Jasmine Nayak

Objectives: Widely used organophosphorus pesticides, methyl parathion (MePa), alter the reproductive functions in various animals and humans by induction of oxidative stress on augmented release of reactive oxygen species (ROS). MePa affects semen quality by inducing DNA damage through spermatogenic stages. Several heat shock proteins (HSPs) are expressed in response to environmental stressors particularly the redox-active ones for regulation of protein turnover. Since oxidative stress and sperm motility are implicated in MePa toxicity, studying the expression of HSP90b will unravel the mechanism behind its noxiousness. Material and Methods: Spermatozoa isolated from healthy donors were subjected to various concentrations of MePa (50, 250, 500, and 750 μM) for studying its effect on sperm motility, ROS generation, sperm chromatin integrity, and expression of stress responsive molecular chaperone HSP90b. Results: In vitro exposure of MePa at concentrations ≥500 μM results in a decline in sperm motility and an increased generation of ROS, DNA damage, and HSP90b expression. Conclusion: ROS-mediated modulation of HSP90b expression may affect the structural integrity of client proteins and oxidative injury to membrane lipid, along with DNA integrity resulting in declined sperm motility in response to MePa.

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.

Recent evidence entail paternal factors as plausible contributors in spontaneous recurrent pregnancy loss (RPL). Seminal extracellular vesicles secreted from cells of male reproductive tract carry regulatory proteins and RNAs. They are proposed to regulate sperm maturation and function while their fusion to endometrial stromal cells helps in decidualization. Nevertheless, the mechanism(s) involved in these processes are poorly understood. This study aims at elucidating the molecular basis of paternal contribution by comparative proteomics (label‐free LC‐MS/MS) of isolated seminal extracellular vesicles from fertile men and partners of patients with RPL (n = 21 per group). Bioinformatics analysis revealed the identified differentially expressed proteins to be involved in DNA replication, recombination and repair, gene expression, cellular assembly and organization, cell death, and survival. Major disease pathways affected were identified as developmental, hereditary, and immunological disorders. Of the three identified hub genes regulating the above disease pathways, two (HNRNPC and HNRNPU) are overexpressed while RUVBL1 is underexpressed along with over expression of HIST1H1C, DDX1, surmising defective chromatin packaging, and histone removal in spermatozoa resulting in improper expression in paternal genes thereby leading to abnormal embryo development. Besides, alteration in GSTP1 expression points oxidative predominance in RPL group. Differential expression of C3, C4a/C4b, CFB, and GDF 15 may be involved in altered maternal immune response to paternal antigens resulting in impaired decidualization.

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.

Recurrent pregnancy loss (RPL) is a perplexing problem experienced with two or more consecutive unexplained miscarriages wherein the cause remains unexplained in >50% of cases. However, despite several evidences of involvement of paternal factors on early embryogenesis and placental development, its contribution towards RPL has been largely unexplored. There is augmented lipid peroxidation, protein carbonylation, thionylation and enhanced histone retention in spermatozoa of RPL patients. Differentially expressed proteins in the spermatozoa of RPL patients may contribute towards aberrant embryo development and pregnancy loss. The present study comprised of male partners of RPL patients (n = 16) with the absence of any female factor abnormality and age‐matched fertile healthy donors (n = 20). Pooled sperm samples from each group were subjected to high‐throughput liquid chromatography–tandem mass spectrophotometry (LC‐MS/MS) and subsequent bioinformatic analysis that identifies key proteins to be differentially expressed (DEPs). A total of 23 DEPs were identified with ≥2.0 fold change were considered to be significant. A key finding of the study was clusterin (CLUS), a predominant oxidative stress protein that takes part in an array of pre‐ and post‐fertilisation molecular processes, found to be underexpressed as it was confirmed by Western blot analysis. This pilot study supports contributions of paternal oxidative predominance in RPL and encourages further investigation.