Human sperm proteome reveals the effect of environmental borne seminal polyaromatic hydrocarbons exposome in etiology of idiopathic male factor infertility.

Introduction: Polyaromatic hydrocarbons (PAHs) are considered as redox active environmental toxicants inducing oxidative stress (OS) mediated injury to cells. Oxidative predominance is reported in 30%-80% of idiopathic male infertility (IMI) patients. Hence, this work aims to unravel correlation, if any, between seminal PAH exposome and sperm function in IMI patients through a proteomic approach. 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. Results and 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

Comparative proteome profiling of seminal components reveal impaired immune cell signalling as paternal contributors in recurrent pregnancy loss patients.

PROBLEM: Recurrent pregnancy loss (RPL) is usually evaluated from a women's perspective, however, recent evidence implies involvement of male factors as paternally expressed genes predominate placenta. During fertilization, prior to implantation the immune system purposefully produces early pregnancy factors with potent immunomodulatory properties for adaptation to antigenically dissimilar embryo. Therefore, it is hypothesized that paternal immunological factors play a role in RPL. METHOD OF STUDY: Comparative proteome profiling (label free liquid chromatography mass spectroscopy: LC-MS/MS) of the seminal extracellular vesicles (SEVs), extracellular vesicle free seminal plasma (EVF-SP) and spermatozoa was carried out in semen of RPL patients (n = 21) and fertile donors (n = 21). This was followed by pathway and protein-protein interaction analysis, and validation of key proteins' expression (western blot). RESULTS: A total of 68, 28 and 49 differentially expressed proteins in SEVs, EVF-SP and spermatozoa of RPL patients, respectively, were found to be involved in inflammatory response, immune cell signalling and apoptosis. In SEVs, underexpressed GDF-15 and overexpressed C3 imply distorted maternal immune response to paternal antigens leading to impaired decidualization. Dysregulated TGFß signalling in EVF-SP surmises defective modulation of inflammatory response and induction of immune tolerance to seminal antigens in the female reproductive tract through generation of regulatory T cells. Retained histone variants in spermatozoa construe defective expression of early paternal genes, while underexpressed PTN may inflict defective angiogenesis resulting in expulsion of decidua. CONCLUSIONS: Impaired modulation of immune response and improper placental development due to altered cytokine levels in seminal components may be the contributing paternal factors in RPL.

Harnessing the potential of dialdehyde alginate-xanthan gum hydrogels as niche bioscaffolds for tissue engineering.

Biomimetic hydrogels composed of natural polysaccharides have invariably blossomed as niche biomaterials in tissue engineering applications. The prospects of creating an extracellular matrix (ECM)-like milieu from such hydrogels has garnered considerable importance. In this study, we have fabricated bioscaffolds comprising dialdehyde alginate and xanthan gum and explored their potential use in tissue regeneration. The fabricated scaffolds displayed an interconnected porous network structure that is highly desirable for the aforesaid application. The scaffolds were endowed with good mechanical properties, thermostability, protein adsorption efficacy and degradability. Curcumin-loaded hydrogels exhibited appreciable antibacterial activity against E. coli. In vitro cytocompatibility studies revealed that the scaffolds promoted adhesion and proliferation of 3T3 fibroblast cells. The Western blot analysis of p53 gene indicated no growth arrest or apoptosis in 3T3 cells thus, signifying the non-toxic nature of the scaffolds. Furthermore, the ECM formation was confirmed via SDS-PAGE analysis. The overall results clearly validated these scaffolds as effectual biomaterials for tissue engineering applications.

Paternal contributors in recurrent pregnancy loss: Cues from comparative proteome profiling of seminal extracellular vesicles.

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.

Proteomic Signatures in Spermatozoa Reveal the Role of Paternal Factors in Recurrent Pregnancy Loss.

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.

Quantitative proteomics decodes clusterin as a critical regulator of paternal factors responsible for impaired compensatory metabolic reprogramming in recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) is a perplexing problem experienced with two or more consecutive 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.