Separating the chaff from the wheat: antibody-based removal of DNA-fragmented sperm.

STUDY QUESTION: Is it possible to remove sperm with damaged DNA from a semen sample? SUMMARY ANSWER: By using immunomagnetic cell sorting that targets the sperm head-bound epididymal sperm-binding protein 1 (ELSPBP1), it was possible to produce an ELSPBP1(-) sperm fraction characterized by consistently lower levels of sperm DNA fragmentation (SDF). WHAT IS KNOWN ALREADY: In bovines, ELSPBP1 is bound to dead spermatozoa. Human ejaculates with high SDF have increased detected levels of sperm ELSPBP1 when compared to ejaculates with low native SDF. STUDY DESIGN, SIZE, DURATION: We recruited 267 patients who were referred to the clinic for conjugal infertility. After applying exclusion criteria, such as fever within 90 days of the study, history of systemic diseases, alterations or surgical interventions to the genital tract and use of cigarette or drugs, a total of 133 patients were included. A total of 52 samples were used for the evaluation of sperm ELSPBP1 levels (Sub-study 1), 41 samples for determination of ELSPBP1 location in human sperm (Sub-study 2), and 40 samples for immunomagnetic cell sorting targeting ELSPBP1, to produce ELSPBP1(-) (without ELSPBP1) and ELSPBP1(+) (with ELSPBP1) fractions (Sub-study 3). Samples were collected between July 2016 and September 2019. PARTICIPANTS/MATERIALS, SETTING, METHODS: In Sub-study 1, sperm ELSPBP1 levels were assessed by western blotting. For Sub-study 2, ELSPBP1 was localized in sperm by immunocytochemistry. Finally, for Sub-study 3, sperm were selected based on incubation of semen samples with antibody-coated magnetic microspheres targeting ELSPBP1. Two fractions were produced (with or without ELSPBP1), and these sub-populations were submitted to an alkaline Comet assay for determination of SDF. MAIN RESULTS AND THE ROLE OF CHANCE: Men with high SDF presented higher sperm ELSPBP1 levels when compared to the control group (low SDF), while no difference between groups was observed in seminal plasma. ELSPBP1 was located in the head region of human sperm. The ELSPBP1(+) fractions presented high and variable levels of SDF, while their paired ELSPBP(-) fractions presented consistently low SDF. LIMITATIONS, REASONS FOR CAUTION: This work did not validate the levels of ELSPBP1 in other functional alterations of sperm, such as acrosome integrity or mitochondrial activity. Moreover, this is still a pre-clinical study, intended to demonstrate proof-of-concept that ELSPBP1 selects sperm with low DNA fragmentation; further investigation is warranted to demonstrate safety for use in ART. Sperm fractions were not assessed for sperm vitality. A clinical trial is still necessary for these findings to be extrapolated to outcomes in ART. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that ELSPBP1 is associated with sperm with higher levels of DNA fragmentation. The finding that the sperm membrane can reflect alterations in DNA integrity could give rise to a novel molecular method for sperm preparation prior to use of assisted reproductive procedures. Moreover, the detection of sperm-bound ELSPBP1 could serve as an indirect method for the determination of DNA fragmentation. STUDY FUNDING/COMPETING INTEREST(S): L.B.B. was a recipient of a Ph.D. scholarship from the Sao Paulo Research Foundation-FAPESP (process number 2016/05487-3). R.P.B. is a recipient of a Scientific Productivity scholarship from the Brazilian National Council for Scientific and Technological Development-CNPq (process number 306705/2017-6). The authors have no conflict of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.

Towards the identification of reliable sperm biomarkers for male infertility: A sperm proteomic approach.

Male infertility evaluation is mainly based on semen analysis. Thus, identification of additional diagnostic methods is valuable. The aim of this study was to analyse the sperm proteome of infertile men to identify the underlying mechanisms and reliable diagnostic biomarkers. This cross-sectional study consisted of 16 infertile men and seven proven fertile men. An LC-MS/MS approach was performed in five pooled samples of each group (proven fertile men, primary infertility and secondary infertility). Differentially expressed proteins were used for functional enrichment analyses, and the most central proteins involved in altered functions in both infertile groups and the testis-specific proteins were validated using Western blotting and immunocytochemistry. In total, 1,305 sperm proteins were identified, of which 102 were underexpressed and 15 were overexpressed proteins in both infertile groups. Underexpressed proteins were mostly related to protein post-translational modification and folding, especially BAG6, HSPA2 and SPA17. Validation analysis revealed an underexpression of BAG6 in infertile men, whereas HSPA2 and SPA17 expressions did not differ between the groups. No differences were observed in the sperm localisation of these proteins. An overexpression of HIST1H2BA-a testis-specific protein-was observed in both proteomic approaches. Therefore, BAG6 and HIST1H2BA are potential candidates for male infertility biomarkers.

Cysteine-rich secretory protein 3: inflammation role in adult varicocoele.

BACKGROUND: Cysteine-rich secretory protein (CRISP-3), a protein involved in inflammatory response, is highly increased in seminal plasma of adolescents with varicocoele and altered semen analysis, but not in adolescents with varicocoele and normal semen. It is not known, however, whether this increased seminal concentration occurs as an acute marker during the initial stages of varicocoele or whether this persists as an altered protein pathway. OBJECTIVE: The purpose of this study, thus, was to test the hypothesis that this inflammatory state persists through adulthood and the correction of varicocoele could correct this state, by identifying the levels of CRISP-3 in seminal plasma. MATERIALS AND METHODS: This study was carried out in two substudies: (i) to verify the effect of varicocoele and (ii) to verify the effect of varicocelectomy on seminal plasma CRISP-3 levels. Seminal plasma CRISP-3 levels (29 and 31 kDa isoforms) were assessed for each provided sample using standard Western blotting. RESULTS: The varicocoele group presented higher seminal levels of CRISP-3 when compared to controls, with a 67.5-fold increase in the unglycosylated isoform (29 kDa) and a 5.2-fold increase in the glycosylated isoform (31 kDa). In contrast, CRISP-3 levels decreased following varicocelectomy, both in the unglycosylated (5.6-fold decrease) and in the glycosylated (4.3-fold decrease) isoforms. DISCUSSION: CRISP-3, a protein involved in inflammation, is increased in seminal plasma of men with varicocoele and this is partially reversed by varicocelectomy. Monitoring its seminal levels may be useful for assessing inflammation-related alterations to fertility in men with varicocoele. CONCLUSION: We conclude that, in the presence of varicocoele, there is a marked increase in seminal CRISP-3 levels. Surgical intervention (varicocelectomy) decreases CRISP-3 levels and improves semen quality.

Molecular pathways of varicocele and its repair - A paired labelled shotgun proteomics approach.

Varicocelectomy is associated to improved semen quality and sperm functional quality, but individual response is highly variable. Thus, a prospective study was performed including 25 men who collected a semen sample before and 12 months after subinguinal microsurgical varicocelectomy. Semen analysis, sperm functional analysis, and seminal plasma proteomic analysis was performed before and 12 months after varicocelectomy, and according to improvement or not of semen quality (positive and negative outcome). Varicocelectomy led to an increase in semen volume and sperm count, morphology, and mitochondrial activity. In the pre- vs. post-samples, 698 proteins were quantified - 91 differentially expressed after varicocelectomy. In the positive vs. negative outcome analysis, 647 proteins were identified - 151 differentially expressed in the negative outcome group and 30 differentially expressed in the positive outcome group. Tripeptidyl peptidase-1 offered a predictive value for outcome, with an area under a ROC curve of 84.5%. It seems TPP1 is an outcome predictor for varicocelectomy in adults. More importantly, this study demonstrates that the seminal plasma proteome is different in men with varicocele when compared to post-treatment samples from the same individuals. Understanding and monitoring the molecular mechanisms of semen may further establish therapeutic options for these men. SIGNIFICANCE: Although several large-scale studies have demonstrated varicocele is unequivocally associated to male infertility, these same studies have also demonstrated that varicocele is not a determinant of male infertility. We have yet to answer the question of why don't all men with varicocele present with infertility. Varicocele treatment improves semen quality, but its results are variable, and one cannot know who will and who will not benefit from surgical treatment. Results from this study strongly advance a concept that our previous studies have shown: that men with varicocele present an inflammatory semen profile. We have further demonstrated that men operated for varicocele present a decrease in this inflammatory profile, and that when they do not, semen quality remains unaltered. Trypeptidil peptidase-1, a seminal protein, was 3-fold higher in men with a positive outcome after the procedure, when compared to men with a negative outcome. Therefore, inflammation seems to be a central point to varicocele-derived male infertility.