Infertility has recently become a growing social and economic world problem. Genital mycoplasmas, such as
Mycoplasma hominis and M. genitalium, are most frequently associated with several adverse effects on men’s fertility. The present study aimed to determine the prevalence of M. hominis and M. genitalium in the semen samples in thenortheast of Iran. During thiscross-sectional study from February to May, ۲۰۱۸, ۱۰۰ semen samples were collected from ۱۰۰ infertile men in Mashhad, Khorasan Razavi province, northeast of Iran. The presence of M. hominis and M. genitalium was detected by cultivation, polymerase chain reaction (PCR), and Multiplex PCR assays. The colony of mycoplasma was confirmed by Diene’s stain; moreover, arginine hydrolysis, glucose, and urea utilization were evaluated. The following semen indices were analyzed according to World Health Organization guidelines for semen analysis: color, volume, appearance, liquefaction, viscosity, concentration, pH, leukocyte concentration, progressive motility, morphological normality, motile sperm concentration, functional sperm concentration, sperm motility index, and functional sperm. The gene of ۱۶SrRNA (GPO۱& MGSO primers) was used as the target gene of the Mycoplasma genus in PCR assay. Multiplex-PCR was performed with a specific primer for conserved regions in the ۱۶SrRNA gene for M. hominis (RNAH۱& RNAH۲ primers) and the ۱۴۰-kDa Adhesion Protein Gene for M. genitalium (MG۱ & MG۲ primers).According to the results,۹ (۹%) samples were PCR-positive for Mycoplasma spp , while there were ۷ (۷%) cases isolated by cultivation. M. hominis was detected in ۸ (۸%) samples by Multiplex PCR, while there was no evidence for M. genitalium. The mean age scores of all infertile and infected men were obtained at ۳۱ and ۳۰ years, respectively. The study could not show any statistical correlation between mycoplasma infection and abnormal semen parameters. The heterogeneity of mycoplasma prevalence in the reports can be ascribed to differences in geographic areas, the sensitivity of the identification method, condition of the group (fertile/infertile), sample size, and operator proficiency. Various results have been reported in numerous studies conducted on the relationship between mycoplasma infection and abnormal semen parameters.