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Pregabalin (PGB) is a γ-aminobutyric acid (GABA) alkylated analog prescribed to treat neuropathic pain, fibromyalgia, and postherpetic neuralgia. Using analytical, spectroscopic methods and molecular docking and molecular dynamics (MD) simulations, a detailed experimental and theoretical investigation was conducted into the binding process and interactions between PGB and double-stranded fish sperm deoxyribonucleic acid (dsDNA). It was evident from the collected experimental results that PGB binds with ds-DNA. PGB attaches to dsDNA via minor groove binding, as demonstrated by the results of electrochemical studies, UV-Vis absorption spectroscopy, and replacement study with ethidium bromide and Hoechst-32588. PGB's binding constant (K b ) with dsDNA, as determined by the Benesi-Hildebrand plot, is 2.41×10 4 ± 0.30 at 298 K. The fluorescence investigation indicates that PGB and dsDNA have a binding stoichiometry (n) of 1.21 ± 0.09. Molecular docking simulations were used in the research to computational determination of the interactions between PGB and dsDNA. The findings demonstrated that minor groove binding was the mechanism by which PGB interacted with dsDNA. Based on the electrochemically responsive PGB-dsDNA biosensor, we developed a technique for low-concentration detection of PGB utilizing differential pulse voltammetry (DPV). The voltammetric analysis of the peak current decrease in the deoxyadenosine oxidation signals resulting from the association between PGB and dsDNA enabled a sensitive estimation of PGB in pH 4.80 acetate buffer. The deoxyguanosine oxidation signals exhibited a linear relationship between 2 and 16 μM PGB. The values for the limit of detection (LOD) and limit of quantitation (LOQ) were 0.57 μM and 1.91 μM, respectively., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Aysegul Golcu reports financial support was provided by Istanbul Technical University., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The main goal of our study is to demonstrate the applicability of the PPy-cryogel-modified electrodes for electrochemical detection of DNA. First, a polysaccharide-based cryogel was synthesized. This cryogel was then used as a template for chemical polypyrrole synthesis. This prepared polysaccharide-based conductive cryogel was used for electrochemical biosensing on DNA. Carrageenan (CG) and sodium alginate (SA) polysaccharides, which stand out as biocompatible materials, were used in cryogel synthesis. Electron transfer was accelerated by polypyrrole (PPy) synthesized in cryogel networks. A 2B pencil graphite electrode with a diameter of 2.00 mm was used as a working electrode. The prepared polysaccharide solution was dropped onto a working electrode as a support material to improve the immobilization capacity of biomolecules and frozen to complete the cryogelation step. PPy synthesis was performed on the electrodes whose cryogelation process was completed. In addition, the structures of cryogels synthesized on the electrode surface were characterized by thermogravimetric analysis (TGA), Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Surface characterization of the modified electrodes was performed by energy-dispersive X-ray spectroscopy (EDX) analysis. Electrochemical determination of fish sperm DNA (fsDNA) was performed using a PPy-cryogel-modified electrode. The use of a porous 3D cryogel intermediate material enhanced the signal by providing a large surface area for the synthesis of PPy and increasing the biomolecule immobilization capacity. The detection limit was 0.98 µg mL -1 in the fsDNA concentration range 2.5-20 µg mL -1 . The sensitivity of the DNA biosensor was estimated to 14.8 µA mM -1  cm -2 . The stability of the biosensor under certain storage conditions was examined and observed to remain 66.95% up to 45 days., (© 2024. The Author(s).)

About 18% of reproductive-age adults worldwide are affected by infertility. In vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are widely used assisted reproductive technologies (ARTs) aimed at improving clinical outcomes. Efficient and noninvasive selection and isolation of highly motile sperm with intact DNA are essential for the success of IVF and ICSI and can potentially impact the therapeutic efficacy and the health of the offspring. Compared to traditional methods, microfluidic technology offers significant advantages such as low sample consumption, high efficiency, minimal damage, high integration, similar microenvironment, and high automation, providing a new platform for ARTs. Here, we review the current situation of microfluidic technology in the field of sperm motility screening and evaluation and IVF research. First, we focus on the working principle, structural design, and screening results of sperm selection microfluidic platforms. We then highlight how the multiple steps of the IVF process can be facilitated and integrated into a microfluidic chip, including oocyte capture, sperm collection and isolation, sperm sorting, fertilization, and embryo culture. Ultimately, we summarize how microfluidics can complement and optimize current sperm sorting and IVF protocols, and challenges and possible solutions are discussed., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Nucleic acid aptamers have been used in the past for the development of diagnostic methods against a number of targets such as bacteria, pesticides, cancer cells etc. In the present study, six rounds of Cell-SELEX were performed on a ssDNA aptamer library against X-enriched sperm cells from Sahiwal breed cattle. Sequencing was used to examine the aptamer sequences that shown affinity for sperm carrying the X chromosome in order to find any possible X-sperm-specific sequences. Out of 35 identified sequences, 14 were selected based on bioinformatics analysis like G-Score and Mfold structures. Further validation of their specificity was done via fluorescence microscopy. The interaction of biotinylated-aptamer with sperm was also determined by visualizing the binding of streptavidin coated magnetic beads on the head region of the sperm under bright field microscopy. Finally, a real-time experiment was designed for the validation of X-sperm enrichment by synthesized aptamer sequences. Among the studied sequences, aptamer 29a exhibited a higher affinity for X sperm compared to Y sperm in a mixed population of sperm cells. By using aptamer sequence 29a, we obtained an enrichment of 70% for X chromosome bearing sperm cells.

Background: Sperm selection, a key step in assisted reproductive technology (ART), has long been restrained at the preliminary physical level (morphology or motility); however, subsequent fertilization and embryogenesis are complicated biochemical processes. Such an enormous "gap" poses tough problems for couples dealing with infertility, especially patients with severe/total asthenozoospermia ., Methods: We developed a biochemical-level, automatic-screening/separation, smart droplet-TO-hydrogel chip (BLASTO-chip) for sperm selection. The droplet can sense the pH change caused by sperm's respiration products and then transforms into a hydrogel to be selected out., Findings: The BLASTO-chip system can select biochemically active sperm with an accuracy of over 90%, and its selection efficiency can be flexibly tuned by nearly 10-fold. All the substances in the system were proven to be biosafe via evaluating mice fertilization and offspring health. Live sperm down to 1% could be enriched by over 76-fold to 76%. For clinical application to patients with severe/total asthenozoospermia, the BLASTO-chip could select live sperm from human semen samples containing 10% live but 100% immotile sperm. The rates of fertilization, cleavage, early embryos, and blastocysts were drastically elevated from 15% to 70.83%, 10% to 62.5%, 5% to 37.5%, and 0% to 16.67%, respectively., Conclusions: The BLASTO-chip represents a real biochemical-level technology for sperm selection that is completely independent of sperm's motility. It can be a powerful tool in ART, especially for patients with severe/total asthenozoospermia., Funding: This work was funded by the Ministry of Science and Technology of China, the Ministry of Education of China, and the Shenzhen-Hong Kong Hetao Cooperation Zone., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

This study is the first to identify bovine blastocysts through in vitro fertilization (IVF) of matured oocytes with a large quantity of high-quality sperm separated from a biomimetic cervix environment. We obtained high-quality sperm in large quantities using an IVF sperm sorting chip (SSC), which could mimic the viscous environment of the bovine cervix during ovulation and facilitates isolation of progressively motile sperm from semen. The viscous environment-on-a-chip was realized by formulating and implementing polyvinylpyrrolidone (PVP)-based solutions for the SSC medium. Sperm separated from the IVF-SSC containing PVP 1.5% showed high motility, normal morphology and high DNA integrity. As a result of IVF, a higher rate of hatching blastocysts, which is the pre-implantation stage, were observed, compared to the conventional swim-up method. Our results may significantly contribute to improving livestock with superior male and female genetic traits, thus overcoming the limitation of artificial insemination based on the superior genetic traits of existing males.

Increasing evidence of sperm RNA's role in fertilization and embryonic development has provided impetus for its isolation and thorough characterization. Sperm are considered tough-to-lyse cells due to the compact condensed DNA in sperm heads. Lack of consensus among bovine sperm RNA isolation protocols introduces experimental variability in transcriptome studies. Here, we describe an optimized method for total RNA isolation from bovine sperm using the TRIzol reagent. This study critically investigated the effects of various lysis conditions on sperm RNA isolation. Sperm suspended in TRIzol were subjected to a combination of mechanical treatments (sonication and passage through a 30G needle and syringe) and chemical treatments (supplementation with reducing agents 1,4-dithiothreitol and tris(2-carboxyethyl) phosphine hydrochloride (TCEP)). Microscopic evaluation of sperm lysis confirmed preferential sperm tail versus sperm head lysis. Interestingly, only TCEP-supplemented TRIzol (both mechanical treatments) had progressive sperm head lysis and consistently yielded total sperm RNA. Furthermore, RNA integrity was confirmed based on the electrophoresis profile and an absence of genomic DNA and somatic cells (e.g., epithelial cells, spermatids, etc.) with RT-qPCR. Our findings highlighted the importance of sperm lysis, specifically of the sperm head using TCEP with mechanical treatment, in total RNA isolation and presented a bovine-specific sperm RNA isolation method to reduce experimental variabilities., Competing Interests: The authors declare there are no competing interests.

Following the fertilization of an egg by a single sperm, the egg coat or zona pellucida (ZP) hardens and polyspermy is irreversibly blocked. These events are associated with the cleavage of the N-terminal region (NTR) of glycoprotein ZP2, a major subunit of ZP filaments. ZP2 processing is thought to inactivate sperm binding to the ZP, but its molecular consequences and connection with ZP hardening are unknown. Biochemical and structural studies show that cleavage of ZP2 triggers its oligomerization. Moreover, the structure of a native vertebrate egg coat filament, combined with AlphaFold predictions of human ZP polymers, reveals that two protofilaments consisting of type I (ZP3) and type II (ZP1/ZP2/ZP4) components interlock into a left-handed double helix from which the NTRs of type II subunits protrude. Together, these data suggest that oligomerization of cleaved ZP2 NTRs extensively cross-links ZP filaments, rigidifying the egg coat and making it physically impenetrable to sperm., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

A prominent technical barrier when imaging swimming sperm is capturing a singular sperm cell's head and tail position simultaneously at a high resolution to understand their relationship in different stages of the sperm tail beating cycle. This is due to the sperm's high beating frequency, rotational movement, and the large difference in diameter between the head and tail. These intricacies increase the complexity of determining the position of a dynamic subcellular structure in the sperm neck, such as the centriole. We have developed a way to obtain this information by snap freezing mobile sperm at different stages of the sperm tail beating cycle and then analyzing them with super-resolution microscopy. This method captures the position of both the sperm head and tail at the microscale and centriolar substructure details at the nanoscale. This chapter describes the detailed procedures for the selection, preparation, antibody staining, 3D N-STORM imaging, and image quantification of bovine spermatozoa., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Human semen, consisting of spermatozoa (sperm) and seminal plasma, represents a special clinical sample type in human body fluid. Protein glycosylation in sperm and seminal plasma plays key roles in spermatogenesis, maturation, capacitation, sperm-egg recognition, motility of sperm, and fertilization. In this study, we profiled the most comprehensive O-glycoproteome map of human sperm and seminal plasma using our recently presented Glycoproteomics based on Two Complementary Fragmentation Methods (GlycoTCFM). We showed that sperm and seminal plasma contain many novel and distinctive O-glycoproteins, which are mostly located in the extracellular region (seminal plasma) and sperm membrane, enriched in the biological processes of cell adhesion and angiogenesis, and mainly involved in multiple biological functions including extracellular matrix structural constituents and binding. Based on GlycoTCFM, we created a comprehensive human sperm and seminal plasma O-glycoprotein database that contains 371 intact O-glycopeptides and 202 O-glycosites from 68 O-glycoproteins. Interestingly, 105 manually confirmed O-glycosites from 25 O-glycoproteins were reported for the first time, and they were mainly modified by core 1 O-glycans. We also found that three highly abundant, highly complex, and highly O-glycosylated proteins (semenogelin-1, semenogelin-2, and equatorin) may play important roles in sperm or seminal plasma composition and function. These data deepen our knowledge about O-glycosylation in sperm and seminal plasma and lay the foundation for the functional study of O-glycoproteins in male infertility.

To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5., Competing Interests: Declaration of interests We declare that one or more authors have a competing interest as defined by Nature Portfolio. The Krogan Laboratory has received research support from Vir Biotechnology, F. Hoffmann-La Roche, and Rezo Therapeutics. N.J.K. has previously held financially compensated consulting agreements with the Icahn School of Medicine at Mount Sinai, New York, and Twist Bioscience Corp. He currently has financially compensated consulting agreements with Maze Therapeutics, Interline Therapeutics, Rezo Therapeutics, and GEn1E Lifesciences, Inc. He is on the Board of Directors of Rezo Therapeutics and is a shareholder in Tenaya Therapeutics, Maze Therapeutics, Rezo Therapeutics, and Interline Therapeutics., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Sperm chromatin dispersion test is a common and inexpensive technique to assess sperm DNA fragmentation, but its subjectivity in assessing a small number of spermatozoa is a disadvantage., Objectives: To study the efficacy of a new sperm chromatin dispersion test kit (R10) combined with an artificial intelligence-aided halo-evaluation platform (X12) and compare the results to those of existing sperm DNA fragmentation testing methods., Materials and Methods: Semen samples from normozoospermic donors (n = 10) and infertile men with abnormal semen parameters (n = 10) were enrolled. DNA fragmentation indices were examined by multiple assays, including R10, Halosperm G2 (G2), sperm chromatin structure assay, and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling. In R10 assay, the DNA fragmentation indices were obtained both manually (manual R10) and by X12 (AI-R10). The obtained DNA fragmentation indices were analyzed by agreement analyses., Results: The DNA fragmentation indices obtained by manual R10 and those obtained by AI-R10 showed a strong significant correlation (r = 0.97, p < 0.001) and agreement. The number of spermatozoa evaluated by AI-R10 was 2078 (680-5831). The DNA fragmentation indices obtained by manual R10 and AI-R10 both correlated with those of G2 (r = 0.90, p < 0.001; r = 0.88, p < 0.001). Between the AI-R10 and G2 results, Passing-Bablok regression showed no systematic or proportional difference, and Bland-Altman plots revealed overall agreement and a mean bias of 6.3% with an SD of 6.9% (95% limit of agreement: -7.2% to 19.9%). AI-R10 and sperm chromatin structure assays showed systematic differences with a mean bias of -1.9%, while AI-R10 and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling revealed proportional differences with a mean bias of -10.7%., Conclusions: The novel sperm chromatin dispersion kit and artificial intelligence-aided platform demonstrated significant correlation and agreement with existing sperm chromatin dispersion methods by assessing greater number of spermatozoa. This technique has the potential to provide a rapid and accurate assessment of sperm DNA fragmentation without technical expertise or flow cytometry., (© 2023 The Authors. Andrology published by Wiley Periodicals LLC on behalf of American Society of Andrology and European Academy of Andrology.)

The determination of cellular origin of DNA is a useful method in forensic genetics and complements identification of the DNA donor by STR analysis, since it could provide helpful information for the reconstruction of crime scenes and verify or disprove the descriptions of involved people. There already exist several rapid/pre-tests for several secretions (blood, sperm secretion, saliva, and urine), RNA-based expression analyses (blood, menstrual blood, saliva, vaginal secretion, nasal secretion, and sperm secretion), or specific CpG methylation analyses (nasal blood, blood, saliva, vaginal secretion, nasal secretion, and sperm secretion) for determining the cell type.To identify and to discriminate seven different body fluids and mixtures thereof in a simple workflow from each other, assays based on specific methylation patterns at several CpGs combined with pre-/rapid tests were set up in this study. For each of the seven secretions listed above, we selected the CpG marker achieving the highest possible discrimination (out of 30 markers tested). Validation studies confirmed a definite identification for saliva, vaginal secretion, and semen secretion in 100% of samples as well as discrimination from all other secretions. Moreover, the unambiguously correctly determined proportion of nasal samples, blood and menstrual blood varied between 61% (nasal blood) and 85% (nasal secretion).In summary, our workflow proved to be an easy and useful tool in forensic analysis for the identification and discrimination of seven different body fluids often found at a crime scene., (© 2023. The Author(s).)

The newly characterized sperm-specific Na + /H + exchanger stands out by its unique tripartite domain composition 1,2 . It unites a classical solute carrier unit with regulatory domains usually found in ion channels, namely, a voltage-sensing domain and a cyclic-nucleotide binding domain 1,3 , which makes it a mechanistic chimera and a secondary-active transporter activated strictly by membrane voltage. Our structures of the sea urchin SpSLC9C1 in the absence and presence of ligands reveal the overall domain arrangement and new structural coupling elements. They allow us to propose a gating model, where movements in the voltage sensor indirectly cause the release of the exchanging unit from a locked state through long-distance allosteric effects transmitted by the newly characterized coupling helices. We further propose that modulation by its ligand cyclic AMP occurs by means of disruption of the cytosolic dimer interface, which lowers the energy barrier for S4 movements in the voltage-sensing domain. As SLC9C1 members have been shown to be essential for male fertility, including in mammals 2,4,5 , our structure represents a potential new platform for the development of new on-demand contraceptives., (© 2023. The Author(s).)

Voltage-sensing domains control the activation of voltage-gated ion channels, with a few exceptions 1 . One such exception is the sperm-specific Na + /H + exchanger SLC9C1, which is the only known transporter to be regulated by voltage-sensing domains 2-5 . After hyperpolarization of sperm flagella, SLC9C1 becomes active, causing pH alkalinization and CatSper Ca 2+ channel activation, which drives chemotaxis 2,6 . SLC9C1 activation is further regulated by cAMP 2,7 , which is produced by soluble adenyl cyclase (sAC). SLC9C1 is therefore an essential component of the pH-sAC-cAMP signalling pathway in metazoa 8,9 , required for sperm motility and fertilization 4 . Despite its importance, the molecular basis of SLC9C1 voltage activation is unclear. Here we report cryo-electron microscopy (cryo-EM) structures of sea urchin SLC9C1 in detergent and nanodiscs. We show that the voltage-sensing domains are positioned in an unusual configuration, sandwiching each side of the SLC9C1 homodimer. The S4 segment is very long, 90 Å in length, and connects the voltage-sensing domains to the cytoplasmic cyclic-nucleotide-binding domains. The S4 segment is in the up configuration-the inactive state of SLC9C1. Consistently, although a negatively charged cavity is accessible for Na + to bind to the ion-transporting domains of SLC9C1, an intracellular helix connected to S4 restricts their movement. On the basis of the differences in the cryo-EM structure of SLC9C1 in the presence of cAMP, we propose that, upon hyperpolarization, the S4 segment moves down, removing this constriction and enabling Na + /H + exchange., (© 2023. The Author(s).)

The spermadhesin AQN-3 is a major component of porcine seminal plasma. While various studies suggest that this protein binds to boar sperm cells, its attachment to the cells is poorly understood. Therefore, the capacity of AQN-3 to interact with lipids was investigated. For that purpose, AQN-3 was recombinantly expressed in E. coli and purified via the included His-tag. Characterizing the quaternary structure by size exclusion chromatography revealed that recombinant AQN-3 (recAQN-3) is largely present as multimer and/or aggregate. To determine the lipid specificity of recAQN-3, a lipid stripe method and a multilamellar vesicle (MLV)-based binding assay were used. Both assays show that recAQN-3 selectively interacts with negatively charged lipids, like phosphatidic acid, phosphatidylinositol phosphates, and cardiolipin. No interaction was observed with phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or cholesterol. The affinity to negatively charged lipids can be explained by electrostatic interactions because binding is partly reversed under high-salt condition. However, more factors have to be assumed like hydrogen bonds and/or hydrophobic forces because the majority of bound molecules was not released by high salt. To confirm the observed binding behavior for the native protein, porcine seminal plasma was incubated with MLVs comprising phosphatidic acid or phosphatidyl-4,5-bisphosphate. Attached proteins were isolated, digested, and analyzed by mass spectrometry. Native AQN-3 was detected in all samples analyzed and was - besides AWN - the most abundant protein. It remains to be investigated whether AQN-3, together with other sperm associated seminal plasma proteins, acts as decapacitation factor by targeting negative lipids with signaling or other functional roles in fertilization., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The acrosome located within the mammalian sperm head is essential for successful fertilization, as it enables the sperm to penetrate the extracellular layers of the oocyte and fuse with oolemma. However, the mammalian acrosomal vesicle is no longer considered to contain only hydrolytic enzymes. Using label-free nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics, we identified a total of 885 proteins in the acrosome isolated from spermatozoa obtained from cauda epididymis of free-living house mice Mus musculus musculus contains a total of 885 proteins. Among these, 334 proteins were significantly enriched in the acrosome thus representing 27.3% of the whole proteome of the intact sperm. Importantly, we have detected a total of nine calycins while eight of them belong to the lipocalin protein family. In mice, lipocalins are involved in multi-level chemical communication between individuals including pheromone transport and odor perception. Using an indirect immunofluorescence assay, we demonstrated that lipocalin 5 (LCN5) is expressed in the mouse germ cells, and after completing spermatogenesis, it remains localized in the sperm acrosome until the last step of the extratesticular maturation, the acrosome reaction. The presence of lipocalins in the acrosome and acrosome-reacted sperm suggests their original role as chelators of organic and potentially toxic compounds resulting from ongoing spermiogenesis. Along with this evidence, detected mitochondrial (e.g., a subunit of the cytochrome c oxidase MTCO1) and proteasomal proteins (subunits of both 20 S core proteasome [PSMA2, PSMBs] and 19 S regulatory particle [PSMDs]) in acrosomes provide further evidence that acrosomes could also function as `waste baskets` after testicular sperm maturation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

This review presents information on biochemical features of spermatozoa bearing X or Y chromosome, enabling production of a sperm fraction with pre-defined sex chromosome. The almost only technology currently used for such separation (called sexing) is based on the fluorescence-activated cell sorting of sperm depending on DNA content. In addition to the applied aspects, this technology made it possible to analyze properties of the isolated populations of spermatozoa bearing X or Y chromosome. In recent years, existence of the differences between these populations at the transcriptome and proteome level have been reported in a number of studies. It is noteworthy that these differences are primarily related to the energy metabolism and flagellar structural proteins. New methods of sperm enrichment with X or Y chromosome cells are based on the differences in motility between the spermatozoa with different sex chromosomes. Sperm sexing is a part of the widespread protocol of artificial insemination of cows with cryopreserved semen, it allows to increase proportion of the offspring with the required sex. In addition, advances in the separation of X and Y spermatozoa may allow this approach to be applied in clinical practice to avoid sex-linked diseases.

Objective: To determine whether the levels of sperm very long-chain polyunsaturated fatty acids (VLC-PUFAs) are correlated with sperm parameters and the outcome of live birth after conventional therapy for unexplained infertility., Design: Cohort analysis of the Reproductive Medicine Network's Assessment of Multiple Intrauterine Gestations from Ovarian Stimulation randomized controlled trial., Setting: Multicenter randomized controlled trial., Patients: Male partners from 185 couples with unexplained infertility who provided baseline semen samples for analysis., Intervention: We determined the levels of VLC-PUFAs in total lipid isolated from sperm membranes using liquid chromatography-mass spectrometry/mass spectrometry analyses., Main Outcome Measures: Sperm concentration, motility, morphology, total motile count (TMC), and live birth after standard treatment for unexplained infertility., Results: Total VLC-PUFA percentage was positively correlated with sperm concentration (Spearman's rank correlation (r s ) 0.56, P<.0001), TMC (r s = 0.40, P<.0001), and morphology (r s = 0.26, P=.0005). After adjustment for male body mass index, age, and race, a one-standard-deviation increase in the percentage of total VLC-PUFA was associated with a 62% increase in the geometric mean (GM) of sperm concentration (GM Ratio: 1.62 [95% confidence intervals {CI}: 1.45, 1.82]) and a 43% increase in the geometric mean of TMC (GM Ratio: 1.43 [95% CI; 1.24, 1.63]). Although no evidence of association was observed for sperm motility, a positive relationship was also observed between the percentage of total VLC-PUFA and sperm morphology [adjusted incidence rate ratio (IRR) for one-standard-deviation increase in total VLC-PUFA: 1.18 (95% CI; 1.02, 1.36)]. After adjustment for female age and treatment group, the probability of a live birth outcome was 72% more likely among those in the third tertile of hydroxylated VLC-PUFA percentage than in the first tertile (RR 1.72 [95% CI; 1.01, 2.94])., Conclusions: The positive correlation between sperm VLC-PUFAs percentage and sperm parameters, as well as the significant association between hydroxylated VLC-PUFA percentage and the outcome of live birth, strongly suggest that this class of fatty liquid chromatography-mass spectrometry/mass spectrometry acids is essential for normal sperm structure and function., (Published by Elsevier Inc.)

Objective: To investigate the correlation of the severity of teratospermia and the age of the patient with sperm DNA fragmentation index (DFI) and high DNA stainability (HDS) in male infertility patients., Methods: We collected semen samples from 1 393 infertile males from July to December 2021. Based on the WHO Laboratory Manual for the Examination and Processing of Human Semen (5th Ed), we performed sperm morphology analysis, examined perm DFI and HDS by flow cytometry, and analyzed the impacts of the severity of teratospermia and the age of the patients on sperm DFI and HDS., Results: Among the 1 393 male infertility patients, 124 (8.90%) were found with extremely severe, 214 (15.36%) with severe, 235 (16.87%) with moderate, 163 (11.70%) with mild teratospermia, and 657 (47.16%) with morphologically normal sperm (MNS), with statistically significant differences in sperm DFI and HDS among the five groups, and 822 (59.00%) were aged <35 years, 306 (21.97%) 35－<40 years, 223 (16.01%) 40－<45 years and 42 (3.02%) ≥45 years, with statistically significant differences in sperm DFI and HDS among different age groups (P < 0.05). Sperm DFI and HDS were correlated negatively with the percentage of MNS (P > 0.05), but positively with the age of the patients (P < 0.05)., Conclusion: Increased severity of teratospermia and age of the patient can increase sperm DFI and HDS, and sperm nuclear chromatin integrity and maturity are important indicators of male fertility.

The present research assessed how the physical and chemical changes associated with decomposition affect the detection and identification of blood and semen evidence, as well as subsequent DNA analysis. A feeder pig (postmortem interval (PMI) < 3 h) was placed within the Boston University Outdoor Research Facility for a period of 22 days. Human blood and semen were individually dispensed onto multiple areas of two cotton t-shirts; one layer of fabric was placed above and below the pig and a control. One of each sample type was collected per day for a period of 22 days from each location. It was observed that both sample types when collected from beneath the pig exhibited the greatest decline in enzymatic activity over the course of testing, followed by samples from beneath the control, which can be inferred from the increase in negative screening results compared to the other samples. Spermatozoa were observed in nearly all semen samples, even when all screening results were negative, which lead to the generation of comparable DNA profiles for nearly all semen samples typed. Genetic typing of the blood samples beneath the pig and control rarely yielded comparable data while the samples from above yielded full profiles for all but a few samples tested., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Cryopreservation process negatively affects spermatozoa functions. Humanin, a small polypeptide encoded in the mitochondrial genome, is well known for its role in cell survival., Objective: To quantify the endogenous levels of humanin in seminal plasma of crossbred Frieswal bulls and to study its role in cryoprotection. The presence of humanin in bull spermatozoa was also investigated., Materials and Methods: A total of 40 semen samples were separated into two groups based on the initial progressive motility (IPM): Good (IPM >70%) and Poor (IPM <50%) groups; and/or based on the post-thaw motility (PTM): Freezable (PTM>50%) and Non-freezable (PTM < 50%) groups. Humanin concentration in seminal plasma (SP-HN) was quantified using ELISA., Results: SP-HN concentration ranged from undetectable to 67.6 pg/mL with a median level of 35.2 pg/mL. SP-HN level was significantly higher in the good quality semen group than in the poor quality semen group (p<0.001), and also significantly higher in the freezable group than in the non-freezable group (p<0.001). SP-HN level was positively correlated with initial progressive motility, post-thaw semen motility, viability, acrosome intactness and plasma membrane integrity, but negatively correlated the level of reactive oxygen species and malondialdehyde content. Immunochemical localization showed the presence of humanin in the proximal region of the middle piece of spermatozoa., Conclusion: Endogenous humanin level had significant correlation with semen quality and might protect sperm cells against freeze-induced oxidative stress. doi.org/10.54680/fr22510110712.

The root Rhynchosia volubilis was widely used for contraception in folk medicine, although its molecular mechanism on antifertility has not yet been revealed. In human sperm, it was reported that the cation channel of sperm, an indispensable cation channel for the fertilization process, could be regulated by various steroid-like compounds in plants. Interestingly, these nonphysiological ligands would also disturb the activation of the cation channel of sperm induced by progesterone. Therefore, this study aimed to explore whether the compounds in R. volubilis affect the physiological regulation of the cation channel of sperm. The bioguided isolation of the whole herb of R. volubilis has resulted in the novel discovery of five new prenylated isoflavonoids, rhynchones A - E (1:  - 5: ), a new natural product, 5'- O -methylphaseolinisoflavan (6: ) ( 1 H and 13 C NMR data, Supporting Information), together with twelve known compounds (7:  - 18: ). Their structures were established by extensive spectroscopic analyses and drawing a comparison with literature data, while their absolute configurations were determined by electronic circular dichroism calculations. The experiments of intracellular Ca 2+ signals and patch clamping recordings showed that rhynchone A (1: ) significantly reduced cation channel of sperm activation by competing with progesterone. In conclusion, our findings indicat that rhynchone A might act as a contraceptive compound by impairing the activation of the cation channel of sperm and thus prevent fertilization., Competing Interests: The authors declare that they have no conflict of interest., (The Author(s). This is an open access article published by Thieme under the terms of the Creative Commons Attribution-NonDerivative-NonCommercial License, permitting copying and reproduction so long as the original work is given appropriate credit. Contents may not be used for commecial purposes, or adapted, remixed, transformed or built upon. (https://creativecommons.org/licenses/by-nc-nd/4.0/).)

We aimed to explore the relationship between intakes of food-derived antioxidants (zinc, vitamin E, β-carotene, and selenium) and semen parameters in an infertile male population. This is a cross-sectional study among infertile men (> 18 years) referred to Isfahan Fertility and Infertility Center in Iran from March 2019 to October 2019. The main outcome measures were semen volume, sperm count, concentration, total motility, and morphology. Micronutrients considered in this analysis were zinc, selenium, β-carotene, and vitamin E. A total of 350 infertile men with a mean (SE) age of 34.77 (0.42) were included in this study. Selenium intake tended to be positively associated with higher semen volume (β = 0.79; 95% CI: - 0.01, 1.59) and sperm total motility after adjustment for main confounders (β = 2.64; 95% CI: - 3.61, 8.89). Intake of β-carotene was only associated with higher sperm total motility (β = 5.46; 95% CI: - 0.84, 11.77). No significant association was detected between intakes of zinc and vitamin E and semen parameters.In a population of infertile adult men, selenium intake was associated with higher sperm total motility and semen volume, and in the case of β-carotene, better sperm total motility. Further, prospective studies are warranted., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The study's objective was to adapt the Sperm Chromatin Dispersion (SCD) protocol to evaluate sperm DNA fragmentation and implement a fragmentation control in dogs. Correlation between DNA status and routine sperm parameters was also analysed. To adapt the SCD, two different mercaptoethanol (ME) concentrations were assayed (2.5% and 5%) in fourteen ejaculates from seven dogs and semen incubation with 0.3 M NaOH for 15 min at room temperature was assayed as a control for sperm DNA fragmentation. Data were analysed using a Mann-Whitney test and either Pearson's or Spearman's correlation. The selected ME concentration to use in the SCD test was 5%, as it produced the largest DNA dispersion halo while preserving the core nucleus structure. Four DNA halo patterns were identified as follows: large dispersion halos, medium halos, small halos and nuclei without halos. Semen incubated with NaOH showed 100% sperm without halos (damaged DNA). A significant positive correlation was observed between sperm with fragmented DNA and sperm with coiled tails. Thus, it was possible to adapt the SCD protocol to evaluate dog sperm DNA fragmentation in raw semen without using a commercial kit and establish incubation with NaOH as a DNA fragmentation control. Only coiled tails showed correlation with DNA fragmentation., (© 2022 Wiley-VCH GmbH.)

SP17 is a mammalian protein found in the testis and spermatozoa that have been identified as a tumor-associated antigen in a range of human cancers. A unique method for fabricating the first ultrasensitive, selective, and label-free immunosensor for the detection of SP17, a new cancer biomarker in complicated serum samples, is presented in this paper. This immunosensor was also the first biosensor built using a disposable ITO sheet modified with an aminosilane known as APTMS as an immobilization platform for fabricating the SP17 biosensor. The immobilization of chemical and biological species onto the electrode surface was cross-verified by various analytical and morphological techniques. Stepwise modifications done on the immunoelectrodes were also studied using electrochemical techniques. Selective interaction between anti-SP17 and SP17 with varying concentrations (100-5000 pg mL -1 ) was measured with the DPV technique. The immunosensor exhibited low LOD and LOQ of 70.07 and 233.57 pg mL -1 , respectively, with a sensitivity of 0.013 μA mL pg -1 cm -2 . The fabricated immunosensor performance was analyzed by quantifying the SP17 concentrations in patient serum samples. The data obtained from the developed immunosensor demonstrated excellent reproducibility, repeatability, and selectivity among various interferants, including cancer biomarkers. Further, the observed results have been validated via ELISA, which showed good agreement with the electrochemical results. This could establish a new platform for detecting other cancer biomarkers and can be employed for clinical diagnostics applications., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: This study aimed to determine (i) associations between levels of the polycyclic aromatic hydrocarbon (PAH) mixture with 16 targeted PAH compounds in the personal breathing zone area and sperm oxidative DNA damage, (ii) associations between levels of individual PAH compounds and sperm oxidative DNA damage, (iii) oxidative stress as the mode of action for the genotoxic effects on sperm, and (iv) any dose-response relationship between exposure to the PAH mixture and/or individual PAH compounds and sperm oxidative DNA damage., Methods: Sixteen targeted PAH compounds in the personal breathing zone area of 38 coke-oven workers and 24 control subjects were quantified using gas chromatography-mass spectrometry. Sperm oxidative damage and status were evaluated by measuring levels of sperm 7,8-dihydro-8-oxoguanie (8-oxodGuo), seminal malondialdehyde (MDA) and seminal reactive oxygen species (ROS). Bayesian kernel machine regression with hierarchical variable selection process was employed to determine associations of the PAH mixture and the biomarkers of sperm oxidative damage. A novel grouping approach needed for the hierarchical variable selection process was developed based on PAH bay region and molecular weight., Results: The PAH mixture exhibited a positive trend with increased sperm 8-oxodGuo levels at their lower percentiles (25th-50th). The exposure of the PAH mixture was associated with increased MDA levels in sperm. Bay and bay-like regions of the PAH mixture were the most important group for estimating the associations between the PAH mixture and sperm oxidative stress status. Benzo[a]anthracene was the main individual PAH compound that was associated with increased MDA levels., Conclusion: Sperm oxidative DNA damage induced by occupational exposure to the PAH mixture had a suggestive association with increased MDA levels in coke-oven workers. Finally, the study identified that the individual PAH compound, benzo[a]anthracene, was the primary driver for the suggestive association between the PAH mixture and sperm oxidative damage., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Occupational Hygiene Society.)

Objective: To study the contagiousness of sperm and its influence on fertility after recovery from COVID-19 infection., Design: Prospective cohort study., Setting: University medical center., Patient(s): One hundred twenty Belgian men who had recovered from proven COVID-19 infection., Intervention(s): No intervention was performed., Main Outcome Measure(s): Semen quality was assessed using the World Health Organisation criteria. DNA damage to sperm cells was assessed by quantifying the DNA fragmentation index and the high density stainability. Finally antibodies against SARS-CoV2 spike-1 antigen, nuclear and S1-receptor binding domain were measured by Elisa and chemilumenscent microparticle immunoassays, respectively., Result(s): SARS-CoV-2 RNA was not detected in semen during the period shortly after infection nor at a later time. Mean progressive motility was reduced in 60% of men tested shortly (<1 month) after COVID-19 infection, 37% of men tested 1 to 2 months after COVID-19 infection, and 28% of men tested >2 months after COVID-19 infection. Mean sperm count was reduced in 37% of men tested shortly (<1 month) after COVID-19 infection, 29% of men tested 1 to 2 months after COVID-19 infection, and 6% of men tested >2 months after COVID-19 infection. The severity of COVID-19 infection and the presence of fever were not correlated with sperm characteristics, but there were strong correlations between sperm abnormalities and the titers of SARS-CoV-2 IgG antibody against spike 1 and the receptor- binding domain of spike 1, but not against nucleotide, in serum. High levels of antisperm antibodies developed in three men (2.5%)., Conclusion(s): Semen is not infectious with SARS-CoV-2 at 1 week or more after COVID-19 infection (mean, 53 days). However, couples with a desire for pregnancy should be warned that sperm quality after COVID-19 infection can be suboptimal. The estimated recovery time is 3 months, but further follow-up studies are under way to confirm this and to determine if permanent damage occurred in a minority of men., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) packing various molecules play vital roles in intercellular communication. Non-coding RNAs (ncRNAs) are important functional molecules and biomarkers in EVs. A comprehensive investigation of ncRNAs expression in EVs under different conditions is a fundamental step for functional discovery and application of EVs. Here, we curated 2030 small RNA-seq datasets for human EVs (1506 sEV and 524 lEV) in 24 conditions and over 40 diseases. We performed a unified reads dynamic assignment algorithm (RDAA) considering mismatch and multi-mapping reads to quantify the expression profiles of seven ncRNA types (miRNA, snoRNA, piRNA, snRNA, rRNA, tRNA and Y RNA). We constructed EVAtlas (http://bioinfo.life.hust.edu.cn/EVAtlas), a comprehensive database for ncRNA expression in EVs with four functional modules: (i) browse and compare the distribution of ncRNAs in EVs from 24 conditions and eight sources (plasma, serum, saliva, urine, sperm, breast milk, primary cell and cell line); (ii) prioritize candidate ncRNAs in condition related tissues based on their expression; (iii) explore the specifically expressed ncRNAs in EVs from 24 conditions; (iv) investigate ncRNA functions, related drugs, target genes and EVs isolation methods. EVAtlas contains the most comprehensive ncRNA expression in EVs and will be a key resource in this field., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Terminally differentiated mammalian sperm are exposed to gradients of viscosity, pH, and osmolality both in the male and female reproductive tract during their perilous journey to quest the ovum. The complex physicochemical factors play an integral role in preparing sperm for the fertilization process., Objectives: To elucidate the influence of the reproductive tract microenvironment especially viscosity, pH, and osmolality in regulating sperm functional and fertilization competence., Materials and Methods: The data used in this review were collected from the research papers and online databases focusing on the influence of viscosity, pH, and osmolality on sperm function., Discussion: The gradients of viscosity, pH, and osmolality exist across various segments of the male and female reproductive tract. The changes in the viscosity create a physical barrier, pH aid in capacitation and hyperactivation, and the osmotic stress selects a progressive sperm subpopulation for accomplishing fertilization. The sperm function tests are developed based on the concept that the male genotype is the major contributor to the reproductive outcome. However, recent studies demonstrate the significance of sperm genotype-environment interactions that are essentially contributing to reproductive success. Hence, it is imperative to assess the impact of physicochemical stresses and the adaptive ability of the terminally differentiated sperm, which in turn would improve the outcome of the assisted reproductive technologies and male fertility assessment., Conclusion: Elucidating the influence of the reproductive tract microenvironment on sperm function provides newer insights into the procedures that need to be adopted for selecting fertile males for breeding, and ejaculates for the assisted reproductive technologies., (© 2021 American Society of Andrology and European Academy of Andrology.)

In alleged sexual assault cases, identification of the presence of spermatozoa at the crime scene, or on items of eventual significance, or associated with the body of the victim, is integral to the forensic investigation to support or refute the proposition that sexual act has occurred. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) system has been developed previously to identify spermatozoa based on the presence or absence of DNA methylation. This assay showed that 0.1 ng of DNA from a semen extract was sufficient to identify the presence of spermatozoa even when there was excessively more DNA isolated from vaginal fluid than DNA from a semen extract (80 ng/0.1 ng) or a mix of the menstrual blood/semen DNA (5 ng/0.1 ng). In this study, we combine spermatozoa detection with co-amplification of 23 Y-STR loci. We perform standard validation steps to present a novel test that saves time and uses the same sample for both DNA typing and spermatozoa detection in the same reaction. The combined assay can identify Y-STR and spermatozoa simultaneously using just 0.1 ng semen DNA, even in the presence of 5 ng of DNA from a female (male/female:1/50). No other body fluid tested, such as saliva, gave a result for the presence of spermatozoa. A total of 9 non-probative forensic samples from 7 sexual assault cases were tested by this co-amplification system. In all cases, the same sperm-positive data were obtained, concordant with our previous study analyzed by only 3-plex MSRE-PCR, and the Y-STR results were also consistent with that analyzed by only PowerPlex® Y23 kit. The co-amplification will be beneficial for the limited samples in many criminal cases., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: To investigate the intraindividual agreement of the sperm chromatin dispersion (SCD) assay results to assess sperm DNA fragmentation (SDF) in men with infertility., Design: Diagnostic test reliability study., Setting: Andrology laboratories., Patient(s): A total of 219 men with infertility., Intervention(s): Sperm DNA fragmentation assessment in two ejaculates of the same subjects within a 3-month interval, using the SCD assay performed and analyzed by the same observers under similar testing conditions., Main Outcome Measure(s): Cohen's κ statistics to assess the degree of agreement between the pairs of results after converting the nominal SCD values into categorical data, that is, normal (<20%), intermediate (21%-29%), and high (≥30%) SDF rates. We also assessed the pairs of results using reliability measures for numerical variables (intraclass correlation coefficient for consistency using the two-way mixed-effects model and Bland-Altman plots)., Result(s): The degree of agreement in classifying patients according to normal and pathological SDF classes was overall substantial (κ = 0.632; 95% confidence interval [CI], 0.546-0.718). A total of 76.7% of individuals were classified under the same class using paired ejaculates. The agreement rate was highest (approximately 80%) in ejaculates initially classified as either normal or high and lowest (approximately 60%) among those with intermediate SDF levels. The frequency of intermediate SDF ejaculates downgraded to normal or upgrade to high SDF classes in the second test was similar (approximately 20%). The intraclass correlation coefficient was 0.856 (95% CI, 0.812-0.887), and the mean difference between the pairs of observations was 0.80% (95% CI, -0.72 to 2.23), indicating no systematic difference between paired observations., Conclusion(s): Our study indicates a substantial intraindividual agreement of paired SCD assay results to classify men with infertility into three SDF categories: normal, intermediate, and high. The reliability of the SCD assay was adequate and exceeded 0.80 using two ejaculates analyzed within a 3-month interval under similar conditions. Although this evidence overall supports a single SCD test for patient classification using predefined SDF thresholds, particularly when the first test shows normal or high SDF levels, one in four men will have discordant values in paired ejaculates. Clinicians should be judicious when using SDF test results in decision-making., (Copyright © 2021 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Background/aims: Epigenetic regulation is considered the main molecular mechanism underlying the developmental origin of health and disease's (DOHAD) hypothesis. Previous studies that have investigated the role of paternal exercise on the metabolic health of the offspring did not control for the amount and intensity of the training or possible effects of adaptation to exercise and produced conflicting results regarding the benefits of parental exercise to the next generation. We employed a precisely regulated exercise regimen to study the transgenerational inheritance of improved metabolic health., Methods: We subjected male mice to a well-controlled exercise -training program to investigate the effects of paternal exercise on glucose tolerance and insulin sensitivity in their adult progeny. To investigate the molecular mechanisms of epigenetic inheritance, we determined chromatin markers in the skeletal muscle of the offspring and the paternal sperm., Results: Offspring of trained male mice exhibited improved glucose homeostasis and insulin sensitivity. Paternal exercise modulated the DNA methylation profile of PI3Kca and the imprinted H19/Igf2 locus at specific differentially methylated regions (DMRs) in the skeletal muscle of the offspring, which affected their gene expression. Remarkably, a similar DNA methylation profile at the PI3Kca, H19, and Igf2 genes was present in the progenitor sperm indicating that exercise-induced epigenetic changes that occurred during germ cell development contributed to transgenerational transmission., Conclusion: Paternal exercise might be considered as a strategy that could promote metabolic health in the offspring as the benefits can be inherited transgenerationally.

To date, there is limited information about the presence of SARS-CoV-2 in semen especially in the acute phase of the infection. While available data from cohort studies including a total of 342 patients in the acute or recovery phase of the infection are reassuring, one study mentioned detecting virus in the semen of 6/38 COVID-19 patients. Here we assessed SARS-CoV-2 presence in the semen of COVID-19 positive patients in the acute stage of infection, within 24 hours of the positive nasopharyngeal swabs. Semen, seminal plasma and spermatozoa pellet were screened for SARS-CoV-2 and manual or airborne contamination during semen sampling. Among the 32 COVID-19 volunteers, the median interval from the onset of symptoms to semen collection was 4 days [IQR: 0-8]. Only one presented positive SARS-CoV-2 PCR in semen and seminal plasma fractions, although the spermatozoa pellet was negative. Viral cultures were all negative. We observed slightly higher concentrations of bacterial DNA in the SARS-CoV-2 positive specimen than in all negative samples. The bacteria identified neither confirm nor rule out contamination by oropharyngeal secretions during collection. SARS-CoV-2 was rarely present in semen during the acute phase of the disease. This very rare situation could be connected to oral or manual contamination during semen collection. The possible presence of SARS-CoV-2 in semen calls for nasopharyngeal viral testing and strict hygiene protocols during semen collection before assisted reproductive attempts., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To examine the association between modifiable lifestyle factors and the main semen parameter values, the number of qualified sperm donors, and to provide some sensible guidance for sperm donors., Methods: Healthy men screened as potential sperm donors were recruited in the Hunan Province Human Sperm Bank of China from March 2019 to December 2019. Participants were invited to complete interviewer-assisted questionnaires on eleven items of information. Univariate and multivariate analyses were conducted to analyze which lifestyle factors collected by the questionnaire had an impact on the eligibility and main semen parameters of sperm donors., Results: The eligibility of men as sperm donors was strongly influenced by the duration of abstinence (P = 0.002). The rate of eligibility sperm donors increased significantly with the number of days of abstinence. In addition, semen volume increased with abstinence time (P = 0.000). Exercise frequency (P = 0.025) and abstinence time (P = 0.000) were positively correlated with sperm concentration, and masturbation frequency was negatively correlated with sperm concentration (P = 0.013). Progressive sperm motility was significantly affected by abstinence time (P = 0.000) and bedtime (P = 0.047)., Conclusions: Abstinence time was highly associated with semen parameters and donor qualification. Increase the abstinence time before donation may be meaningful in improving the proportion of eligible sperm donors., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: What is the trend in sperm parameters in a group of men attending a single reproductive center, over a 10-year period?, Methods: A retrospective study was conducted on 12,188 semen samples obtained from unique individuals who attended a university reproductive clinic from 2009 to 2018, inclusively. Semen analysis was done using computer-assisted sperm analysis and verified by an andrologist. Analysis was done after dividing the dataset into two groups: above WHO 2010 lower reference limits (ARL) (N = 6325) and below the reference limits (BRL) (N = 5521)., Results: Volume increased slightly (ARL, p = 0.049) before returning to baseline or was stable (BRL, p = 0.59). Sperm concentration and total count of the BRL and ARL group declined initially and then recovered slightly (p < 0.0001, in all cases). Although these changes were statistically significant, this was due to the large study population; clinically, these changes were quite mild and would not have been significant for fertility. Sperm total motility and progressive motility of both the BRL group and the ARL group increased slightly from 2009 until 2015 and then decreased back to baseline (p < 0.0001). This change offset the decrease in count seen in those years. A spurious change was observed with sperm morphology that declined after the first 2 years and remained stable thereafter (p < 0.0001, in both groups). However, this change was attributed to a contemporaneous change in the method of analyzing strict morphology which happened when the change occurred., Conclusion: While statistically significant changes were found, clinically, these changes were quite mild and would not have been significant for fertility., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background and Objectives: Cancer is one of the leading causes of death in the world affecting millions of people. The commercially available anticancer drugs lack the selectivity and show several undue side effects during the biologically targeted therapy, thus calling for the exploration of wider chemical space to furnish new structural leads with promising anticancer potential. In this endeavor, we synthesized a series of coumarinyl thiazolotriazoles with diverse functional group tolerance and will be tested for their anticancer properties against cancer cell lines (HeLa and MCF-7) and a normal cell line (BHK-21)., Materials and Methods: To overcome such complications, in the current study, we evaluated the cytotoxic effects of coumarinyl thiazolotriazole hybrids on human breast adenocarcinoma (MCF-7), cervical adenocarcinoma (HeLa) cells and normal cells i.e., Baby Hamster Kidney cells (BHK-21) using MTT (dimethyl-2-thiazolyl- 2,5-diphenyl-2H-tetrazolium bromide) assay. DNA binding studies of compound 6c was performed on Herring- Sperm DNA (HS-DNA) and docking studies were also carried out. The mechanistic studies were performed on potent compounds by fluorescent microscopic studies, release of Lactate Dehydrogenase (LDH) and mitochondrial membrane potential, activation of caspase-9 and -3 and flow cytometric analysis., Results: As revealed by MTT assay, compounds 6m and 6c were identified as the most potent derivatives among the tested series with IC 50 values of 5.64 and 29.1 μM against HeLa and MCF cells, respectively as compared to cisplatin which gave IC 50 values of 11.3 and 6.20 μM, respectively. DNA binding studies of compound 6c showed the binding of compound in DNA with Gibbs free energy of ‒17 KJ/mol and docking studies validated the DNA binding studies. Fluorescent microscopic studies using 4',6-diamidino-2-phenylindole (DAPI) and Propidium Iodide (PI) staining confirmed the occurrence of apoptosis in HeLa cells treated with the most active compound 6m. Moreover, compounds 6m and 6c also triggered the release of Lactate Dehydrogenase (LDH) in treated HeLa and MCF-7 cells while a luminescence assay displayed a remarkable increase in the activity of caspase-9 and -3. Moreover, flow cytometric results revealed that compound 6m caused G 0 /G 1 arrest in the treated HeLa cells., Conclusion: Our results suggested that the compound possesses chemotherapeutic properties against breast cancer and cervical adenocarcinoma cells, thus warranting further research to test the anticancer efficacy of this compound at clinical level., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Spermatogenesis is a multifaceted and meticulously orchestrated process involving meiosis, chromatin build up, transcriptional and translational hushing, and spermiogenesis. Male germ cell lines GC-1spg (GC-1) and GC-2(spd)ts (GC-2) provide a useful resource to comprehend the molecular events occurring during such a tightly regulated process. Using cDNA microarray, expression profiling of GC-1 and GC-2 cell lines was done to precisely understand their characteristics and uniqueness. Our observations indicate that whilst both the cell lines are indeed of testicular origin, GC-2 is not haploid as was originally thought. Data analysis of the 23,351 transcripts detected in GC-1 and 20,992 in GC-2 cell lines demonstrates an 80% transcript overlap between GC-1 and GC-2 cells and ~ 40% similarity of both with the primary spermatocyte transcriptome. 3152 and 793 transcripts exclusive to GC-1 and GC-2, respectively, were identified. The presence of transcripts for 36 genes was validated in these cell lines including those showing testis-specific expression, as well as genes not reported previously. Overall, this study provides the transcriptome database of GC-1 and GC-2 cells. Analysis of the data demonstrates the transcriptomic transitions between GC-1 and GC-2 thus providing a glimpse to the process of germ cell differentiation from type B spermatogonium into preleptotene spermatocyte., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Mammals exhibit a tremendous amount of variation in sperm morphology and despite the acknowledgement of sperm structural diversity across taxa, its functional significance remains poorly understood. Of particular interest is the sperm of rodents. While most Eutherian mammal spermatozoa are relatively simple cells with round or paddle-shaped heads, rodent sperm are often more complex and, in many species, display a striking apical hook. The function of the sperm hook remains largely unknown, but it has been hypothesized to have evolved as an adaptation to inter-male sperm competition and thus has been implicated in increased swimming efficiency or in the formation of collective sperm movements. Here we empirically test these hypotheses within a single lineage of Peromyscus rodents, in which closely related species naturally vary in their mating systems, sperm head shapes, and propensity to form sperm aggregates of varying sizes. We performed sperm morphological analyses as well as in vitro analyses of sperm aggregation and motility to examine whether the sperm hook (i) morphologically varies across these species and (ii) associates with sperm competition, aggregation, or motility. We demonstrate inter-specific variation in the sperm hook and then show that hook width negatively associates with sperm aggregation and sperm swimming speed, signifying that larger hooks may be a hindrance to sperm movement within this group of mice. Finally, we confirmed that the sperm hook hinders motility within a subset of Peromyscus leucopus mice that spontaneously produced sperm with no or highly abnormal hooks. Taken together, our findings suggest that any adaptive value of the sperm hook is likely associated with a function other than inter-male sperm competition, such as interaction with ova or cumulous cells during fertilization, or migration through the complex female reproductive tract.

Research Question: The semen harbours a diverse range of microorganisms. The origin of the seminal microbes, however, has not yet been established. Do testicular spermatozoa harbour microbes and could they potentially contribute to the seminal microbiome composition?, Design: The study included 24 samples, comprising a total of 307 testicular maturing spermatozoa. A high-throughput sequencing method targeting V3 and V4 regions of 16S rRNA gene was applied. A series of negative controls together with stringent in-silico decontamination methods were analysed., Results: Between 50 and 70% of all the detected bacterial reads accounted for contamination in the testicular sperm samples. After stringent decontamination, Blautia (P = 0.04), Cellulosibacter (P = 0.02), Clostridium XIVa (P = 0.01), Clostridium XIVb (P = 0.04), Clostridium XVIII (P = 0.02), Collinsella (P = 0.005), Prevotella (P = 0.04), Prolixibacter (P = 0.02), Robinsoniella (P = 0.04), and Wandonia (P = 0.04) genera demonstrated statistically significant abundance among immature spermatozoa., Conclusions: Our results indicate that the human testicle harbours potential bacterial signature, though in a low-biomass, and could contribute to the seminal microbiome composition. Further, applying stringent decontamination methods is crucial for analysing microbiome in low-biomass site., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Chromatin condensation is one of the main factors essential for sperm function. Evaluation of chromatin condensation by current methods render the assessed sperm unsuitable for assisted reproduction. We examined the Raman spectra of normal morphology sperm to determine whether a non-invasive confocal Raman spectroscopy can detect spectral differences between groups having different levels of chromatin condensation. Semen samples from 85 donors who underwent ICSI were obtained. Chromomycin A3, aniline blue and acridine orange staining were performed to evaluate the protamine deficiency, histone retention and DNA fragmentation respectively. Raman spectra were obtained from 50 normal morphology sperm for each donor. Spectral analysis was performed using home written programs in LabVIEW software and samples were grouped based on chromomycin A3 staining. Raman peaks intensities at 670 cm -1 , 731 cm -1 , 785 cm -1 , 858 cm -1 , 1062 cm -1 , 1098 cm -1 , 1185 cm -1 , 1372 cm -1 , 1424 cm -1 , 1450 cm -1 , 1532 cm -1 , 1618 cm -1 and 1673 cm -1 were significantly correlated with at least one of the sperm staining methods. The median intensity of the Raman peaks at 670 cm -1 , 731 cm -1 , 785 cm -1 , 1062 cm -1 , 1098 cm -1 , 1185 cm -1 , 1372 cm -1 , 1424 cm -1 , 1450 cm -1 , 1532 cm -1 , 1618 cm -1 and 1673 cm -1 show a significant difference between the CMA3≤41 and CMA3>41groups. The Raman spectroscopic measurements represent a promising diagnostic tool that has the ability to label-free detect sperm with chromatin abnormalities, such as improper chromatin condensation and DNA fragmentation to a certain degree similar to that of the existing staining techniques at the individual cell level., (© 2021. The Author(s).)

One hundred fifty years ago, Friedrich Miescher discovered DNA when he isolated "Nuclein"-as he named it-from nuclei of human pus cells. Miescher recognized his isolate as a new type of molecule equal in importance to proteins. He realised that it is an acid of large molecular weight and high phosphorus content. Subsequently, he discovered Nuclein also in the nuclei of other cell types, realised that it chemically defines the nucleus, and speculated on its role in proliferation, heredity and fertilisation. While now universally recognised as the discoverer of DNA, whether Miescher also discovered RNA has not yet been addressed. To determine whether his isolation also yielded RNA, we first reproduced his historic protocols. Our resulting modern Nuclein contained a significant percentage of RNA. Encouraged by this result, we then analysed a sample of Nuclein isolated by Miescher from salmon sperm. Assuming that the RNA present in this sample had degraded to nucleobases, we tested for the presence of uracil in the historic Nuclein. Detection of significant levels of uracil by LC-UV-MS demonstrates that Miescher isolated both forms of nucleic acid-DNA and RNA-and underlines the fundamental nature of his discovery for the field of molecular genetics., (© 2021 Andreas Thess et al., published by De Gruyter, Berlin/Boston.)

Background: Male infertility has been related to an increased sperm DNA fragmentation index (DFI). Nutritional factors may improve sperm nuclear DNA integrity and thus pregnancy rates. Objective: To evaluate the effect of micronutrient supplementation on sperm DNA integrity in subfertile men and subsequent pregnancy rates., Methods: In this retrospective comparative study 339 subfertile males were included on whom a sperm chromatin dispersion test (SCD) was performed as a method to detect DNA fragmentation, as well as an initial semen analysis. Of all, n  = 162 received a nutritional management program for three months, consisting of two daily capsules of a standardized combined micronutrient formulation together with a guidance to diet modification and to lifestyle changes (study group). Each capsule contained L-carnitine, L-arginine, coenzyme Q10, zinc, vitamin E, folic acid, glutathione, and selenium. The control group consisted of those patients who did not receive the active treatment ( n  = 177), yet were instructed to engage in a healthy lifestyle, including a modification of their regular diet. The SCD test was repeated for both groups after three months. As part of the routine follow up, pregnancy rate was assessed six months after the second SCD test. Males with complete follow up and healthy female partners (aged 18 to 40 years) where included., Results: Data of men with an initial mean DFI of >15% were analyzed first ( n  = 81;46 study and 35 control patients). After three months, both groups displayed a significant decrease of mean DFI values; however, the mean percent difference was higher in the study group (10.46 ± 1.20 % vs. 5.29 ± 0.57 %; p <  .001). Then, the entire population was considered ( n  = 339). After three months, only the study group displayed a significant decrease of mean DFI initial values (10.48 ± 7.76 % to 6.51 ± 4.61%; p  < .001); and the percent difference was higher in the study group (3.97 ± 0.28 % vs. 0.91 ± 0.28 %; p <  .001). At six months follow-up, pregnancy rate was higher in the study group (27.78% vs. 15.25%, p  = .002)., Conclusion: Both regimes significantly reduced sperm DNA fragmentation among subfertile men with a DFI >15%; however, when any baseline DFI value was considered, only micronutrient supplementation achieved a better result on DFI and thus pregnancy rate was higher.

A rapid and sensitive electrochemical sensing platform is reported based on bimetallic gold-platinum nanoclusters (AuPtNCs) dispersed on reduced graphene oxide (rGO) for the simultaneous detection of guanine and adenine using square wave voltammetry (SWV). The synthesis of AuPtNCs-rGO nanocomposite was achieved by a simultaneous reduction of graphene oxide (GO) and metal ions (Au 3+ and Pt 4+ ) in an aqueous solution. The developed AuPtNCs-rGO electrochemical sensor with the optimized 50:50 bimetallic (Au:Pt) nanoclusters exhibited an outstanding electrocatalytic performance towards the simultaneous oxidation of guanine and adenine without the aid of any enzymes or mediators in physiological pH. The electrochemical sensor platform showed low detection limits of 60 nM and 100 nM (S/N = 3) for guanine and adenine, respectively, with high sensitivity and an extensive linear range from 1.0 μM to 0.2 mM for both guanine and adenine. The interference from the most common electrochemically active interferents, including ascorbic acid, uric acid, and dopamine, was almost negligible. The simultaneous sensing of guanine and adenine in denatured Salmon Sperm DNA sample was successfully achieved using the proposed platform, showing that the AuPtNCs-rGO nanocomposite could provide auspicious clinical diagnosis and biomedical applications., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Background: It has been previously demonstrated that cholesterol content and cholesterol/phospholipid ratio were significantly higher in asthenozoospermia and oligoasthenoteratozoospermia. The majority of published studies have investigated the fatty acid composition of phospholipids rather than lipids themselves. This study evaluated the lipid composition of asthenozoospermic and normozoospermic spermatozoa, and identified the exact lipid species that correlated with sperm motility., Methods: A total of 12 infertile asthenozoospermia patients and 12 normozoospermia subjects with normal sperm motility values were tested for semen volume, sperm concentration, count, motility, vitality and morphology. High-coverage targeted lipidomics with 25 individual lipid classes was performed to analyze the sperm lipid components and establish the exact lipid species that correlated with sperm motility., Results: A total of 25 individual lipid classes and 479 lipid molecular species were identified and quantified. Asthenozoospermic spermatozoa showed an increase in the level of four lipid classes, including Cho, PE, LPI and GM3. A total of 48 lipid molecular species were significantly altered between normozoospermic and asthenozoospermic spermatozoa. Furthermore, the levels of total GM3 and six GM3 molecular species, which were altered in normozoospermic spermatozoa versus asthenozoospermic spermatozoa, were inversely correlated with sperm progressive and total motility., Conclusions: Several unique lipid classes and lipid molecular species were significantly altered between asthenozoospermic and normozoospermic spermatozoa, revealing new possibilities for further mechanistic pursuits and highlighting the development needs of culture medium formulations to improve sperm motility.

Background: Active smoking has been reported among 7% of teenagers worldwide, with ages ranging from 13 to 15 years. An epidemiological study suggested that preconceptional paternal smoking is associated with adolescent obesity in boys. We developed a murine adolescent smoking model before conception to investigate the paternal molecular causes of changes in offspring's phenotype., Method: Male and female C57BL/6J mice were exposed to increasing doses of mainstream cigarette smoke (CS) from onset of puberty for 6 weeks and mated with room air (RA) controls., Results: Thirteen miRNAs were upregulated and 32 downregulated in the spermatozoa of CS-exposed fathers, while there were no significant differences in the count and morphological integrity of spermatozoa, as well as the proliferation of spermatogonia between CS- and RA-exposed fathers. Offspring from preconceptional CS-exposed mothers had lower body weights (p = 0.007). Moreover, data from offspring from CS-exposed fathers suggested a potential increase in body weight (p = 0.062)., Conclusion: We showed that preconceptional paternal CS exposure regulates spermatozoal miRNAs, and possibly influences the body weight of F1 progeny in early life. The regulated miRNAs may modulate transmittable epigenetic changes to offspring, thus influence the development of respiratory- and metabolic-related diseases such as obesity, a mechanism that warrants further studies for elaborate explanations.

The acetic acid-urea polyacrylamide gel electrophoresis system could separate very similar basic proteins on differences in size and effective charge. This system has been used for many years to analyse histones and their post-translational modifications and widely used in the study of mammal protamines. Two types of protamine have been described, the protamine 1 (P1) and the protamine 2 (P2) family members, which are synthetized by PRM1 and PRM2 genes. The ratio of P1 and P2 is important for predicting fertility in humans and mice. Therefore, the quantification of protamines is a fundamental step in order to establish the ratio between P1 and P2 in these species. In other mammals, studies linking sperm protamination and the protamine ratio with fertility are increasing. So, the use of an effective technique to separate and quantify protamines is important to study sperm P1/P2 ratio. Therefore, this article describes in detail a feasible and useful procedure to isolate bovine sperm protamines, to perform pre-electrophoresis with PEG solution and finally to carry out acid-urea polyacrylamide gel electrophoresis in reverse polarity. This technique allows a clear separation and efficient detection of bovine sperm protamines., (© 2021 Wiley-VCH GmbH.)

We disclose a peculiar rotational propulsion mechanism of Ray sperms enabled by its unusual heterogeneous dual helixes with a rigid spiral head and a soft tail, named Heterogeneous Dual Helixes (HDH) model for short. Different from the conventional beating propulsion of sperm, the propulsion of Ray sperms is from both the rotational motion of the soft helical tail and the rigid spiral head. Such heterogeneous dual helical propulsion style provides the Ray sperm with high adaptability in viscous solutions along with advantages in linearity, straightness, and bidirectional motion. This HDH model is further corroborated by a miniature swimming robot actuated via a rigid spiral head and a soft tail, which demonstrates similar superiorities over conventional ones in terms of adaptability and efficiency under the same power input. Such findings expand our knowledge on microorganisms' motion, motivate further studies on natural fertilization, and inspire engineering designs., Competing Interests: The authors declare no competing interest.

Bacterial resistance to antibiotics due to increased efficiency of the efflux is a serious problem in clinics of infectious diseases. Knowledge of the factors affecting the activity of efflux pumps would help to find the solution. For this, fast and trustful methods for efflux analysis are needed. Here, we analyzed how the assay conditions affect the accumulation of efflux indicators ethidium (Et + ) and tetraphenylphosphonium in Salmonella enterica ser. Typhimurium cells. An inhibitor phenylalanyl-arginyl-β-naphtylamide was applied to evaluate the input of RND family pumps into the total efflux. In parallel to spectrofluorimetric analysis, we used an electrochemical assessment of Et + concentration. The results of our experiments indicated that Et + fluorescence increases immediately after the penetration of this indicator into the cells. However, when cells bind a high amount of Et + , the intensity of the fluorescence reaches the saturation level and stops reacting to the accumulated amount of this indicator. For this reason, electrochemical measurements provide more trustful information about the efficiency of efflux when cells accumulate high amounts of Et + . Measurements of Et + interaction with the purified DNA demonstrated that the affinity of this lipophilic cation to DNA depends on the medium composition. The capacity of DNA to bind Et + considerably decreases in the presence of Mg 2+ , Polymyxin B or when DNA is incubated in high ionic strength media.