Background: Infections are one of the correctable causes of infertility with low cost and cost effective treatment. The 50% of infertile cases is related to men in some way, and 30% of them are absolutely related to them. Mycoplasmas are the smallest microorganisms with capability of DNA replication. Present study is planned to compare the mycoplasma infection in infertile men and men with established fertility.Materials and Methods: 45 Semen samples were collected from case and control persons who reffered to Royan Infertility and Fertility Institute between 2004 and 2005 and stored in -20°C until time of test. DNA was extracted from semen using phenol chloroform. PCR reaction was done by mycoplasma specific primers.Results: Mycoplasma genitalium gene was amplified in 6 (40%) cases from 15 infertile semen samples and 11 (36.6%) from 30 control semen samples.Conclusion: Probability of genital infection, at least, in these studies group, is very lower than other communities' reports

Bandehpour, Mojgan

Hosseiny, Seyed Jalil

Kazemi, Bahram

Khorramizadeh, Mohammad Reza

Safavifar, Farnoosh

Shahverdi, Abdolhossein

English

Journals Portal, Shahid Beheshti University of Medical Sciences

  Novelty in Biomedicine NBM 25   Novelty in Biomedicine 2015, 1, 25-9 Original Article   Mycoplasma Infection in Pyospermic Infertile and Healthy Fertile Men   Farnoosh Safavifar1, Mojgan Bandehpour1,2, Seyed Jalil Hosseiny3, Mohammad Reza Khorramizadeh4, Abdolhossein Shahverdi5, Bahram Kazemi1,2*  1 Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2 Biotechnology Department, Shahid Beheshti University of Medical Sciences, Tehran, Iran 3 Urology Department, Shohada Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran  4 Pathobiology Department, School of Public Health, Tehran Medical University, Tehran, Iran 5 Royan Institute, Tehran, Iran Abstract Background: Infections are one of the correctable causes of infertility with low cost and cost effective treatment. The 50% of infertile cases is related to men in some way, and 30% of them are absolutely related to them. Mycoplasmas are the smallest microorganisms with capability of DNA replication. Present study is planned to compare the mycoplasma infection in infertile men and men with established fertility. Materials and Methods: 45  Semen samples were collected from case and control persons who reffered to Royan Infertility and Fertility Institute between 2004 and 2005 and stored in -20°C until time of test. DNA was extracted from semen using phenol chloroform. PCR reaction was done by mycoplasma specific primers. Results: Mycoplasma genitalium gene was amplified in 6 (40%) cases from 15 infertile semen samples and 11 (36.6%) from 30 control semen samples. Conclusion: Probability of genital infection, at least, in these studies group, is very lower than other communities' reports. Keywords: Mycoplasma, pyospermic infertile men, fertile men  *Corresponding Author: Bahram Kazemi. Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran. Tel:  +98-21-22439957, Fax: +98-21-22439956; Email: bahram_14@yahoo.com or kazemi@sbmu.ac.ir  Please cite this article as: Safavifar F, Bandehpour M, Hosseiny SJ, Khorramizadeh MR, Shahverdi A, Kazemi B. Mycoplasma Infection in Pyospermic Infertile and Healthy Fertile Men. Novel Biomed. 2015;3(1):25-9.    Introduction As well as attempts to control population and development of contraceptive methods by the WHO in the past decades, it is essential to discover and dissolve the infertility problems. Today, 13-18% of couples in the productive age are infertile all over the world1. Thus, mental health’s of these persons, which consist of considerable percentage of population, are at risk. Also infertility and in correct non-scientific and non-realistic encounter to it, resulted in destruction of families as the community health columns. Solutions for infertility problems not only associated with many risks, but also with high costs2 and are not available for all levels and regions in the world, finally success percentage of these methods are not complete. Thus, attempts to find low cost methods are explained. Infections are one of the correctable causes of infertility with low cost and cost effective treatment. The 50% of infertile cases is related to men in some way, and 30% of them are absolutely related to them1. Safavifar et al.                                                        Mycoplasma Infection in Pyospermic Infertile and Healthy Fertile Men NBM                                                                            26                                   Novelty in Biomedicine 2015, 1, 25-9 The 50% of infertile men are curable. So, researcher’s concern are discover of infertility etiology in men, specially discover of correctable causes3-5.  Gram negative infections can results in occlusion of epididymis and cause to sperm motility impairments6. Also, Chlamydia trachomatis and mycoplasma infections are involved in infertility and many researches have done to establish the role of these micro organisms3- 5. Mycoplasmas are the smallest microorganisms with capability of DNA replication. These organisms should enter to the host cells because they don’t have cell wall and their genome is very small so need some host enzymes to replication. In the other word, they are intracellular parasites. Their slow growing on media results in difficult replication in laboratory condition7. Previous methods for detection of mycoplasma, which are involved in infertility, were: serological and microbe culture methods have no sufficient specificity and sensitivity. The PCR is the gold standard method for diagnosis of mycoplasma with high speed and accuracy.  Present study is planned to compare the mycoplasma infection in infertile men and men with established fertility. Methods This case control study was performed on Royan Infertility and Fertility Institute on 45 samples, in Tehran capital city of Iran, between 2004 To 2005. Semen samples were collected from 15 cases and 30 control persons and stored in -20oC until time of test.  Case group: This group consists of 15 men who were referred by physician to the Royan Infertility and Fertility Institute with complaint of infertility. Diagnosis process was performed in this method: first the women visited by the gynecological specialist and then with an administration of spermogram from their husbands, they were referred to the urologist who was record the identification information, history, physical exam and fertility laboratory tests results in the man’s sheet. Characteristics of this group were, refer to Royan Institute with complaint of infertility, existence of pyospermia and rule out of other causes of infertility in the couples in the primary investigations. Control group: This group was selected among those men that: first fertility was established in them and no problem was seen in their semen analysis.  Meaning full difference between case and control groups can suggest the role of mycoplasma in infertility. Thus, control group were selected among men referring to Shahid Jafari Health Center in order to family management and after consultation, in the primary steps, candidate for vasectomy. Because of positive history of fertility in these men, their semen samples should be tested three times. This group was matched with the case group in regard of age.  DNA extraction and PCR amplification: DNA was extracted from semen using phenol chloroform as previously described8. PCR reaction was done by Mycoplasma specific primers.  PCR reaction was done using: primers MgeniF  5'-CCT   TAT   CGT   TAG   TTA   CAT   TGT   TTA  A –3  and  MgeniR 5`- TGA   CAT   GCG    CTT    CCA    ATA   AA  -3'  witch were amplified 340 bp of Mycoplasma genitalium genome. PCR reaction was included 2 micro liters of DNA (containing 100 ng), 150 µM dNTP, and 40 Picomoles each of forward and reverse primers, 1.5 mM MgCI2, 1 X PCR buffer and 1.25 units of Taq DNA polymerase in 30 µL final volume. PCR amplification was performed by following parameters: denaturing at 940C for 30 second, annealing at 62oC for 30 second and extension at 72oC for 30 second. These processes were repeated for 30 cycles. Reaction was settled at 94oC and 72oC for 5 minutes before and after PCR cycling, respectively.   Detection of PCR product: PCR product was electrophoresed on 2% agarose gel and DNA band was observed by UV Transilluminator after ethidium bromide staining. Results Mycoplasma genitalium gene was amplified in 6 (40%) cases from 15 infertile semen samples and 11 (36.6%) from 30 controls semen samples (Table 1). From these data, it’s suggested that no considerable statistically significant difference between these two groups. For above table, odds ratio can be:  Odds ratio = 15.1119196. Mycoplasma Infection in Pyospermic Infertile and Healthy Fertile Men                                                     Safavifar et al. NBM 27 Novelty in Biomedicine 2015, 1, 25-9 Discussion Mycoplasma is the smallest microorganisms with capability of DNA replication. These organisms should enter to the host cell because they don’t have cell wall and their genome is very small (obligate intra cellular parasite). Their growth in culture media is very slow for laboratory detection and they needed a reliable diagnostic method in routine diagnostic laboratory7. The rates of Mycoplasma species infection in men with non-gonococci urethritis witch detected by culture in case and control groups were 19.2% and 7.2% for Ureaplasma urealyticum, 7.2% and 0.8% for Mycoplasma genitalium, and 2.4% and 1.6% for Mycoplasma hominis respectively9. The rate of infection assayed by indirect hemagglutination in China for immoral persons was 25.47% for Ureaplasma urealyticum, 18.22% for Chlamydia trachomatis and 8.80% for Mycoplasma hominis that is higher than of infection in healthy controls10.  The prevalence of infection in patients with non-gonococcal urethritis in Japan was 47.7% for Chlamydia trachomatis, 17.0% for Mycoplasma genitalium, 16.3% for Ureaplasma urealyticum (biovar 2), 7.8% for Ureaplasma parvum (biovar 1) and 2.6% for Mycoplasma hominis11. Mycoplasma genitalium infection was reported at 5.3% in men with symptomatic urethritis in Bristol12 and 13.8% 13 and 6.3% 14 for Swedish women and 4.1%13 and 6%14 for Swedish men. Chlamydia trachomatis was reported at 4.7%13 and 4.5%14 for Swedish women and 9.1%13 and 5.4%14 for Swedish men. If we concern that prevalence of disease caused by Mycoplasma species in our country is the same as those witch other researches were done, it should be expected that in infertile men with positive test of Mycoplasma, 42% are infected with mycoplasma genus and 21% of control group are infected. Correlation between pyospermia and infertility: Because of selection of case group from infertile men with pyospermia, attention to the correlation of pyospermia and infection in urogenital tract and relation of them with infertility seems to be essential. There are different reports from prevalence of pyospermia in infertile men. The prevalence of Mycoplasma hominis and Ureaplasma urealyticum were reported as 7% and 20% respectively in patients with sterile pyuria in Austria15. Its prevalence is reported from 6 to 23 percentages16. Important concern in diagnosis of pyospermia is that immature germ cells in simple staining are resembled to leukocytes, in the form that, they called “Round cell” in overall. 1:3 of patients with “Round cell” in their semen, were involved in pyospermia, and in others, just germ cell was seen. The most suitable differential test is peroxide test that is used in laboratory for report of results17,18. Although both of infection and infertility suggested to be attributed to pyospermia, one should attend that many of pyospermia patients had no infection in their genital tract. Other case-control studies showed that there were not considerable differences between fertile and infertile couples exist. This can be attributed with direct or indirect existence of WBC in semen samples. Laboratory results of case group showed that 60% of them had other various seminal disorders except pyospermia19. Table 1: Results of mycoplasma genitalium PCR reaction for infertile and healthy men.         Result Sample type Positive by PCR Negative by PCR  Frequency  Percent  Frequency  Percent  Total Infertile (case) 6 40 9 60 15 Fertile  (control) 11 36.6 19 63.4 30 Total 17  28  45  Safavifar et al.                                                        Mycoplasma Infection in Pyospermic Infertile and Healthy Fertile Men NBM                                                                            28                                   Novelty in Biomedicine 2015, 1, 25-9 Impact of sexual behavior on prevalence of genital Mycoplasma: Prevalence pattern of genital infections in a community is greatly contributed to sexual behavior20 and genital Mycoplasma are not exceptions in this regard. Multiple partners, especially with someone who are multi partner themselves, resulted in grant spread of genital infections that is called “ping pong” phenomenon. Because of differences between comminute in regard of believes, demography and population that are contributed to sexual behaviors, we cannot consider the prevalence of genital infection of other country as the same as ours, in the other hand, both case and control group, using in this study, are not good statistic population to reflect the sexual behavior of our community.  Control group has the fertile men who referring to health center in order to vasectomy, and all of them are married that decided to this, due to responsibility to their families' condition. From the referral cases of Royan institution, number of cases with more than one partner was rare and no cases with sexual behavior beyond ethical frame exist. Relationship between immunity and pathogenesis of Mycoplasma:  One of the special characteristic of Mycoplasma is the interaction of one’s immunity and its pathogenesis21,22. Mycoplasma pneumonia was first extracted from HIV positive patients23-25. Mycoplasma pneumonia is important etiology of pneumonia in 5-15 ages and with the maturation of immunity system, its pathogenesis reduced. So, pathogenesis of Mycoplasma and its other pathogen species is contributed to the immunity quality. Immunity quality, itself, is contributed genetic and innate characteristics, environmental pollutions, underlying disease and even emotional stress21,22. Therefore, in interpretation of Mycoplasma prevalence in infertile patients, the quality of immunity should be considered. One solution to remove this configuring factor, is matching of case and control groups in regard of immunity level in the future study which should be supported with some immunologic assay for determining immunity quality and measuring tests. Conclusion From all of above, we can conclude that probability of genital infection, at least, in these studies group, is very lower than other communities' reports. (If cannot absolutely considered for all community of Iran). Acknowledgments This study was supported by the Vice Chancellor for Research of Shahid Beheshti University of Medical Sciences and was done in Cellular and Molecular Biology Research Center We appreciates the expertise and assistance of members. References 1. www.IVF.com/male.html 2. Blandford JM, Gift TL. Productivity losses attributable to untreated chlamydial infection and associated pelvic inflammatory disease in reproductive-aged women.  Sex Transm Dis.  2006;33(10 Suppl):117-21. 3. Paavonen  J,  Eggert- Kruse W. Chlamydia trachomatis: impact on human reproduction. Hum reprod Update. 1999; 5(5): 433-47. 4. Yoshida K, Kobayashi N,  Negishi T. Chlamydia trachomatis infection in the semen of asymptomatic infertile men: detection of the antigen by in situ hybridization. Urol Int.  1994;53(4):217-21. 5. Ochsendorf FR, Ozdemir K, Rabenau H, Fenner T, Oremek R, Milbradt R, et al. Chlamydia trachomatis and male infertility: chlamydia-IgA antibodies in seminal plasma are C. trachomatis specific and associated with an inflammatory response. J Eur Acad Dermatol Venereol. 1999;12(2):143-52. 6. Gonzalez-jimenez MA, Villanueva-Diaz CA. Epididymal stereocilia in semen of infertile men: evidence of chronic epididymitis?  Andrologia.  2006;38(1):26-30. 7. Blattner RJ. Mycoplasma infection. J Pediatr.  1968;72(4):554-6. 8. Köchl S, Niederstätter H, Parson W. DNA extraction and quantitation of forensic samples using the phenol-chloroform method and real-time PCR. Methods Mol Biol.  2005;297:13-30. 9. Salari MH, Karimi A. Prevalence of ureaplasma urealyticum and mycoplasma genitalium in men with non-gonococcal urethritis. East Mediterr Health.  2003;9(3):291-5.  10. Zhao J, Xu C, Wang N. Epidemiological study on the infections of Mycoplasma and Chlamydia in immoral persons and healthy controls in seven areas of China Zhonghua Liu Xing Bing Hue Za Zhi.  1994;15(5): 263-6. 11. Maeda S, Deguchi T, Ishiko H, Matsumoto T, Naito S, Kumon H, et al. Detection of mycoplasma genitalium, mycoplasma hominis, ureaplasma parvum (biovar 1) and ureaplasma urealyticum (biovar 2) in patients with non-gonococcal urethritis using polymerase chain reaction-microtiter plate hybridization. Int J Urol.  2004;11(9):750-4. 12. Leung A, Fastick K Haddon LE, Horn CK, Ahuja D,  Horner PJ. Mycoplasma genitalium is associated with symptomatic urethritis. Int J STD.  2006;17(5):285-8. 13. Mellenius H, Boman J, Lundgvist EN, Jensen JS. Mycoplasma genitalium should be suspected in unspecific urethritis and cervicitis. A study from Vasterbotten confirms the high prevalence of the bacteria. Lakartidningen. 2005;102(47):3538, 3540-1. Mycoplasma Infection in Pyospermic Infertile and Healthy Fertile Men                                                     Safavifar et al. NBM 29 Novelty in Biomedicine 2015, 1, 25-9 14. Anagrius C, Lore B, Jensen JS. Mycoplasma genitalium: prevalence, clinical significance, and transmission. Sex Transm Infect.  2005;81(6):458-62. 15. Daxboeck F, Zitta S, Stadler M, Iro E, Krause R. Mycoplasma hominis and ureaplasma urealyticum in patients with sterile pyuria. J Infect. 2005;51(1):54-8. 16. Jarvi K, Noss MB. Pyospermia and male infertility. Can J Urol. 1994;1(2):25-30. 17. Sigman M, Lopes L. The correlation between round cells and white blood cells in the semen.  J Urol.  1993;149(5 Pt 2):1338-40. 18. Arata de Bellabarba G, Tortolero I, Villarroel V, Molina CZ, Bellabarba C, et al. (2000).  Nonsperm cells in human semen and their relationship with semen parameters. Arch Androl.  2000;45(3):131-6. 19. Campbell M, Patric C. Campbell`s Urology, 8th edition, Philadelphia Saunders. 2002;1047–8. 20. Tsunoe H, Tanaka M, Nakayama H, Sano M, Nakamura G, Shin T, et al.  High prevalence of Chlamydia trachomatis, Neisseria gonorrhoeae and Mycoplasma genitalium in female commercial sex workers in Japan. Int J STD AIDS.  2000;12:790-4. 21. Atkinson TP, Balish MF, Waites KB. Epidemiology, clinical manifestations, pathogenesis and laboratory detection of Mycoplasma pneumoniae infections. FEMS Microbiol Rev. 2008;32(6):956-73. 22. Waites KB, Balish MF, Atkinson TP. New insights into the pathogenesis and detection of Mycoplasma pneumonia Infections. Future Microbiol.  2008;3(6):635–48.  23. Miller-Catchpole R, Shattuck M, Kandalaft P, Variakojis D, Anastasi J, Abrahams C. The incidence and distribution of Mycoplasma fermentans (incognitus strain) in the Chicago AIDS autopsy series: an immunohistochemical study. Chicago Associated Pathologists. Mod Pathol.  1991;4(4):481-6. 24. Watson ME Jr, Storch GA. Recurrent Mycoplasma pneumoniae infection in a human immunodeficiency virus-positive child. Pediatr Infect Dis J.  2008;27(11):1037-8. 25. Shankar EM, Kumarasamy N, Vignesh R, Balakrishnan P, Solomon SS, Murugavel KG, et al.  Epidemiological studies on pulmonary pathogens in HIV-positive and -negative subjects with or without community-acquired pneumonia with special emphasis on Mycoplasma pneumoniae. Jpn J Infect Dis.  2007;60(6):337-41. 

Mycoplasma Infection in Pyospermic Infertile and Healthy Fertile Men

Background: Infections are one of the correctable causes of infertility with low cost and cost effective treatment. The 50% of infertile cases is related to men in some way, and 30% of them are absolutely related to them. Mycoplasmas are the smallest microorganisms with capability of DNA replication. Present study is planned to compare the mycoplasma infection in infertile men and men with established fertility.Materials and Methods: 45 Semen samples were collected from case and control persons who reffered to Royan Infertility and Fertility Institute between 2004 and 2005 and stored in -20°C until time of test. DNA was extracted from semen using phenol chloroform. PCR reaction was done by mycoplasma specific primers.Results: Mycoplasma genitalium gene was amplified in 6 (40%) cases from 15 infertile semen samples and 11 (36.6%) from 30 control semen samples.Conclusion: Probability of genital infection, at least, in these studies group, is very lower than other communities' reports.</p

Farnoosh Safavifar

Mojgan Bandehpour

Seyed Jalil Hosseiny

Mohammad Reza Khorramizadeh

Abdolhossein Shahverdi

Bahram Kazemi

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Background: Infections are one of the correctable causes of infertility with low cost and cost effective treatment. The 50 % of infertile cases is related to men in some way, and 30 % of them are absolutely related to them. Mycoplasmas are the smallest microorganisms with capability of DNA replication. Present study is planned to compare the mycoplasma infection in infertile men and men with established fertility. Materials and Methods: 45 Semen samples were collected from case and control persons who reffered to Royan Infertility and Fertility Institute between 2004 and 2005 and stored in-20°C until time of test. DNA was extracted from semen using phenol chloroform. PCR reaction was done by mycoplasma specific primers. Results: Mycoplasma genitalium gene was amplified in 6 (40%) cases from 15 infertile semen samples and 11 (36.6%) from 30 control semen samples. Conclusion: Probability of genital infection, at least, in these studies group, is very lower than other communities &apos; reports

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Mycoplasma Infection in Pyospermic Infertile and Healthy Fertile Men

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