. According to WHO recommendations, 1 million leukocytes in 1 ml of ejaculate should be considered as the limit of leukocytospermia. However, many studies have not found a correlation between the number of leukocytes and the number of sperm progenitor cells or their function [6].
The most common method of identifying a bacterial infection of the male urogenital tract is microbiological culture of seminal fluid or prostate juice. If after classical sowing the number of colonies of pathogens is large, there is a version of the probable damage to the spermatogenic epithelium or sperm by free oxygen radicals. They are always in excess contained in infected biological fluids because in the inflammatory process they migrate phagocytes with activated oxygen-dependent enzymes. However, the question whether the detected infectious factors always causes azoospermia is not straightforward [9]. It cannot be ruled out, that it is only a combination of asymptomatic infection with another (underlying) pathology.
The most common method of identifying a bacterial infection of the male urogenital tract is microbiological culture of seminal fluid or prostate fluid. If after laboratory diagnostics there is the number of colonies of pathogens is large, there is a version of the probable damage to the spermatogenic epithelium or sperm by free oxygen radicals. They are always in excess contained in infected biological fluids because in the inflammatory process they migrate phagocytes with activated oxygen-dependent enzymes. In practice, however, the questions whether the infectious factors detected always causes azoospermia is not straightforward [9]. It cannot be ruled out, that it is only a combination of asymptomatic infection with another (underlying) pathology.
It should be noted that the infection caused by Ureaplasma urealyticum is the most dangerous for male reproductive function [6]. Our studies revealed the highest percentage of this pathogen among other microorganisms, in men with NOA - 12.3%, and in OA -
11.7%. This is the most common microorganism that infects the male reproductive system. This pathogen changes various characteristics of the process of sper-matogenesis, as well as spermatogenic epithelium and sperm. Also affects the concentrations of cytokines such as IL-6, IL-8, TNF-a, INF-y.
Conclusion. So, the results obtained indicate that infections of the male urogenital tract and infection of seminal fluid or prostate juice are important factors, causing the development of NOA and OA.
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