Discussion
Of the 16 standards toxic PAH, 13 were detected in semen. Impact of the different PAHs on fertility are Anthracene < benzo (a) pyrene < benzo [b] fluoranthene < Fluoranthene < benzo (a) anthracene <indol (123CD) pyrene < pyrene < naphthalene < dibenzo (AH) anthracene < fluorene < 2bromonaphthalene < chrysene < benzo (GH1) perylene as revealed by ROC Curve analysis (AUCROC). Benzo [a] pyrene is invariably present in all infertile patients while naphthalene is present in both groups. Of the total 773 detected proteins (Control: 631 and PAH: 717); 71 were differentially expressed (13 underexpressed, 58 overexpressed) in IMI patients. Enrichment analysis revealed them to be involved in mitochondrial dysfunction and oxidative phosphorylation, DNA damage, Aryl hydrocarbon receptor (AHR) signaling, xenobiotic metabolism and induction of NRF-2 mediated OS response. Increased 4-hydroxynonenal and nitrosylated protein adduct formation, and declined antioxidant defense validates induction of OS. Increased GSH/GSSG ratio in patients may be an adaptive response for PAH metabolism via conjugation as evidenced by over-expression of AHR and Heat shock protein 90 beta (HSP90β) in patients. Seminal PAH concentrations, particularly benzo (a) pyrene can be used as a marker to distinguish IMI from fertile ones with 66.67% sensitivity and 100% specificity (95% confidence interval) along with oxidative protein modification and expression of AHR and HSP90β.
Methods
Seminal PAH exposome was analyzed in 43 fertile donors and 60 IMI patients by HPLC and receiver operating characteristic (ROC) curve was applied to find out the cut-off limits. Spermatozoa proteome was analyzed by label free liquid chromatography mass spectroscopy (LC-MS/MS) followed by molecular pathway analysis using bioinformatic tools. Validation of key proteins' expression and protein oxidative modifications were analyzed by western blot.