Rapid detection and simultaneous identification of the Mycoplasma and Ureaplasma species by real-time PCR and melt curve analysis among fertile and infertile females

Document Type : Original Article


1 Student, Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Obstetrics and Gynecology, Isfahan University of Medical Sciences, Isfahan, Iran

3 Department of Genetics and Molecular Biology, Isfahan University of Medical Sciences, Isfahan, Iran

4 Department of Microbiology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran


Objective(s): Mycoplasma and Ureaplasma species threaten reproductive health and fertility worldwide. Due to the lack of sensitive, accurate, and affordable diagnostic tools, the simultaneous contributions of these agents in infertility have been overlooked. This study aims to detect and identify Mycoplasma and Ureaplasma species in the genital tracts of fertile and infertile females simultaneously.
Materials and Methods: In a case-control study, cervicovaginal clinical samples were collected from patients referred to two teaching hospitals in Isfahan from July 2019 to February 2019. The initial screening was by using Real-time PCR and designed primer to evaluate the presence of Mycoplasma and Ureaplasma species including fertile and infertile women. The bacteria species were then detected and differentiated by using the melt curve and sequenced to confirm and identify. Finally, the standard curve was used to measure and compare the copy number of each species in each group. The isolates also were detected in clinical samples using the commercial PCR method.
Results: The frequencies of Mycoplasma genitalium and Mycoplasma hominis were (0.0, 10.0%) in the fertile group and (4.3%, 34.3%) in the infertile group, respectively. Ureaplasma parvum and Ureaplasma urealyticum species in the fertile group (7.1%, 5.7%) and in the infertile group (32.9%, 24.3) were determined, respectively. The comparison of the results obtained from PCR and Real-time PCR showed that the recent technique has the ability to track 101–103 copy numbers.
Conclusion: The present method allows differential diagnosis and quantification of Mycoplasma and Ureaplasma species in a short time and simultaneously. 


Main Subjects

1. Kumar N, Singh AK. Trends of male factor infertility, an important cause of infertility: A review of literature. J Hum Reprod Sci. 2015;8:191-196.
2. Vander Borght M, Wyns C. Fertility and infertility: Definition and epidemiology. Clin Biochem. 2018;62:2-10.
3. Ruggeri M, Cannas S, Cubeddu M, Molicotti P, Piras GL, Dessole S, et al. Bacterial agents as a cause of infertility in humans. New Microbiol. 2016;39:206-209.
4. Bai S, Li Y, Hu M-H, Wu L, Shui L-J, Wang X-H, et al. Association of sexually transmitted infection with semen quality in men from couples with primary and secondary infertility. Asian J Androl 2022; 24: 317-322.    
5. Safarkar R, Mehrabadi JF, Noormohammadi Z, Mirnejad R. Development a rapid and accurate multiplex real time PCR method for the detection Chlamydia trachomatis and Mycoplasma hominis. J Clin Lab Anal 2017;31:e22126.
6. Zhu X, Li M, Cao H, Yang X, Zhang C. Epidemiology of Ureaplasma urealyticum and Mycoplasma hominis in the semen of male outpatients with reproductive disorders. Exp Ther Med 2016;12:1165-70.
7. Farahani¹ MT, Hoseini F, Minai-Tehrani A, Novin MG. The effect of infection with genital Mycoplasma hominis and the presence of antisperm antibodies in iranian women with unexplained infertility. Int J Women’s Health Reprod Sci 2016;4:18-22.
8. Waites KB, Xiao L, Paralanov V, Viscardi RM, Glass JI. Mycoplasma and Ureaplasma. Molecular Typing in Bacterial Infections: Springer; 2013. p. 229-281.
9. Seifoleslami M, Safari A, Khameneie MK. Prevalence of Ureaplasma urealyticum and Mycoplasma hominis in high vaginal swab samples of infertile females. Iran Red Crescent Med J 2015;17.
10. Zanotta N, Campisciano G, Morassut S, Castro‐Silva E, Luksa V, Zito G, et al. Emerging role for Ureaplasma parvum serovar 3: Active infection in women with silent high‐risk human papillomavirus and in women with idiopathic infertility. J Cell Physiol 2019;234:17905-11791.
11. Rekha S, Nooren M, Kalyan S, Mohan M, Bharti M, Monika R, et al. Occurrence of Mycoplasma genitalium in the peritoneal fluid of fertile and infertile women with detailed analysis among infertile women. Microb Pathog 2019;129:183-186.
12. Yoshida T, Maeda S-I, Deguchi T, Ishiko H. Phylogeny-based rapid identification of mycoplasmas and ureaplasmas from urethritis patients. J Clin Microbiol 2002;40:105-110.
13. Horner P, Donders G, Cusini M, Gomberg M, Jensen J, Unemo M. Should we be testing for urogenital Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum in men and women?–a position statement from the European STI Guidelines Editorial Board. J Eur Acad Dermatol Venereol 2018;32:1845-1851.
14. Moosavian M, Ghadiri A, Amirzadeh S, Rashno M, Afzali M, Ahmadi K. Investigating Chlamydia trachomatis and genital Mycoplasma prevalence and apoptosis markers in infertile and fertile couples. Jundishapur J Microbiol 2019;12:e84954.
15. Paavonen J, Miettinen A, Stevens CE, Chen KC, Holmes KK. Mycoplasma hominis in nonspecific vaginitis. Sex Transm Dis 1983:271-275.
16. Lau A, Bradshaw CS, Lewis D, Fairley CK, Chen MY, Kong FY, et al. The efficacy of azithromycin for the treatment of genital Mycoplasma genitalium: A systematic review and meta-analysis. Clin Infect Dis 2015;61:1389-1399.
17. Strus M, Pilarczyk-Żurek M, Adamski P, Janeczko J, Heczko PB, Tomusiak A. Bacterial infections of the lower genital tract in fertile and infertile women from the southeastern Poland. Ginekol Pol 2013;84:352-358.
18. Clausen HF, Fedder J, Drasbek M, Nielsen PK, Toft B, Ingerslev HJ, et al. Serological investigation of Mycoplasma genitalium in infertile women. Hum Reprod Title 2001;16:1866-1874.
19. Tantengco OAG, de Castro Silva M, Velayo CL. The role of genital mycoplasma infection in female infertility: A systematic review and meta‐analysis. Am J Reprod Immunol 2021;85:e13390.
20. Kornats’ ka A. Local humoral immunity in women with combined forms of infertility. Likars’ ka Sprava. 1998; 4:82-84.
21. Mousavi A, Farhadifar F, Mirnejad R, Ramazanzadeh R. Detection of genital mycoplasmal infections among infertile females by multiplex PCR. Iran J Microbiol 2014;6:398-403.
22. Mahdi Shirzad M. Fertility behaviour of Iranian women: A community-based, cross-sectional study. Arch Iran Med 2015;18:2.
23. Maleki S, Motamedi H, Moosavian SM, Shahbaziyan N. Frequency of Mycoplasma hominis and Ureaplasma urealyticum in females with urogenital infections and habitual abortion history in Ahvaz, Iran; using multiplex PCR. Jundishapur J Microbiol 2013;6:1M.
24. Naserpour Farivar T, Aslanimehr M, Peymani A, Dabaghi Ghaleh T, Jahanihashemi H, Saadat S. Prevalence of Ureaplasma urealyticum in endocervical specimens of female patients in Qazvin, Iran. Biotech Health Sci 2016; 3:e39599.
25. Gupta A, Gupta A, Gupta S, Mittal A, Chandra P, Gill AK. Correlation of mycoplasma with unexplained infertility. Arch Gynecol Obstet 2009;280:981-985.
26. Mbah CE, Jasani A, Aaron KJ, Akoachere J-F, Tita AT, Geisler WM, et al. Association between Chlamydia trachomatis, Neisseria gonorrhea, Mycoplasma genitalium, and Trichomonas vaginalis and secondary infertility in cameroon: A case-control study. PLoS One 2022;17: e0263186.
27. Rajkumari N, Kaur H, Roy A, Gupta N, Dhaliwal LK, Sethi S. Association of Mycoplasma genitalium with infertility in North Indian women. Indian J Sex Transm Dis 2015;36:144-148.
28. Contini C, Rotondo JC, Magagnoli F, Maritati M, Seraceni S, Graziano A, et al. Investigation on silent bacterial infections in specimens from pregnant women affected by spontaneous miscarriage. J Cell Physiol 2019;234:100-107.
29. Huang Y, Pan H, Xu X, Lv P, Wang X, Zhao Z. Droplet digital PCR (ddPCR) for the detection and quantification of Ureaplasma spp. BMC Infectious Diseases. 2021;21:1-9.
30. Cao X, Wang Y, Hu X, Qing H, Wang H. Real-time TaqMan polymerase chain reaction assays for quantitative detection and differentiation of Ureaplasma urealyticum and Ureaplasma parvum.  Diagn Microbiol Infect Dis 2007;57:373-378.