Purpose To identify the paternal factors responsible for aberrant embryo development leading to loss of foetus in recurrent pregnancy loss (RPL) through proteomic analysis of ejaculated spermatozoa. Materials and Methods: This prospective study consisted of male partners of RPL patients (n=16) experienced with two or more consecutive unexplained miscarriages and with no female factor abnormality as revealed by gynaecologic investigation including karyotyping and age matched fertile healthy volunteers (n-20). All samples were collected during 2013 to 2015 after getting institutional ethical approval and written consent from the participants. Seminal ejaculates were collected by masturbation after 2 to 3 days of sexual abstinence and analyzed according to World Health Organization 5th criteria 2010. Two-dimensional difference gel electrophoresis followed by mass spectrophotometric analysis was used to identify differentially expressed proteins (DEPs). Western blotting was used for validation of the key proteins. Results: The data identified 36 protein spots to be differentially expressed by more than 2-fold change with p<0.05 considered as significant. Matrix-assisted laser desorption/ionization time of flight/mass spectrometry identified GPx4, JIP4, ZN248 to be overexpressed while HSPA2, GSTM5, TF3C1, CC74A was underexpressed in RPL group. Western blot analysis confirmed the differential expression of key redox associated proteins GPx4 and HSPA2 in the RPL group. Functional analysis revealed the involvement of key biological processes that includes spermatogenesis, response to oxidative stress, protein folding and metabolic process. Conclusions: The present study provides a snapshot of the altered protein expression levels consistent with the potential involvement of the sperm chromatin landscape in early embryonic development.

Oxidative stress (OS) is implicated in 80% of idiopathic male infertility (IMI) patients where exposure to redox active environmental toxicants such as polyaromatic hydrocarbons (PAHs) may play. In the present study the seminal exposome of various PAH was analyzed in two separate cohorts including 43 fertile donors and 60 IMI patients by HPLC and receiver operator characteristic curve was applied to find out the cut-off limits. Furthermore, spermatozoa from both the groups were subjected to label free liquid chromatography mass spectroscopy (LC-MS/MS) followed by bioinformatics analysis to elucidate the molecular mechanism(s) involved and the key proteins from the affected pathways were validated by western blot along with oxidative modification of proteins. Of the 16 standard PAH 13 were detected in the semen. Receiver Operator Character (ROC) Curve analysis (AUC _ROC ) revealed the PAHs having most significant effect on fertility are of the following order 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. Benzo[a] pyrene is invariably present in all infertile patients while naphthalene is present in both fertile and IMI group. Of the total 773 detected proteins (Control: 631 and PAH: 717); 71 were differentially expressed (13 underexpressed, 58 overexpressed) in IMI patients resulting in impaired mitochondrial dysfunction and oxidative phosphorylation, DNA damage, Aryl hydrocarbon receptor (AHR) signalling, xenobiotic metabolism and induction of NRF-2 mediated oxidative stress response (increased in 4-hydroxynonenal and nitrosylated protein adduct formation, and declined antioxidant defence). The increased GSH/GSSG ratio in patients may be an adaptive response to metabolize the xenobiotics via conjugation as evidenced by overexpression of AHR and Heat shock protein 90 beta (HSP90β) in patients. Seminal PAH concentrations, oxidative protein modification along with protein markers (e.g. AHR and HSP90β) may help in better prediction and management of IMI. Contribution of environment borne PAH in semen should not undermined in infertility evaluation.