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.

Scintillation light detection in the 6-m drift-length ProtoDUNE Dual Phase liquid argon TPC.

DUNE is a dual-site experiment for long-baseline neutrino oscillation studies, neutrino astrophysics and nucleon decay searches. ProtoDUNE Dual Phase (DP) is a 6  ×  6  ×  6 m 3 liquid argon time-projection-chamber (LArTPC) that recorded cosmic-muon data at the CERN Neutrino Platform in 2019-2020 as a prototype of the DUNE Far Detector. Charged particles propagating through the LArTPC produce ionization and scintillation light. The scintillation light signal in these detectors can provide the trigger for non-beam events. In addition, it adds precise timing capabilities and improves the calorimetry measurements. In ProtoDUNE-DP, scintillation and electroluminescence light produced by cosmic muons in the LArTPC is collected by photomultiplier tubes placed up to 7 m away from the ionizing track. In this paper, the ProtoDUNE-DP photon detection system performance is evaluated with a particular focus on the different wavelength shifters, such as PEN and TPB, and the use of Xe-doped LAr, considering its future use in giant LArTPCs. The scintillation light production and propagation processes are analyzed and a comparison of simulation to data is performed, improving understanding of the liquid argon properties.

The influence of hydration on the architectural rearrangement of normal and neoplastic human breast tissues.

In adult women, the water-content represents between 50% and 70% of the mass in normal breast tissues and this percentage is increased within diseased tissues. Water molecules play an essential role in the structural organization of biological tissues such as breast. Then, in this study, we have investigated the influence of the water molecules on the breast tissue organization and their role on the hierarchical tissue rearrangement promoted by tumor growth. SAXS and WAXS techniques were used to analyze healthy, benign and malignant human breast samples in native and lyophilized conditions. The scattering profiles in SAXS and WAXS regime of each tissue type in both conditions were compared in order to identify the structural transformation in these tissues and verify the water influence on the morphological arrangement of normal and pathological human breast tissues. From SAXS, changes at the axial periodicity of collagen fibrils were revealed. Additionally, when the water content has removed a peak at q = 4.17 nm-1 (that was present only in pathological samples) shifted in opposite directions within benign and malignant lesions. From WAXS, water and fatty acids were identified within native samples. However, after freeze-drying, only the fat component was observed in the scattering profiles. Therefore, when the water molecules were removed from the samples, structural changes associated with pathological progression were visible. From this, insights about their influence over the changes promoted by the tumor growth have been proposed. Finally, the findings of this study have the potential to provide valuable information to the development of new target therapy.

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.

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.

Effect of in vitro vitamin E (alpha-tocopherol) supplementation in human spermatozoon submitted to oxidative stress.

The aim of this study was to evaluate the antioxidant effect of in vitro supplementation with vitamin E in human spermatozoon incubated with an oxidative stress inducer. In this study, semen samples from 30 patients were collected and with one aliquot we performed semen analysis according to WHO. The remaining volume was divided into four aliquots: group C: incubated with BWW medium; group I: incubated with 5 mmol 1-1 hydrogen peroxide; group A: incubated with 40 µmol 1-1 vitamin E; and group AI: incubated with both them. After incubations, sperm functional analyses were performed and included: evaluation of oxidative stress, acrosome integrity, mitochondrial activity and DNA fragmentation. Groups were compared using a Friedman test with Bonferroni post hoc (α = 5%). In this study, we observed that in group I there was a decrease in acrosome integrity and mitochondrial activity, and an increase in DNA fragmentation, when compared to group C. Group AI showed an increase in acrosome integrity and mitochondrial activity when compared with group I. Based on our findings, we conclude that the vitamin E supplementation had a positive effect in protecting human spermatozoon from induced oxidative stress.

Compton scattering spectrum as a source of information of normal and neoplastic breast tissues' composition.

In this work we measured X-ray scatter spectra from normal and neoplastic breast tissues using photon energy of 17.44 keV and a scattering angle of 90°, in order to study the shape (FWHM) of the Compton peaks. The obtained results for FWHM were discussed in terms of composition and histological characteristics of each tissue type. The statistical analysis shows that the distribution of FWHM of normal adipose breast tissue clearly differs from all other investigated tissues. Comparison between experimental values of FWHM and effective atomic number revealed a strong correlation between them, showing that the FWHM values can be used to provide information about elemental composition of the tissues.

Study of electron densities of normal and neoplastic human breast tissues by Compton scattering using synchrotron radiation.

Electron densities of 33 samples of normal (adipose and fibroglangular) and neoplastic (benign and malignant) human breast tissues were determined through Compton scattering data using a monochromatic synchrotron radiation source and an energy dispersive detector. The area of Compton peaks was used to determine the electron densities of the samples. Adipose tissue exhibits the lowest values of electron density whereas malignant tissue the highest. The relationship with their histology was discussed. Comparison with previous results showed differences smaller than 4%.

Response functions of Si(Li), SDD and CdTe detectors for mammographic x-ray spectroscopy.

In this work, the energy response functions of Si(Li), SDD and CdTe detectors were studied in the mammographic energy range through Monte Carlo simulation. The code was modified to take into account carrier transport effects and the finite detector energy resolution. The results obtained show that all detectors exhibit good energy response at low energies. The most important corrections for each detector were discussed, and the corrected mammographic x-ray spectra obtained with each one were compared. Results showed that all detectors provided similar corrected spectra, and, therefore, they could be used to accurate mammographic x-ray spectroscopy. Nevertheless, the SDD is particularly suitable for clinic mammographic x-ray spectroscopy due to the easier correction procedure and portability.

Preliminary study of human breast tissue using synchrotron radiation combining WAXS and SAXS techniques.

Using synchrotron radiation, we combined simultaneously wide angle X-ray scattering (WAXS) and small angle X-ray scattering (SAXS) techniques to obtain the scattering profiles of normal and neoplastic breast tissues samples at the momentum transfer range 6.28 nm(-1)