Detection of Ureaplasma spp. serovars in genital tract of infertile males

Background The colonization of Ureaplasma species in genital tract is related with male infertility. However, it has been postulated based upon limited study that virulence is related to serotype specificity. The aim of this study was to determine the distribution of Ureaplasma serovars in genital tract of infertile males and analyze their role in male infertility. Methods A total of 358 urethral swabs samples were obtained from infertile males. The culture of Ureaplasma species were performed using a commercially available Mycoplasma IST 2 kit. Serovars were determined by real‐time polymerase chain reaction (real‐time PCR). Results A total of 92 (25.7%) infertile males were positive for Ureaplasma spp; among them, Ureaplasma parvum (UPA) was detected in 73 (79.3%) isolates, and Ureaplasma urealyticum (UUR) was detected in 19 (20.7%) isolates. Serovars 1, 6, or in combination accounted for 63.0% (46/73) of UPA isolates. Serovar 9 (alone and in combination of other serovars) was the most common serovar in UUR (47.4%, 9/19). Multiple serovars were detected in 21 (22.8%) isolates, and serovars 4, 5, 7, and 12 were not detected in any sample. Conclusion The distribution of 14 Ureaplasma serovars in genital tract of infertile males was identified for the first time by real‐time PCR assay. UPA serovars 1 and 6, and UUR serovar 9 are the most common serovars colonization in urogenital tract of infertile males.


Funding information
This study was supported by a grant from Science and technology Bureau of Zhejiang Province (grant number LGD19H080002) and grant from Education Bureau of Zhejiang Province (grant number Y201636005) and grant from Health Bureau of Zhejiang Province (grant number 2016KYB154, 2017KY419).

Background:
The colonization of Ureaplasma species in genital tract is related with male infertility. However, it has been postulated based upon limited study that virulence is related to serotype specificity. The aim of this study was to determine the distribution of Ureaplasma serovars in genital tract of infertile males and analyze their role in male infertility.
Methods: A total of 358 urethral swabs samples were obtained from infertile males.
The culture of Ureaplasma species were performed using a commercially available Mycoplasma IST 2 kit. Serovars were determined by real-time polymerase chain reaction (real-time PCR).

Conclusion:
The distribution of 14 Ureaplasma serovars in genital tract of infertile males was identified for the first time by real-time PCR assay. UPA serovars 1 and 6, and UUR serovar 9 are the most common serovars colonization in urogenital tract of infertile males.
infertility. The aim of this study was to determine the distribution of Ureaplasma serovars in genital tract of infertile males, and we have analyzed the role of Ureaplasma serovars in male infertility by comparing results with other studies.

| MATERIAL S AND ME THODS
A total of 358 males, aged 24-39 (mean31) years, selected from infertile couples attended the fertility clinic at Sir Run Run Shaw hospital (Zhejiang CN) from January 2013 to October 2013 were enrolled to participate in the present study. All patients had no pregnancy after more than one year of unprotected regular intercourse. The infertility caused by female partners and males who were symptomatic for any genital tract infections in the previous two weeks were excluded from the study. Genital sample collection and culture for Ureaplasma spp were performed according to our previous study. 4 The culture of Ureaplasma species was performed by using a commercially available Mycoplasma IST 2 kit (bioMérieux, Marcy L'etoile, France). Genomic DNA was extracted by the proteinase K method as described in our previous study. 10 Briefly, a total of 0.5 mL of Ureaplasma spp. broth culture of each Ureaplasma spp. strain was harvested by centrifugation at 12 000 g for 10 minutes. The cell was resuspended in 50 μL of lysis buffer (10 mmol/L Tris-HCl, pH 8.0; 50 mmol/L KCl; 2.5 mmol/L MgCl 2 ; and 0.5% Tween 20) and proteinase K (10 mg/mL), and incubated at 55°C for 1 hour. Then, the sample was heated at 95°C for 10 minutes and centrifuged at 10 000 g for 1 minute to remove debris. The supernatant was utilized immediately or stored a −80°C for future use.

| D ISCUSS I ON
During the past decades,the role of Ureaplasma spp. in male infertility has been controversial. Some investigators failed to show any significant difference in the fertilizing ability of the sperm with a positive culture or any influence on pregnancy rates or outcome, [13][14][15][16] while others reported that Ureaplasma spp. infection may negatively influence semen quality, for example, concentration, activity, motility and/or morphology. [5][6][7][8][9]17,18 Due to their differential pathogenicity, effort has gone into assignment of isolates into serovars and attempting to correlate specific serovars with specific clinical outcomes, while the ability to differentiate the Ureaplasma spp. serovars in clinical samples presented a great challenge for investigators. is in contrast to other study, which found UUR to be more common among infertile men. 23 As to particular serovar and their relationship with the male infertility has been examined in only a few studies. 24 As shown in Figure 1, we found the most common serovars alone and in combinations with other serovars were UPA serovars 1 and 6, and UUR serovar 9. This is contrary to the previous view that the as one subgroup may contribute to this difference. Compared with serotypes of 169 vaginal swabs from healthy pregnant females in previous study. 25 The results indicated that serovar 1, alone and in combination, was significantly increased in infertile males (40/92, 44%) compared with healthy pregnant females (33/169, 20%); serovar 3, alone and in combination, was significantly decreased in infertile males (26% vs 51%); but serovar 6 and 9 showed no significant difference. However, whether the difference is caused by regional factors or pathogenicity differences still needs to be further verified.
In the present study, multiple serovars were detected in 21 (22.8%) isolates, while 21.1% isolates could not be assigned to any serovar in UUR. To determine whether those untypeable isolates represent new serovars or loss of markers needs further investigation.
In conclusion, the distribution of 14 Ureaplasma serovars in genital tract of infertile males was described for the first time in China.
UPA serovars 1 and 6, and UUR serovar 9 are the most common serovars identified in urogenital tract of infertile males.

I N FO R M E D CO N S E NT
All recruited patients provided written informed consent prior to data collection.

E TH I C A L A PPROVA L
All procedures were evaluated and approved by the Institutional Ethics Committee of Sir Run Run Shaw Hospital School of Medicine, Zhejiang University.