Semen Quality Research Articles

Correlation of Seminal Plasma EMMPRIN Levels with Sperm Quality and the Efficacy of Activation Techniques

Background: Infertility is a major global health challenge, impacting 15% to 20% of couples globally. Male infertility, which is typically associated with issues such as low sperm motility, has increased during the last two decades. Male fertility relies heavily on the development of extracellular matrix metalloproteinase inducer (EMMPRIN), a glycosylated transmembrane protein involved in sperm function and embryo implantation. Understanding how sperm preparation techniques affect sperm quality and EMMPRIN expression is crucial for achieving optimal results in assisted reproductive technologies (ART). Objective: To investigate EMMPRIN expression in human sperm and compare the impact of two in vitro sperm preparation procedures, “direct swim-up (DSU) and pellet swim-up (PSU), on various sperm characteristics. To determine the optimum technique for improving sperm quality in ART. Materials and Methods: Masturbation was employed to collect sperm samples from 44 participants (aged 21 to 45). Following liquefaction, samples were evaluated macroscopically and microscopically in accordance with WHO 2021 guidelines. The samples were separated into three aliquots for ELISA assessment of EMMPRIN levels and sperm processing with DSU and PSU techniques. Sperm chromatin immaturity (SCI) was assessed using Aniline Blue staining. Statistical investigations were carried out to determine the correlations between EMMPRIN levels, semen parameters, and the impacts of the two in vitro sperm preparation techniques. Results: Sperm concentration significantly decreased from control (40.25 ± 2.30) to DSU (15.51 ± 1.74) and PSU (9.99 ± 1.16) conditions. However, motility, morphology, and SCI significantly improved, particularly with PSU, which showed a greater reduction in SCI % compared to DSU, indicating its effectiveness in selecting higher-quality sperm. There were no significant associations detected between EMMPRIN levels with semen metrics, although PSU exhibited a slightly higher association with improved sperm parameters. Conclusion: Seminal plasma EMMPRIN levels do not appear to directly influence semen parameters or sperm chromatin maturity. However, the choice of sperm activation technique, particularly PSU, significantly impacts sperm quality. More research is needed to better understand the importance of EMMPRIN in ART outcomes and its potential as a sperm quality indicator.

Investigation of optimal sperm storage conditions for short-term storage

Objective The quality of sperm cells is important role in the success rates of assisted reproduction technology (ART) treatments. The quality of the sperm cells shows variations depending on the temperature of short-term semen storage as well as the methods of semen preparation. Thus, this study aimed to investigate the sperm viability, motility and DNA fragmentation following different sperm preparation methods and short-term storage conditions, respectively. Materials and Methods A total of 25 semen samples were evaluated. In the first part of this study, different incubation temperatures were investigated in two groups, in such the first group involved the semen samples and the second group involved the sperm cells separated by density gradient centrifugation method, respectively. The samples in each group were incubated at 4°C, room temperature (21°C) and 37°C for 24 hours, respectively. The sperm cell qualities were evaluated by mobility analysis, DNA fragmentation by acridine orange staining and sperm cell viability by propidium iodide staining. Results and Discussion The analysis outcome demonstrated that the mobility, DNA fragmentation and viability of the sperm cells were statistically different when incubated at RT (21°C) in both groups. Furthermore, samples prepared by the density gradient centrifugation method were shown to have better quality. The optimum short-term storage temperature was detected to be the room temperature. Conclusion The conclusion of this investigation is crucial to assess storage conditions in ART clinics. This study provides essential data for short-term sperm storage and preparation methods to improve the success rates of ART clinics. Bangladesh Journal of Medical Science Vol. 23 No. 04 October’24 Page : 1137-1141

Pulmonary Arterial Hypertension Affects Sperm Quality and Epididymis Function in Sedentary and Exercised Wistar Rats.

Pulmonary arterial hypertension (PAH) is a disease that affects millions of people worldwide. Besides the effects on the lungs and heart, PAH can affect other organs, including the liver, kidneys, brain, glands, and testis. This study aimed to evaluate the impact of PAH and physical resistance training (RT), a complementary treatment for hypertension, on epididymis morphology and function and sperm parameters. Wistar rats were divided into four experimental groups (n = 8/ group): sedentary control, sedentary PAH, RT control, and RT + PAH. PAH was induced using monocrotaline injections on Day 1 and 7 of the experiment. Sixteen rats from RT groups underwent RT training for 30days, while rats from sedentary groups did not exercise. The epididymis was processed and analyzed using microscopic, biochemical, and functional approaches. Sperm were harvested from the cauda epididymis and evaluated for morphology and motility. Our results showed that PAH compromised the epididymis antioxidant defense system and reduced NO levels, leading to an imbalance in the organ's mineral content. These alterations affected the epididymis morphology and reduced the sperm transit time in the proximal epididymis, resulting in an increase in abnormal sperm morphology in the cauda region. Unfortunately, RT was not a good therapy against the PAH effect on the epididymis. PAH negatively affected epididymis functions with consequences to male gametes. Dysfunctions in the post-testicular environment may lead to male infertility due to the disturbance of spermatozoa fecundity.

Effects of astaxanthin supplementation during vitrification and liquid nitrogen vapor freezing on motility, morphology, survival, reactive oxygen species (ROS), and DNA fragmentation of post-cryopreserved human sperm.

To investigate the effect of astaxanthin supplementation in cryopreservation media on post-thawed sperm motility, viability, morphology, reactive oxygen species (ROS), and DNA fragmentation in two cryopreservation techniques using vitrification and liquid nitrogen vapor freezing. Thirty normozoospermic semen samples were used in the study. Post-prepared semen samples were divided into 1) non-cryopreserved control, 2) and 3) vitrified without (V) and with astaxanthin 0.5 µM (V+ATX), 4) and 5) frozen in liquid nitrogen vapor without (L) and with astaxanthin 0.5 µM (L+ATX). Cryopreservation using vitrification and liquid nitrogen vapor freezing significantly decreased sperm motility and viability and increased ROS levels. However, no changes were seen in sperm morphology or DNA fragmentation. The addition of astaxanthin in cryopreservation media significantly increased post-thawed motility in both vitrification (77.6±8.9% vs. 69.0±9.5% in V+ATX and V) and vapor freezing (57.0±13.3% vs. 47.7±14.6% in L+ATX and L); it significantly increased sperm viability in vitrification (75.0±11.9% vs. 65.9±11.1% in V+ATX and V), and significantly decreased ROS level in both vitrification (4.7 (2.6-8.3) RLU/sec/106 vs. 10.6 (9.4-16.0) RLU/sec/106 in V+ATX and V) and vapor freezing (4.6 (3.3-10.5) RLU/sec/106 vs. 10.3 (7.9-18.6) RLU/ sec/106 in L+ATX and L). Astaxanthin supplementation in cryopreservation media did not affect sperm morphology or DNA fragmentation. Astaxanthin supplementation improved post-cryopreserved sperm motility, decreased ROS levels in both vitrification and liquid nitrogen vapor freezing and improved sperm viability only in the vitrification technique.

Quantitative proteomics reveal the protective effects of Qiang Jing decoction against oligoasthenospermia via modulating spermatogenesis related-proteins.

According to the current situation, the treatment of oligoasthenospermia with Western medicine is still full of challenges. Qiang Jing decoction (QJD) is used for treating male infertility as a traditional Chinese medicine (TCM) prescription. In this study. we will try to uncover the molecular mechanism of QJDs in treating oligoasthenospermia based on the proteomics technology. In this study, sperm quality from oligoasthenospermia model, QJD treatment, and control rats were analyzed. Tandem mass tag (TMT) quantitative proteomics was used for the comparative analysis of protein abundance in rat testis of different treatment. The therapeutic effect of QJDs on spermatogenesis function by improving sperm quality was confirmed in the present experiment. In addition, our results showed that the majority of proteins related to spermatogenesis were significantly downregulated in oligoasthenospermia model rats compared to the control group, whereas these proteins' expression levels were recovered after the QJD treatment. In the meantime, immunohistochemistry was used to determine PI3K/AKT (phosphoinositide 3 kinase/serine-threonine kinase) signaling pathway-related protein expression profiles. In brief, we think QJD improved sperm density and quality by activating spermatogenesis-related protein expression and PI3K/AKT signaling pathway. The results of this study may help to establish a foundation for further functional studies of key protein-mediated for improving sperm quality and alleviating oligoasthenospermia.

Do sperm factors influence embryonic aneuploidy? Long live the oocyte.

What is the impact of male age- and sperm-related factors on embryonic aneuploidy? Using a 3-fold analysis framework encompassing patient-level, embryo-level, and matching analysis, we found no clinically significant interactions between male age and sperm quality with embryo ploidy. While the effect of maternal age on embryo chromosomal aneuploidy is well-established, the impact of male age and sperm quality on ploidy is less well-defined. This retrospective cohort study analyzed autologous preimplantation genetic testing for aneuploidy (PGT-A) and frozen embryo transfer cycles from December 2014 to June 2021. The study involved 11087 cycles from 8484 patients, with a total of 35797 embryos. The aneuploidy rate, calculated as the ratio of aneuploid blastocysts to the total number of blastocysts biopsied in a single treatment cycle, was evaluated. In the embryo-level analysis, the main outcome measure was the ploidy state of the embryos. The study employed a multifaceted analytical approach that included a patient-level analysis using generalized linear mixed models, an embryo-level analysis focusing on chromosomal ploidy, and a propensity score matching analysis contrasting groups with distinct ploidy rates (0% and 100%). There were no interventions as this was an observational study of PGT-A cycles. No clinically relevant factors influencing ploidy rate related to male and sperm quality were revealed. In contrast, female age (coefficient = -0.053), BMI (coefficient = 0.003), prior ART cycle (coefficient = -0.066), and number of oocytes retrieved (coefficient = -0.018) were identified at the patient level. Embryo analysis identified age (coefficient = -0.1244) and ICSI usage (coefficient = -0.0129) as significant factors. Despite these, no significant interactions between male and female assessed factors on the ploidy rate emerged. Propensity score matching between maximal (100% vs 0%) euploid rates did not reveal significant differences of influence by male age and sperm quality. The focus on patients having blastocyst biopsy for PGT-A may not reflect the broader IVF population. Other semen quality parameters like DNA fragmentation were not included. Exclusion of embryo mosaicism from the analysis could affect aneuploidy rate interpretations. There may also be unmeasured influences like lifestyle or environmental factors. Male age and sperm quality parameters were consistent across both maximal and minimal ploidy rate comparisons. No significant clinical characteristics related to the factors assessed for the male-influenced blastocyst ploidy status, confirming the dominancy of the oocyte and female age. The study was not funded. There are no competing interests. N/A.

Meta-Analytical and Meta-Regression Evaluation of Subclinical Hyperthyroidism's Effect on Male Reproductive Health: Hormonal and Seminal Perspectives.

Subclinical hyperthyroidism (SCH) is a subtle thyroid dysfunction marked by decreased serum thyroid-stimulating hormone (TSH) levels while maintaining a normal thyroid hormone profile. Despite its mild nature, SCH can significantly impact various physiological functions, including male reproductive health. However, the effects of SCH on reproductive hormones and semen quality are less understood compared to overt thyroid disorders. This study employed extensive search methods across various databases from January 2000 to February 2024 to explore the relationship between SCH and Hormonal and Seminal Perspectives. Effect sizes, estimated using the standardized mean difference (SMD) and pooled with a Random-effect model, provided significant insights from 748 participants. Included studies adhered to the following criteria: Patients (male individuals with SCH), Intervention (assessment of reproductive hormones and semen quality), Comparison (SCH patients versus healthy controls), and Outcome (changes in reproductive factors). Significant alterations in reproductive hormones were observed in SCH patients, including reduced LH levels (SMD = - 0.20; p = 0.007), elevated FSH levels (SMD = 0.25; p = 0.002), and stable testosterone levels (SMD = - 0.05; p = 0.50). Regarding thyroid profile, SCH was associated with increased FT3 (SMD = 0.15; p < 0.001) and FT4 (SMD = 0.08; p = 0.002) levels, along with decreased TSH levels (SMD = - 2.00; p < 0.001). Adverse effects on semen quality were also observed. These findings underscore the need to incorporate thyroid health assessments in the evaluation of male infertility, recognizing the impact of minor thyroid hormone deviations on reproductive outcomes.

Seasonality analysis on cryopreserved doses from the autochthonous cattle breeds Asturiana de la Montaña and Asturiana de los Valles.

Germplasm banking is a fundamental tool for the preservation of autochthonous breeds. Semen cryopreservation is effective for this task, but protocols are adapted to commercial species, and post-thawing sperm quality could be sensitive to environmental cues. We compared the post-thawing sperm quality in doses from the CBA-SERIDA bank in northern Spain for the Asturiana de la Montaña (AM) and Asturiana de los Valles (AV) autochthonous cattle breeds. Doses from 23 AM and 16 AV bulls (ejaculates from at least three different seasons) were assessed for motility (computer-assisted sperm analysis), physiology and chromatin status (flow cytometry) after thawing and after 5 h at 38°C. Data were analysed using linear mixed-effects and cosinor models for seasonal and breed effects and by correlations with the association of sperm quality with temperature-humidity index (THI), considering the interval of spermatogenesis plus maturation. The breed affected sperm quality, with higher motility for AV and higher apoptotic ratio, mitochondrial activity, reactive oxygen species, DNA fragmentation and chromatin immaturity for AM. However, seasonality effects were minimal, and THI was not associated with sperm quality. In summary, the season seems to be a minor factor in the post-thawing quality of the AM and AV autochthonous breeds, well-adapted to their local environment.

Mapping the Landscape of Obesity Effects on Male Reproductive Function: A Bibliometric Study.

Due to changes in lifestyle and dietary habits, the global population with obesity is increasing gradually, resulting in a significant rise in the number of individuals having obesity. Obesity is caused by an imbalance between energy intake and consumption, leading to excessive fat accumulation, which interferes with normal human metabolism. It is also associated with cardiovascular disease, metabolic syndrome, male reproductive endocrine regulation disorders, systemic and local inflammatory reactions, excessive oxidative stress, and apoptosis. All these factors can damage the internal environment for sperm generation and maturation, resulting in male sexual dysfunction, a decline in sperm quality, and lower fertility. This study analyzes the trends and priorities of the effects of obesity on male reproductive disorders from a bibliometric perspective. This study uses the Web of Science as the statistical source, covering all time spans. Tools like Web of Science, VOSviewer, and CiteSpace are used to analyze countries, institutions, authors, journals, and keywords in the field. Total publications, total citations, and average number of citations are selected for statistics. The results show that the research on the impact of obesity on male reproductive function can be roughly divided into three stages: the initial stage, the slow development stage, and the rapid development stage. Our statistical scope includes 463 highly relevant articles that we have screened. We found that the journal with the most publications in this field is Andrologia, and the institution with the highest total citations is the University of Utah. The most influential countries, institutions, and authors in this field are the United States, the University of Utah, and Carrell, Douglas. Currently, research related to the impact of obesity on male reproduction focuses mainly on three aspects: biochemistry, molecular biology, and reproductive biology. The keyword explosion results indicate that sperm, obesity, and male reproduction are at the forefront and trends of future research in this field. There has been a shift from basic biochemical and molecular research to research on molecular mechanisms relying on omics technologies. However, we have observed that the number of papers published in 2022 is lower than in 2021, indicating a growth interruption during this period. Considering that this deviation may be due to the impact of the COVID-19 pandemic, it may hinder the progress of certain experiments in 2022. In recent years, China has rapidly developed research in this field. However, the average citation rate is relatively low, indicating the need for Chinese scholars to improve the quality of their articles further. Based on our research and in the context of global obesity, men are at risk of increased infertility. Addressing this issue relies on our continued research into the mechanisms of obesity-related male reproductive disorders. Over the past forty-three years, with the contributions of scientists worldwide, research in this field has flourished. The impact of obesity on male reproductive disorders has been extensively studied. Currently, research in this field primarily focuses on male sperm function, sperm quality, and the effects or mechanisms of cells on male reproduction. Future trends in this field should concentrate on the relationship between male fertility and energy metabolism, as well as the endocrine function of adipose tissue. This study comprehensively analyzes the current research status and global trends in obesity and male reproductive disorders. We also discuss the future developments in this field, making it easier for researchers to understand its developmental history, current status, and trends, providing valuable reference for effective exploration in this area.

Damage to Mitochondria During the Cryopreservation, Causing ROS Leakage, Leading to Oxidative Stress and Decreased Quality of Ram Sperm.

Semen cryopreservation can achieve long-term preservation of sperm. Ice crystal damage, as well as oxidative stress, result in mitochondrial dysfunction and a reduction in sperm motility after thawing. However, limited information exists regarding the impact of reactive oxygen species (ROS) and mitochondria on the cryopreservation of ram sperm. The primary objective of this study was to investigate the relationship between ROS and mitochondria concerning sperm quality during the cryopreservation of ram sperm. This investigation assessed sperm motility, kinematic characteristics, membrane integrity, acrosome integrity, mitochondrial membrane potential (MMP), adenosine triphosphate (ATP) levels, expression of mitochondrial respiratory genes (NDUFV2, SDHA, CYC1, and COXIV), ROS levels, malondialdehyde (MDA) content, phosphatidylserine externalisation rate, sperm ultrastructure, mtDNA copy number, expression of apoptosis-related genes (Bax, Caspase-3, and Caspase-8), Cytochrome C, and Caspase-3 content. The results showed the cryopreservation significantly (p < 0.05) decreased motility, kinetic parameters, membrane integrity, acrosome integrity, MMP, ATP, mRNA expression levels of mitochondrial respiratory-related genes, and significantly (p < 0.05) increased ROS levels, MDA content, phosphatidylserine externalisation rate, damage of sperm ultrastructure, mtDNA copy number, mRNA expression levels of apoptosis-related genes, Cytochrome C and Caspase-3 content compared to the fresh semen group. In conclusion, the cryopreservation causes damage to mitochondria, leading to increased ROS and subsequent oxidative stress. This process also initiates mitochondrial dysfunction and interferes with the electron transport chain, ultimately resulting in decreased MMP and ATP production. Furthermore, the liberation of Cytochrome C prompted the increase in Caspase-3 expression and subsequent sperm apoptosis occurred, ultimately leading to a deterioration in sperm quality after thawing.

Dexamethasone and azithromycin enhance goat sperm preservation quality by regulating lipid metabolism

Phospholipase A (PLA) in goat semen aggregates with egg yolk in semen diluent, leading to sperm death. The aim of this study is to address the issue of sperm death caused by the interaction between PLA and egg yolk, and to explore the protective effect and metabolic regulation mechanism of the combination of dexamethasone (DXMS) and azithromycin (AZM) on goat sperm under low temperature conditions. At a low temperature of 4°C, different concentrations of DXMS were added to semen diluents containing 30μg/mL AZM to detect the quality of goat sperm. The optimal concentration of DXMS was determined to be 20μg/mL. On the 5th day of storage, antioxidant capacity, total cholesterol (TC) levels, energy metabolism, and metabolomics analysis were performed on the sperm of the 20μg/mL DXMS group. The results showed that there was no aggregation caused by the interaction between PLA and egg yolk in the group containing 30μg/mL AZM at 4°C. 20 μg/mL DXMS significantly improved sperm motility, plasma membrane integrity, acrosome integrity, glutathione peroxidase (GPX) (P<0.05), catalase (CAT) (P<0.01), and superoxide dismutase (SOD) activity (P<0.01). The content of reactive oxygen species (ROS) and Fe2+ significantly decreased (P<0.01), while the content of ATP (P<0.01) and TC (P<0.05) significantly increased. Through metabolomics analysis, a total of 56 differential metabolites (P<0.05) were screened, including 5a, 6-Anhydrotetracycline, Betamethasone, and 11-Dehydrocorticosterone, mainly enriched in 8 metabolic pathways (P<0.05), including steroid hormone biosynthesis, glycerophospholipid metabolism, and choline metabolism in cancer. Among them, 5 metabolic pathways are related to lipid metabolism. The results indicate that AZM effectively inhibits the aggregation of PLA and yolk, and the combination of AZM and DXMS enhances the preservation quality of goat sperm during low-temperature preservation by regulating lipid metabolism.

The Effect Of Biologically Active Substances On Improving The Quality Of Ram Semen During The NonBreeding Season

Background: The effect of a biologically active preparation derived from cyanobacteria on improving the quality of semen in breeding rams during the non-breeding season was studied. Materials and Methods: Rams in the experimental group were given an additional 7 ml of the biologically active preparation daily, along with their main diet, for 50 days. Results: The introduction of the biologically active preparation derived from cyanobacteria into the diet of rams led to a significant improvement in both qualitative and quantitative semen parameters during the non-breeding season. Sperm concentration increased by 40%, ejaculate volume by 71.4%, and the total number of spermatozoa in the ejaculate increased by 140%. A significant increase in motile spermatozoa was observed, reaching 81.25%, and spermatozoa with straight-line progressive motility increased to 39.25%, indicating a substantial improvement in semen quality. The addition of the preparation to the diet also led to a significant increase in testosterone levels in the experimental group by 5.73% compared to the baseline level. This increase may positively affect the spermatogenesis process and improve the overall reproductive potential of the animals. The use of the biologically active preparation significantly reduced the percentage of spermatozoa with damaged acrosomes at all stages of cryopreservation, confirming its positive effect on maintaining the structural integrity of spermatozoa. This improves the efficiency of cryopreservation and the subsequent use of sperm for insemination.Conclusion: The results showed that the preparation positively influences the quantitative and qualitative semen parameters obtained from the breeding rams in the experimental group

Microfluidics-A novel technique for high-quality sperm selection for greater ART outcomes.

Microfluidics represent a quality sperm selection technique. Human couples fail to conceive and this is so in a significant population of animals worldwide. Defects in male counterpart lead to failure of conception so are outcomes of assisted reproduction affected by quality of sperm. Microfluidics, deals with minute volumes (μL) of liquids run in small-scale microchannel networks in the form of laminar flow streamlines. Microfluidic sperm selection designs have been developed in chip formats, mimicking invivo situations. Here sperms are selected and analyzed based on motility and sperm behavioral properties. Compared to conventional sperm selection methods, this selection method enables to produce high-quality motile sperm cells possessing non-damaged or least damaged DNA, achieve greater success of insemination in bovines, and achieve enhanced pregnancy rates and live births in assisted reproduction-in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). Besides, the concentration of sperm available to oocyte can be controlled by regulating the flow rate in microfluidic chips. The challenges in this technology are commercialization of chips, development of fully functional species-specific microfluidic tools, limited number of studies available in literature, and need of thorough understanding in reproductive physiology of domestic animals. In conclusion, incorporation of microfluidic system in assisted reproduction for sperm selection may promise a great success in IVF and ICSI outcomes. Future prospectives are to make this technology more superior and need to modify chip designs which is cost effective and species specific and ready for commercialization. Comprehensive studies in animal species are needed to be carried out for wider application of microfluidic sperm selection in invitro procedures.

Gone but not forgotten: dynamics of sperm storage and potential ejaculate digestion in the black soldier fly Hermetia illucens.

Understanding the dynamics of sperm storage is essential to unravel the complexity of post-copulatory sexual selection processes in internally fertilized species. This physiological process goes from sperm transfer during copulation to its use for fertilization. In this context, the spatiotemporal dynamics of sperm storage were described in the black soldier fly (BSF) with fluorescence and transmission electron microscopy (TEM). BSF females have compartmentalized spermathecae with a transfer compartment, the fishnet canals, and a storage compartment, the reservoirs. Spermatozoa were counted both during and after mating in the two compartments. In addition to seminal fluids, the male transfers a mass of sperm in the fishnet canals, then only 49% of the transferred spermatozoa reach the reservoirs over two days. TEM observations of the fishnet canals revealed potential digestive functions, explaining the decline in the number and viability of spermatozoa in this compartment but not in the reservoirs. After one mating, females laid up to three fertile clutches, showing no constraints on sperm quantity or quality. Spermatic and ultrastructural investigations strongly suggest that BSF ejaculate acts both as a sperm plug and as a nuptial gift, reinforcing the interest in studying this farming insect as a new model for sexual selection.

Differential abundance of microRNAs in seminal plasma extracellular vesicles (EVs) in Sahiwal cattle bull related to male fertility.

Sahiwal cattle, known for their high milk yield, are propagated through artificial insemination (AI) using male germplasm, largely contingent on semen quality. Spermatozoa, produced in the testes, carry genetic information and molecular signals essential for successful fertilization. Seminal plasma, in addition to sperm, contains nano-sized lipid-bound extracellular vesicles (SP-EVs) that carry key biomolecules, including fertility-related miRNAs, which are essential for bull fertility. The current study focused on miRNA profiling of SP-EVs from high-fertile (HF) and low-fertile (LF) Sahiwal bulls. SP-EVs were isolated using size exclusion chromatography (SEC) and characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). Western blotting detected the EV-specific protein markers TSG101 and CD63. The DLS analysis showed SP-EV sizes of 170-180nm in HF and 130-140nm in LF samples. The NTA revealed particle concentrations of 5.76 × 1010 to 5.86 × 1011 particles/mL in HF and 5.31 × 1010 to 2.70 × 1011 particles/mL in LF groups, with no significant differences in size and concentration between HF and LF. High-throughput miRNA sequencing identified 310 miRNAs in SP-EVs from both groups, with 61 upregulated and 119 downregulated in HF bull. Further analysis identified 41 miRNAs with significant fold changes and p-values, including bta-miR-1246, bta-miR-195, bta-miR-339b, and bta-miR-199b, which were analyzed for target gene prediction. Gene Ontology (GO) and KEGG pathway analyses indicated that these miRNAs target genes involved in transcription regulation, ubiquitin-dependent endoplasmic reticulum-associated degradation (ERAD) pathways, and signalling pathways. Functional exploration revealed that these genes play roles in spermatogenesis, motility, acrosome reactions, and inflammatory responses. qPCR analysis showed that bta-miR-195 had 80% higher expression in HF spermatozoa compared to LF, suggesting its association with fertility status (p < 0.05). In conclusion, this study elucidates the miRNA cargoes in SP-EVs as indicators of Sahiwal bull fertility, highlighting bta-miR-195 as a potential fertility factor among the various miRNAs identified.

Effect of body mass index on semen quality, sperm chromatin integrity and sperm DNA methylation

Obesity represents a growing problem due to its impacts on human health and reproduction. In this study, we analysed semen quality, sperm DNA integrity and gene-specific CpG methylation in 116 healthy men from normal population. The men were divided into three groups according to their body mass index (BMI), and their ejaculates were analysed using standard methods, sperm chromatin structure assay (SCSA), methylation next generation sequencing (NGS) and amplicon sequencing. The sperm methylation NGS revealed six significantly differentially methylated regions (DMRs). Using subsequent targeted amplicon sequencing in 116 men, two of the DMRs were proved as differentially methylated in sperm of men with normal BMI vs. BMI ≥ 25. The DMRs were located in the EPHA8 and ANKRD11 gene. Also, we detected a significant decline in the EPHA8, ANKRD11 and CFAP46 gene methylation in association with increasing BMI values. The genes EPHA8 and ANKRD11 are involved in the nervous system and brain development; the CFAP46 gene plays a role in a flagellar assembly and is associated with sperm motility. Significantly lower rates of motile and progressive motile sperm were observed in men with BMI ≥ 30. Our results show that excess body weight can modify CpG methylation of specific genes, affect sperm motility, and compromise sperm chromatin integrity. These factors can stand behind the observed reduced fertility in men with obesity. The methylation changes might be transmitted to their offspring through sperm, and become a basis for possible developmental and reproductive issues in the next generation.

Resolution of sperm quality impairment following SARS-CoV-2 infection: A prospective study

Objective: To investigate the length of time required to resolve COVID-19 effects on semen quality and DNA integrity. Methods: A prospective cohort study was conducted among 42 men who tested positive for SARS-CoV-2 and underwent semen analysis at baseline and four months’ post-recovery. Semen samples were collected and evaluated for macroscopic and microscopic parameters, sperm chromatin maturation, and DNA fragmentation. Results: The mean age of participants was 37(±7) years, and 14% had normozoospermia at baseline. After a four-month recovery from COVID-19, 48% of patients had normozoospermia. Sperm count, motility, and morphology increased significantly, while sperm DNA fragmentation and sperm chromatin maturation decreased significantly post-recovery from COVID-19. Conclusions: Sperm parameters improve after a four-month recovery from COVID-19. The findings indicate significant improvements in sperm count, motility, morphology, DNA fragmentation, and chromatin maturation after a four-month recovery period.

Denosumab stimulates spermatogenesis in infertile men with preserved Sertoli cell capacity

Sperm production depends on proper Sertoli-germ cell interaction, and we hypothesized that receptor activator of nuclear factor κB ligand (RANKL) activity in Sertoli cells may influence spermatogenesis. Treatment with the RANKL inhibitor denosumab, normally used to treat osteoporosis, increased testicular weight, inhibin B, and germ cell proliferation in exvivo testis cultures and invivo in a humanized RANKL mouse. The effect on germ cell proliferation was positively associated with baseline serum concentrations of anti-müllerian hormone (AMH). In accordance, denosumab increased germ cell proliferation in exvivo human testis cultures with low/moderate but not severe impairment of Sertoli cell function. In a placebo-controlled randomized clinical trial, denosumab had no effect on semen quality but increased sperm concentration in a subgroup of infertile men with serum AMH ≥38 pmol/L at baseline. In conclusion, high serum AMH may increase the probability of a beneficial response to denosumab treatment in infertile men, thus suggesting a possible venue for precision medicine in male infertility.

Influence of free and microencapsulated recombinant rabbit nerve growth factor with chitosan on rabbit sperm quality parameters.

β-nerve growth factor (βNGF) plays a crucial role in reproductive physiology and sperm quality. Enzymatic activity of seminal plasma and vaginal fluids reduces available βNGF and it has been demonstrated that chitosan microspheres could protect rrβNGF from degradation. This study examined the effects of microencapsulated rrbNGF with chitosan on rabbit sperm viability, motility and capacitation status. Results showed that 0.5 and 1 μg/mL of microencapsulated rrβNGF, as well as free rrβNGF or empty microspheres, did not adversely affect sperm viability or total motility after 2 h of incubation. However, the highest progressivity kinetic parameters were observed with 1 μg/mL free rrβNGF, while the highest curvilinear velocity (VCL) occurred with 0.5 μg/mL microencapsulated rrβNGF. Empty chitosan microspheres did not induce acrosome reaction (AR), but both concentrations of free and rrβNGFch favoured AR during invitro incubation. The study suggests that using chitosan spheres did not show any adverse effects on sperm traits, unlike free rβNGF and rrβNGFch promoted capacitation and AR. Further research is needed to explore the potential of rrβNGFch in modifying invitro capacitation and inducing ovulation during artificial insemination.

Impact of semen cryopreservation season on invitro embryo production of prepubertal goat oocytes.

Goat production is affected by reproductive seasonality. Invitro embryo production (IVEP) could overcome this effect. This study aimed to evaluate the impact of the season of semen collection/freezing on IVEP of prepubertal goat oocytes and on sperm quality and functionality concerning capacitation. Semen from six fertile bucks was collected, pooled and cryopreserved in spring and autumn and used for IVEP of oocytes recovered during the breeding season. Oocytes were IVM in TCM-199 with hormones, EGF and cysteamine; fertilized and cultured in BO-IVF and BO-IVC media (IVF Bioscience, UK). Semen samples were assessed at 0 and 3 h after culture in capacitating (BO-IVF, CAP) and non-capacitating conditions for sperm plasma membrane and acrosome integrity, mitochondrial membrane potential (MMP), intracellular calcium and plasma membrane lipid disorder. Blastocyst production was higher with spring sperm compared to autumn (12.0% vs. 2.1%, respectively; p < .05). After CAP, acrosome reaction and intracellular calcium were higher (p < .05) in spring than autumn sperm. No differences were found in other sperm parameters. In conclusion, seasonal variations in the IVEP of prepubertal goats could be linked to differences in sperm ability to undergo invitro capacitation.