All semen specimens were collected by masturbation into sterile containers after 2–7 days of sexual abstinence. The semen was tested for C. trachomatis using immunochromatography and cultured for M. urealyticum and M. hominis. The samples were centrifuged at 1600 ×g for 5 min and supernatant seminal plasma was immediately stored at −80°C. Tandem mass tag (TMT) method followed by mass spectrometry analysis was used to compare the relative expression levels of seminal plasma proteins between the PE and control groups.27 (link)28 (link) Seminal plasma samples (six cases for acquired PE and six matched cases for control) were extracted and digested using trypsin. Later, the proteins were labeled with the TMT reagent. Peptide analysis was performed using the LTQ-Orbitrap instrument (Thermo Finnigan, San Jose, CA, USA) connecting to a Nano AUQUITY UPLC system via a nanospray source. Raw files of proteomics data were processed using MaxQuant (version 1.2.2.5). The false discovery rate (FDR) of the identification was estimated by searching against the databases with the reversed protein sequences. The site, peptide, and protein FDR were all set to 0.05. One-way analysis of variance was used to calculate significant differences in abundance among groups.