Enhancing the Fertility Potential: A Case Report on the Management of Oligoasthenozoospermia Using a Microfluidic Device.

Low sperm count and motility in oligoasthenozoospermia present significant challenges to conception. This case report involves a couple, a 28-year-old female and a 35-year-old male, experiencing secondary infertility for four years. The male partner's habits of alcohol consumption and smoking were potential infertility factors. Semen analysis revealed a total sperm count of 10 million/mL, with total motility at 30% and progressive motility at 5%. The couple underwent intracytoplasmic sperm injection (ICSI), using advanced sperm separation techniques to isolate motile and morphologically normal sperm. Despite the suboptimal sperm parameters, this approach resulted in successful fertilization and pregnancy. The female partner's preparation involved a short antagonist treatment, leading to the retrieval of eight oocytes, seven of which were mature. A positive urine pregnancy test and ultrasound confirmed the pregnancy, with ß-hCG at 798 mIU/mL. This case highlights the potential of individualized treatments in managing oligoasthenozoospermia, emphasizing their promise in improving assisted reproductive outcomes despite mixed research results.

A novel homozygous LRRC6 mutation causes male infertility with asthenozoospermia and primary ciliary dyskinesia in humans.

BACKGROUND: Dysfunction of motile cilia, including respiratory cilia and sperm flagella, typically leads to primary ciliary dyskinesia and male infertility or low fertility in humans. Genetic defects of LRRC6 have been associated with primary ciliary dyskinesia and asthenozoospermia due to abnormal ultrastructure of ciliated axonemes. OBJECTIVES: To identify novel mutations of the LRRC6 gene related to multiple morphological abnormalities of the sperm flagella and male infertility and investigate the underlying molecular mechanisms involved. MATERIALS AND METHODS: The LRRC6 mutations were identified by whole exome sequencing and confirmed with Sanger sequencing. Papanicolaou staining, scanning, and transmission electron microscopy were performed to investigate the morphological and ultrastructural characteristics of spermatozoa. Further tandem mass tagging proteomics analyses were performed to explore the effect of mutations and confirmed by immunostaining and western blotting. Intracytoplasmic sperm injection was applied for the assisted reproductive therapy of males harboring biallelic LRRC6 mutations. RESULTS: In this study, we identified a novel homozygous LRRC6 mutation in a consanguineous family, characterized by asthenozoospermia and primary ciliary dyskinesia. Further Semen parameter and morphology analysis demonstrate that the novel LRRC6 mutation leads to a significant reduction in sperm flagella length, a decrease in sperm progressive motility parameters, and abnormalities of sperm ultrastructure. Specifically, the absence of outer dynein arms and inner dynein arms, and incomplete mitochondrial sheath in the flagellar mid-piece were observed by transmission electron microscopy. In addition, tandem mass tagging proteomics analysis revealed that spermatozoa obtained from patients harboring the LRRC6 mutation exhibited a significant decrease in the expression levels of proteins related to the assembly and function of dynein axonemal arms. Functional analysis revealed that this novel LRRC6 mutation disrupted the function of the leucine-rich repeat containing 6 protein, which in turn affects the expression of the dynein arm proteins and leucine-rich repeat containing 6-interacting proteins CCDC40, SPAG1, and ZMYND10. Finally, we reported a successful pregnancy through assisted reproductive technology with intracytoplasmic sperm injection in the female partner of the proband. DISCUSSION AND CONCLUSION: This study highlights the identification of a novel homozygous LRRC6 mutation in a consanguineous family and its impact on sperm progressive motility, morphology, and sperm kinetics parameters, which could facilitate the genetic diagnosis of asthenozoospermia and offer valuable perspectives for future genetic counseling endeavors.

Identification of potential biomarkers and pathways for asthenozoospermia by bioinformatics analysis and experiments.

Background: Asthenozoospermia, a type of male infertility, is primarily caused by dysfunctional sperm mitochondria. Despite previous bioinformatics analysis identifying potential key lncRNAs, miRNAs, hub genes, and pathways associated with asthenospermia, there is still a need to explore additional molecular mechanisms and potential biomarkers for this condition. Methods: We integrated data from Gene Expression Omnibus (GEO) (GSE22331, GSE34514, and GSE160749) and performed bioinformatics analysis to identify differentially expressed genes (DEGs) between normozoospermia and asthenozoospermia. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted to gain insights into biological processes and signaling pathways. Weighted Gene Co-expression Network Analysis (WGCNA) identified gene modules associated with asthenozoospermia. Expression levels of key genes were assessed using datasets and experimental data. Gene Set Enrichment Analysis (GSEA) and correlation analysis identified pathways associated with the hub gene and explore the relationship between the ZNF764 and COQ9 and mitochondrial autophagy-related genes. Competitive endogenous RNA (ceRNA) networks were constructed, and in vitro experiments using exosome samples were conducted to validate this finding. Results: COQ9 was identified as a marker gene in asthenozoospermia, involved in autophagy, ATP-dependent chromatin remodeling, endocytosis, and cell cycle, etc. The ceRNA regulatory network (LINC00893/miR-125a-5p/COQ9) was constructed, and PCR demonstrated that LINC00893 and COQ9 were downregulated in asthenozoospermia, while miR-125a-5p and m6A methylation level of LINC00893 were upregulated in asthenozoospermia compared to normozoospermic individuals. Conclusion: The ceRNA regulatory network (LINC00893/miR-125a-5p/COQ9) likely plays a crucial role in the mechanism of asthenozoospermia. However, further functional experiments are needed to fully understand its significance.

Changes in seminal plasma microecological dynamics and the mechanistic impact of core metabolite hexadecanamide in asthenozoospermia patients.

Asthenozoospermia (AZS) is a prevalent contributor to male infertility, characterized by a substantial decline in sperm motility. In recent years, large-scale studies have explored the interplay between the male reproductive system's microecology and its implications for reproductive health. Nevertheless, the direct association between seminal microecology and male infertility pathogenesis remains inconclusive. This study used 16S rDNA sequencing and multi-omics analysis to conduct a comprehensive investigation of the seminal microbial community and metabolites in AZS patients. Patients were categorized into four distinct groups: Normal, mild AZS (AZS-I), moderate AZS (AZS-II), and severe AZS (AZS-III). Microbiome differential abundance analysis revealed significant differences in microbial composition and metabolite profiles within the seminal plasma of these groups. Subsequently, patients were classified into a control group (Normal and AZS-I) and an AZS group (AZS-II and AZS-III). Correlation and cross-reference analyses identified distinct microbial genera and metabolites. Notably, the AZS group exhibited a reduced abundance of bacterial genera such as Pseudomonas, Serratia, and Methylobacterium-Methylorubrum in seminal plasma, positively correlating with core differential metabolite (hexadecanamide). Conversely, the AZS group displayed an increased abundance of bacterial genera such as Uruburuella, Vibrio, and Pseudoalteromonas, with a negative correlation with core differential metabolite (hexadecanamide). In vitro and in vivo experiments validated that hexadecanamide significantly enhanced sperm motility. Using predictive metabolite-targeting gene analysis and single-cell transcriptome sequencing, we profiled the gene expression of candidate target genes PAOX and CA2. Protein immunoblotting techniques validated the upregulation protein levels of PAOX and CA2 in sperm samples after hexadecanamide treatment, enhancing sperm motility. In conclusion, this study uncovered a significant correlation between six microbial genera in seminal plasma and the content of the metabolite hexadecanamide, which is related to AZS. Hexadecanamide notably enhances sperm motility, suggesting its potential integration into clinical strategies for managing AZS, providing a foundational framework for diagnostic and therapeutic advancements.

A 2-step remote TUNEL approach for sperm DNA fragmentation assessment. Analysis in donors and patients.

OBJECTIVES: Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters. STUDY DESIGN: A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318). RESULTS: A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients. CONCLUSIONS: Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup.

Success in Parenthood: The In Vitro Maturation Breakthrough in Infertility.

This case report revolves around a 37-year-old woman and her 39-year-old husband, who have been married for seven years and were seeking treatment for infertility. The husband has been diagnosed with asthenozoospermia for the past six years and has been on continued medication, and the woman has been diagnosed with polycystic ovarian syndrome (PCOS). To improve fertility outcomes, this case report enlightens the treatment and medical strategy for people with PCOS. Treatment included low-dose ovarian stimulation for the removal of immature eggs, and then in vitro maturation (IVM) of those oocytes was done. Later, intracytoplasmic sperm injection (ICSI) was performed to form the blast. The formed blasts were later cryopreserved till embryo transfer. This case report highlights the importance of preventing the risk of ovarian hyperstimulation syndrome (OHSS) in patients with PCOS.

Genetic Causes of Qualitative Sperm Defects: A Narrative Review of Clinical Evidence.

Several genes are implicated in spermatogenesis and fertility regulation, and these genes are presently being analysed in clinical practice due to their involvement in male factor infertility (MFI). However, there are still few genetic analyses that are currently recommended for use in clinical practice. In this manuscript, we reviewed the genetic causes of qualitative sperm defects. We distinguished between alterations causing reduced sperm motility (asthenozoospermia) and alterations causing changes in the typical morphology of sperm (teratozoospermia). In detail, the genetic causes of reduced sperm motility may be found in the alteration of genes associated with sperm mitochondrial DNA, mitochondrial proteins, ion transport and channels, and flagellar proteins. On the other hand, the genetic causes of changes in typical sperm morphology are related to conditions with a strong genetic basis, such as macrozoospermia, globozoospermia, and acephalic spermatozoa syndrome. We tried to distinguish alterations approved for routine clinical application from those still unsupported by adequate clinical studies. The most important aspect of the study was related to the correct identification of subjects to be tested and the correct application of genetic tests based on clear clinical data. The correct application of available genetic tests in a scenario where reduced sperm motility and changes in sperm morphology have been observed enables the delivery of a defined diagnosis and plays an important role in clinical decision-making. Finally, clarifying the genetic causes of MFI might, in future, contribute to reducing the proportion of so-called idiopathic MFI, which might indeed be defined as a subtype of MFI whose cause has not yet been revealed.

Global proteomic analyses of lysine acetylation, malonylation, succinylation, and crotonylation in human sperm reveal their involvement in male fertility.

Protein lysine modifications (PLMs) are hotspots of post-translational modifications and are involved in many diseases; however, their role in human sperm remains obscure. This study examined the presence and functional roles of a classical PLM (lysine acetylation, Kac) and three novel PLMs (lysine malonylation, Kmal; lysine succinylation, Ksucc; lysine crotonylation, Kcr) in human sperm. Immunoblotting and immunofluorescence assays revealed modified proteins (15-150 kDa) in the tails of human sperm. An immunoaffinity approach coupled with liquid chromatography/tandem mass spectrometry revealed 1423 Kac sites in 680 proteins, 196 Kmal sites in 118 proteins, 788 Ksucc sites in 251 proteins, and 1836 Kcr sites in 645 proteins. These modified proteins participate in a variety of biological processes and metabolic pathways. Crosstalk analysis demonstrated that proteins involved in the sperm energy pathways of glycolysis, oxidative phosphorylation, the citrate cycle, fatty acid oxidation, and ketone body metabolism were modified by at least one of these modifications. In addition, these modifications were found in 62 male fertility-related proteins that weave a protein-protein interaction network associated with asthenoteratozoospermia, asthenozoospermia, globozoospermia, spermatogenic failure, hypogonadotropic hypogonadism, and polycystic kidney disease. Our findings shed light on the functional role of PLMs in male reproduction. SIGNIFICANCE: Protein lysine modifications (PLMs) are hotspots of posttranslational modifications and are involved in many diseases. This study revealed the presence of a classical PLM (lysine acetylation) and three novel PLMs (lysine malonylation, lysine succinylation, and lysine crotonylation) in human sperm tails. The modified proteins participate in a variety of biological processes and metabolic pathways. In addition, these modifications were found in 62 male infertility-associated proteins and could serve as potential diagnostic markers and therapeutic targets for male infertility.

Elucidating the Role of OXPHOS Variants in Asthenozoospermia: Insights from Whole Genome Sequencing and an In Silico Analysis.

Infertility is a global health challenge that affects an estimated 72.4 million people worldwide. Between 30 and 50% of these cases involve male factors, showcasing the complex nature of male infertility, which can be attributed to both environmental and genetic determinants. Asthenozoospermia, a condition characterized by reduced sperm motility, stands out as a significant contributor to male infertility. This study explores the involvement of the mitochondrial oxidative phosphorylation (OXPHOS) system, crucial for ATP production and sperm motility, in asthenozoospermia. Through whole-genome sequencing and in silico analysis, our aim was to identify and characterize OXPHOS gene variants specific to individuals with asthenozoospermia. Our analysis identified 680,099 unique variants, with 309 located within OXPHOS genes. Nine of these variants were prioritized due to their significant implications, such as potential associations with diseases, effects on gene expression, protein function, etc. Interestingly, none of these variants had been previously associated with male infertility, opening up new avenues for research. Thus, through our comprehensive approach, we provide valuable insights into the genetic factors that influence sperm motility, laying the foundation for future research in the field of male infertility.

Hypoxia enhances autophagy level of human sperms.

The relationship between oxygen sensing and autophagy in human sperms was explored in this study. Health semen and asthenozoospermia (astheno) semen were incubated with hypoxia-inducible factor-1α (HIF-1α) interferents, i.e., lificiguat (YC-1) or cobalt chloride (CoCl2), respectively. Label-free quantitative proteomic technology was used to identify the differentially expressed proteins in human semen under the hypoxia condition. Selected proteins were detected with ELISA. It was found that the autophagy levels of sperm in the YC-1 + health group or CoCl2 + astheno group increased while the vitality decreased. A total of 17, 34 and 35 differentially expressed proteins were observed in the Astheno group, the YC-1 + health group and the CoCl2 + astheno group, respectively. These proteins were primarily associated with protein processing in endoplasmic reticulum, Th17 cell differentiation, progesterone-mediated oocyte maturation, glycolysis/gluconeogenesis, HIF-1 signaling pathway, biosynthesis of amino acids, and carbon metabolism. The expression levels of protein HIF-1α, LC3B, histone H4, cathepsin L and ENO1 changed significantly in the groups. The study suggests that hypoxia can increase sperm autophagy level and reduce their vitality through HIF-1 signaling pathway and glycolysis/gluconeogenesis signaling pathway. Furthermore, proteins histone H4, cathepsin L, glutathione synthetase and ENO1 are proposed as potential biomarkers of autophagy and vitality in asthenozoospermia sperm.

Apoptotic-Related MiRNAs Correlated with Functional and Flow Cytometric Parameters in Asthenozoospermic Holstein Bulls After Freeze-Thaw Process.

Many cellular processes in spermatozoa, including apoptosis and motility, are regulated by miRNA. Different miRNAs and molecular pathways are involved in asthenozoospermia (AS) conditions, which are thought to be one of the causes of infertility with reduced sperm motility. Thirty-two semen samples from four Holstein bulls with normozoospermia (NS), total motility ≥ 70%, and progressive motility ≥ 60%, and 32 semen samples from four bulls with AS, total motility ≤ 40%, and progressive motility ≤ 32% were used to investigate the function of apoptosis-related miRNAs in the AS group. Samples were then aspirated into a 0.5 mL straw after dilution with a Tris-egg yolk extender and frozen at -196°C. After freezing, semen samples were thawed for 2 weeks at 37°C and sperm kinematic parameters, plasma membrane integrity, acrosome integrity, DNA fragmentation, apoptosis status, and expression of apoptosis-related miRNAs (miR-2114, miR-296-3p, miR-455-3p, and miR345-3p) were evaluated. Our results showed that the functional and flow cytometric parameters of the NS group were significantly better than those of the AS group. In the NS group, miR-455-3pp and miR-2412 were upregulated, while miR-345-3p was downregulated compared with the AS group. In the AS group, miR-296-39, miR-2412, and miR-345-3p levels were strongly correlated with membrane integrity, DNA fragmentation, and apoptosis status. The findings demonstrated that the selected miRNAs based on bioinformatic analysis in AS and NS samples had a substantial association with functional and flow cytometry indicators and may be involved in regulating apoptosis and motility in AS samples.

Automatic high-throughput and non-invasive selection of sperm at the biochemical level.

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.

Achilles' heel of male infertility: good LEGO players.

In a recent journal article, Chen et al. identified a germ cell-specific cofactor, STYXL1, associated with male fertility function. Deletion of STYXL1 prevents the LEGO player CCT complex from properly folding key microtubule proteins of the sperm flagellum, which affects sperm motility and male fertility function.

Novel underlying genetic markers for asthenozoospermia due to abnormal spermatogenesis and reproductive organ inflammation.

Asthenozoospermia, a male fertility disorder, has a complex and multifactorial etiology. Moreover, the effectiveness of different treatments for asthenozoospermia remains uncertain. Hence, by using bioinformatics techniques, the present study aimed to determine the underlying genetic markers and pathogenetic mechanisms associated with asthenozoospermia due to abnormal spermatogenesis and inflammation of the reproductive tract. GSE160749 dataset was downloaded from the Gene Expression Omnibus database, and the data were filtered to obtain 1336 differentially expressed genes (DEGs) associated with asthenozoospermia. These DEGs were intersected with the epithelial mesenchymal transition datasets to yield 61 candidate DEGs. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed, and the results revealed that these candidate DEGs were significantly enriched in the enzyme-linked receptor pathway and the thyroid hormone pathway. A protein-protein interaction network was constructed to identify the key genes of asthenozoospermia. A total of five key genes were identified, among which SOX9 was significantly upregulated, while HSPA4, SMAD2, HIF1A and GSK3B were significantly downregulated. These findings were validated by conducting reverse transcription-quantitative PCR for clinical semen samples. To determine the underlying molecular mechanisms, a regulatory network of transcription factors and miRNA-mRNA interactions was predicted. The expression levels of HSPA4, SMAD2 and GSK3B were positively associated with several related etiological genes of asthenozoospermia. In total, five key genes were closely associated with the level and type of immune cells; higher levels of activated B cells and CD8 T cells were observed in asthenozoospermia. Thus, the findings of the present study may provide clues to determine the underlying novel diagnostic genetic markers and treatment strategies for asthenozoospermia.

Both protein and non-protein components in extracellular vesicles of human seminal plasma improve human sperm function via CatSper-mediated calcium signaling.

STUDY QUESTION: What is the significance and mechanism of human seminal plasma extracellular vesicles (EVs) in regulating human sperm functions? SUMMARY ANSWER: EV increases the intracellular Ca2+ concentrations [Ca2+]i via extracellular Ca2+ influx by activating CatSper channels, and subsequently modulate human sperm motility, especially hyperactivated motility, which is attributed to both protein and non-protein components in EV. WHAT IS KNOWN ALREADY: EVs are functional regulators of human sperm function, and EV cargoes from normal and asthenozoospermic seminal plasma are different. Pre-fusion of EV with sperm in the acidic and non-physiological sucrose buffer solution could elevate [Ca2+]i in human sperm. CatSper, a principle Ca2+ channel in human sperm, is responsible for the [Ca2+]i regulation when sperm respond to diverse extracellular stimuli. However, the role of CatSper in EV-evoked calcium signaling and its potential physiological significance remain unclear. STUDY DESIGN, SIZE, DURATION: EV isolated from the seminal plasma of normal and asthenozoospermic semen were utilized to investigate the mechanism by which EV regulates calcium signal in human sperm, including the involvement of CatSper and the responsible cargoes in EV. In addition, the clinical application potential of EV and EV protein-derived peptides were also evaluated. This is a laboratory study that went on for more than 5 years and involved more than 200 separate experiments. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen donors were recruited in accordance with the Institutional Ethics Committee on human subjects of the Affiliated Hospital of Nantong University and Jiangxi Maternal and Child Health Hospital. The Flow NanoAnalyzer, western blotting, and transmission electron microscope were used to systematically characterize seminal plasma EV. Sperm [Ca2+]i responses were examined by fluorimetric measurement. The whole-cell patch-clamp technique was performed to record CatSper currents. Sperm motility parameters were assessed by computer-assisted sperm analysis. Sperm hyperactivation was also evaluated by examining their penetration ability in viscous methylcellulose media. Protein and non-protein components in EV were analyzed by liquid chromatography-mass spectrum. The levels of prostaglandins, reactive oxygen species, malonaldehyde, and DNA integrity were detected by commercial kits. MAIN RESULTS AND THE ROLE OF CHANCE: EV increased [Ca2+]i via an extracellular Ca2+ influx, which could be suppressed by a CatSper inhibitor. Also, EV potentiated CatSper currents in human sperm. Furthermore, the EV-in [Ca2+]i increase and CatSper currents were absent in a CatSper-deficient sperm, confirming the crucial role of CatSper in EV induced Ca2+ signaling in human sperm. Both proteins and non-protein components of EV contributed to the increase of [Ca2+]i, which were important for the effects of EV on human sperm. Consequently, EV and its cargos promoted sperm hyperactivated motility. In addition, seminal plasma EV protein-derived peptides, such as NAT1-derived peptide (N-P) and THBS-1-derived peptide (T-P), could activate the sperm calcium signal and enhance sperm function. Interestingly, EV derived from asthenozoospermic semen caused a lower increase of [Ca2+]i than that isolated from normal seminal plasma (N-EV), and N-EV significantly improved sperm motility and function in both asthenozoospermic samples and frozen-thawed sperm. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This was an in vitro study and caution must be taken when extrapolating the physiological relevance to in vivo regulation of sperm. WIDER IMPLICATIONS OF THE FINDINGS: Our findings demonstrate that the CatSper-mediated-Ca2+ signaling is involved in EV-modulated sperm function under near physiological conditions, and EV and their derivates are a novel CatSper and sperm function regulators with potential for clinical application. They may be developed to improve sperm motility resulting from low [Ca2+]i response and/or freezing and thawing. STUDY FUNDING/COMPETING INTEREST(S): This research was supported by the National Natural Science Foundation of China (32271167), the Social Development Project of Jiangsu Province (BE2022765), the Nantong Social and People's Livelihood Science and Technology Plan (MS22022087), the Basic Science Research Program of Nantong (JC22022086), and the Jiangsu Innovation and Entrepreneurship Talent Plan (JSSCRC2021543). The authors declare no conflict of interest.

Methyltransferase-like 3 mediated RNA m6 A modifications in the reproductive system: Potentials for diagnosis and therapy.

Several studies have highlighted the functional indispensability of methyltransferase-like 3 (METTL3) in the reproductive system. However, a review that comprehensively interprets these studies and elucidates their relationships is lacking. Therefore, the present work aimed to review studies that have investigated the functions of METTL3 in the reproductive system (including spermatogenesis, follicle development, gametogenesis, reproductive cancer, asthenozoospermia and assisted reproduction failure). This review suggests that METTL3 functions not only essential for normal development, but also detrimental in the occurrence of disorders. In addition, promising applications of METTL3 as a diagnostic or prognostic biomarker and therapeutic target for reproductive disorders have been proposed. Collectively, this review provides comprehensive interpretations, novel insights, potential applications and future perspectives on the role of METTL3 in regulating the reproductive system, which may be a valuable reference for researchers and clinicians.

The Association between Abstinence Period and Semen Parameters in Humans: Results in Normal Samples and Different Sperm Pathology.

BACKGROUND: The impact of sexual abstinence on sperm quality, particularly in pathological cases, is a subject of debate. We investigated the link between abstinence duration and semen quality in both normal and pathological samples. METHODS: We analyzed semen samples from 4423 men undergoing fertility evaluation, comprising 1256 samples from healthy individuals and 3167 from those with conditions such as oligozoospermia, asthenozoospermia, teratozoospermia, or a combination of these factors, namely oligoasthenoteratozoospermia (OAT). Parameters including sperm concentration, the percentage of progressively motile spermatozoa, total motile sperm count, and the percentage of spermatozoa with normal morphology were assessed at various abstinence durations (each day, 0-2, 3-7, and >7 days). RESULTS: Extended abstinence correlated with higher sperm concentration overall (p < 0.001), except in oligozoospermia. Longer abstinence reduced progressive motility in normal (p < 0.001) and teratozoospermic samples (p < 0.001). Shorter abstinence was linked to higher morphologically normal sperm in normal samples (p = 0.03), while longer abstinence did so in oligoasthenoteratozoospermic samples (p = 0.013). CONCLUSION: The findings suggest that a prolonged abstinence time is linked to higher sperm concentration, while optimal sperm motility is observed after shorter abstinence periods. However, results regarding morphology remain inconclusive. Recommendations on abstinence duration should be tailored based on the specific parameter requiring the most significant improvement.

Quantitative Analysis of the Human Semen Phosphorometabolome by 31P-NMR.

Phosphorus-containing metabolites occupy a prominent position in cell pathways. The phosphorometabolomic approach in human sperm samples will deliver valuable information as new male fertility biomarkers could emerge. This study analyzed, by 31P-NMR, seminal plasma and whole semen from asthenozoospermic and normozoospermic samples (71% vs. 27% and 45% vs. 17%, total and progressive sperm motility, respectively), and also ejaculates from healthy donors. At least 16 phosphorus-containing metabolites involved in central energy metabolism and phospholipid, nucleotide, and nicotinamide metabolic pathways were assigned and different abundances between the samples with distinct sperm quality was detected. Specifically, higher levels of phosphocholine, glucose-1-phosphate, and to a lesser degree, acetyl phosphate were found in the asthenozoospermic seminal plasma. Notably, the phosphorometabolites implicated in lipid metabolism were highlighted in the seminal plasma, while those associated with carbohydrate metabolism were more abundant in the spermatozoa. Higher levels of phosphocholine, glucose-1-phosphate, and acetyl phosphate in the seminal plasma with poor quality suggest their crucial role in supporting sperm motility through energy metabolic pathways. In the seminal plasma, phosphorometabolites related to lipid metabolism were prominent; however, spermatozoa metabolism is more dependent on carbohydrate-related energy pathways. Understanding the presence and function of sperm phosphorylated metabolites will enhance our knowledge of the metabolic profile of healthy human sperm, improving assessment and differential diagnosis.

Role of sleep in asthenospermia induced by di (2-ethyl-hexyl) phthalate.

Di (2-ethyl-hexyl) phthalate (DEHP) mainly enters the human body through the digestive tract, respiratory tract, and skin. At the same time, it has reproductive and developmental toxicity, neurotoxicity, and so on, which can cause the decrease of sperm motility. Asthenospermia is also known as low sperm motility, and the semen quality of men in some areas of China is declining year by year. Interestingly, previous studies have shown that sleep disorders can also lead to asthenospermia. However, the relationship between sleep, DEHP, and asthenospermia is still unclear. Analysis of the National Health and Nutrition Examination Survey (NHANES) population database showed that DEHP was associated with sleep disorders, and subsequent experiments in mice and Drosophila indicated that DEHP exposure had certain effects on sleep and asthenospermia. Furthermore, we analyzed the Comparative Toxicogenomics Database (CTD) to find out the common signaling pathway among the three: hypoxia-inducible factor 1(HIF-1). Then Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used to screen out the proteins that DEHP affected the HIF-1 pathway: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), serine/threonine-protein kinase (AKT1), epidermal growth factor receptor (EGFR), and finally Western blot analysis was used to detect the expression levels of the three proteins. Compared with the control group, DEHP decreased the protein expression levels of GAPDH and AKT1 in the HIF-1 pathway, and caused sleep disorders and decreased sperm motility. This study provides preliminary evidence for exploring the mechanism among DEHP, sleep disorders, and asthenospermia.

The Role of Chorein Deficiency in Late Spermatogenesis.

VPS13A, also known as chorein, whose loss of function causes chorea-acanthocytosis (ChAc), is characterized by Huntington's-disease-like neurodegeneration and neuropsychiatric symptoms in addition to acanthocytosis in red blood cells. We previously reported that ChAc-model mice with a loss of chorein function exhibited male infertility, with asthenozoospermia and mitochondrial dysmorphology in the spermatozoa. Here, we report a novel aspect of chorein dysfunction in male fertility, particularly its role in spermatogenesis and mitochondrial integrity. An increase in anti-malondialdehyde antibody immunoreaction within the testes, predominantly observed at the advanced stages of sperm formation in chorein-deficient mice, suggests oxidative stress as a contributing factor to mitochondrial dysfunction and impaired sperm maturation. The chorein immunoreactivity in spermatids of wild-type mice accentuates its significance in sperm development. ChAc-model mice exhibit mitochondrial ultrastructural abnormalities, specifically during the late stages of sperm maturation, suggesting a critical timeframe for chorein's action in spermiogenesis. We observed an increase in TOM20 protein levels, indicative of disrupted mitochondrial import mechanisms. The concurrent decrease in metabolic enzymes such as IDH3A, LDHC, PGK2, and ACAT1 suggests a complex chorein-mediated metabolic network that is essential for sperm vitality. Additionally, heightened separation of cytoplasmic droplets from sperm highlights the potential membrane instability in chorein-deficient spermatozoa. Metabolomic profiling further suggests a compensatory metabolic shift, with elevated glycolytic and TCA-cycle substrates. Our findings suggest that chorein is involved in anti-ferroptosis and the maturation of mitochondrial morphology in the late stages of spermatogenesis, and its deficiency leads to asthenozoospermia characterized by membrane instability, abnormal cytosolic glycolysis, abnormal mitochondrial function, and a disrupted TCA cycle. Further analyses are required to unravel the molecular mechanisms that directly link these findings and to elucidate the role of chorein in spermatogenesis as well as its broader implications.