Sperm Research Articles

Vaginal microbiota and fertility: an integrative review

The vaginal microbiota is made up of countless microorganisms that help to make it healthy and resistant to invasion by pathobionts, and studies suggest that there is an association between fertility and the balance/unbalance of this microenvironment. The aim of this study was to learn more about the vaginal microbiota and its influence on female (in)fertility. This is an integrative review guided by the steps of the PRISMA flowchart, searching for scientific articles in the BVS, Nature, PubMed and Scielo databases published in the years 2018-2022, in Portuguese, English and Spanish. Inclusion and exclusion criteria were used, and they were categorized into levels of evidence. This review consisted of twelve articles which show that vaginal dysbiosis causes changes in the metabolome of this environment, triggering inflammatory activation, which impacts on female fertility. In this sense, there is a need for further research into the interaction of microorganisms with each other and with male gametes, in eubiosis and vaginal dysbiosis, in order to facilitate or restrict their passage to the uterus and achieve successful fertilization.

Correlation between sperm DNA fragmentation and methylation in male partners of couples with idiopathic recurrent pregnancy loss

With ∼50% recurrent pregnancy loss cases being termed idiopathic (iRPL), understanding of contribution of male factors to iRPL is still lacking. Higher prevalence of sperm DNA fragmentation index (DFI) and lower sperm 5-methylcytosine (5-mC) levels have been previously reported in male partners of iRPL couples and shed light on importance of the male gamete in maintenance of a successful pregnancy. The present study aimed to determine the serum sex steroid hormone levels, sperm DFI and 5-mC and correlation between them in male partners of fertile and iRPL couples. Further, correlation between sperm DFI and 5-mC with semen parameters and paternal age in both groups were determined. 36 male partners of fertile couples and 45 male partners of women experiencing iRPL were enrolled for this study and semen and blood samples were collected. Serum testosterone and estradiol levels were measured by ELISA; sperm DFI and global 5-mC were determined by TUNEL assay and ELISA respectively. Significantly higher serum testosterone levels were noted in the iRPL group (p = 0.028). Incidence of sperm DNA fragmentation was found to be higher in the iRPL study group but with no significance difference. No significant differences in sperm 5-mC values were noted. Upon correlation analysis within both groups, strong significant negative correlation of sperm DFI % and 5-mC % was observed in the control group (p < 0.001) but not the iRPL group (p = 0.249). Hence, we infer that with lower 5-mC levels in sperm genome, there is a higher incidence of sperm DFI in fertile men. However, this trend is not noted in men of iRPL group which could possibly be due to other underlying epigenetic alterations in genomic regions probably unsusceptible to fragmentation. On the other hand, no significant correlations of semen parameters, testosterone, estradiol and paternal age with sperm DFI and 5-mC were noted in both groups.

Evaluation of structural problems in the application of strict criteria for sperm morphology assessment.

The WHO manual for basic semen analysis and ISO 23162 describe sperm morphology assessment as a standard part of semen analysis. Older studies showed a correlation between morphology results and (artificial) conception. In more recent studies this relationship was less apparent and there is more emphasis on sperm morphology as a marker for healthy spermatogenesis (and general male health). Meantime, many laboratories ceased morphology assessment, probably due to unfamiliarity with this paradigmatic shift and to technical difficulties in the assessment, like the interpretation of morphological criteria. The aim of this study was to identify morphological criteria with high variability in results in the Dutch External Quality Control (EQC) program. Over the period 2015-2020, a total of 72 photos of sperm cells along with dichotomous propositions based on 14 criteria as defined in WHO5 (2010) were distributed in the Dutch EQC program for semen analysis. The EQC results were evaluated for variability per criterion and for trends in time. Between 2015 and 2020, 40 to 60 laboratories assessed the photos. Criteria with low variability between participants were related to acrosomal vacuoles, excessive residual cytoplasm, and tail metrics. In contrast, head ovality, regularity of head and midpiece contours, and alignment of the major axis of the midpiece and head led to the highest variability in outcomes. In general, there was a slightly positive trend (lower variability) in time, except for the criteria with the highest variability (stable or declining trend). This study indicates that there are (high) variabilities in the interpretation of the morphological criteria, leading to inconsistent outcomes of morphology assessment. The results are discussed from the perspective of imperfections in definitions and examples of the criteria as given in the WHO manuals.

Impact of seasonality on the reproductive physiology of diploid and tetraploid eastern oyster, Crassostrea virginica

Aquaculture production of triploid Eastern oyster, Crassostrea virginica, in the Gulf of Mexico (GoM) is reliant on naturally conditioned diploid females and tetraploid males. However, limited knowledge is available on their reproductive biology, including the gametogenic cycle. The aim was to quantify monthly changes in gametogenesis and gamete quality for triploid production in Eastern oyster in the GoM from June 2022 to July 2023. Monthly changes in temperature and salinity were recorded. Shell morphometrics and sex were determined. Sperm density, sperm activity, Spermatogenic Maturity Index (SMI), oocyte diameter, Oogenesis Maturity Index (OMI), and gonad coverage area were quantified. Mean monthly temperature ranged from 15.1 to 31.2 °C and salinity ranged from 12.5 to 27.6 PSU. Shell morphometrics increased over the annual cycle. When gametes were detectable, 48–80% of tetraploids and 45–70% of diploids were males. Hermaphrodites were identified in both ploidies in June; an additional tetraploid hermaphrodite was identified in March. Sperm density exhibited monthly fluctuations with high densities reported in June–July, September, and April–May. Sperm were motile when present, with high motility in June–July. High sperm velocity was also recorded in June–July and March–May. Tetraploids exhibited high SMI from June–September and April–May with increased variability in December–January. Mature sperm cells were predominant in tetraploids in June–July. Gonadal coverage was high from June–September and March–May. OMI was highest from June–September and March–May, while variability was high from August–March. Large oocyte diameters and gonad coverage areas were observed in June–July, September, and April–May. Together, these findings provide novel insights into reproductive dynamics for triploid production of Eastern oyster in GoM.

The absence of canonical respiratory complex I subunits in male-type mitogenomes of three Donax species

Bivalves are an extraordinary class of animals in which species with a doubly uniparental inheritance (DUI) of mitochondrial DNA have been described. DUI is characterized as a mitochondrial homoplasmy of females and heteroplasmy of male individuals where F-type mitogenomes are passed to the progeny with mother egg cells and divergent M-type mitogenomes are inherited with fathers sperm cells. However, in most cases only male individuals retain divergent mitogenome inherited with spermatozoa. Additionally, in many of bivalves, unique mitochondrial features, like additional genes, gene duplication, gene extensions, mitochondrial introns, and recombination, were observed. In this study, we sequenced and assembled male-type mitogenomes of three Donax species. Comparative analysis of mitochondrial sequences revealed a lack of all seven NADH dehydrogenase subunits as well as the presence of three long additional open reading frames lacking identifiable homology to any of the existing genes.

Temporal analysis of cryopreservation effects on human sperm phospholipase C zeta expression profile: Laboratory preliminary findings

Objectives: The objectives of this study were to compare the sperm parameters and sperm phospholipase C zeta (PLC ζ) expression profile before and after vitrification. Materials and Methods: Pre-vitrification and post-vitrification analysis of semen samples of 14 infertile men was carried out for sperm parameters such as motility, vitality, and morphology by standard semen analysis procedures, whereas the proportion of sperms exhibiting PLC ζ and its localization pattern was assessed by indirect immunofluorescence. The temporal analysis was done thrice: over 1 week, 1 month, and 3 months of cryostorage. Statistical Analysis: Statistical analyses were performed using GraphPad Prism version 8.0.1 (San Diego, California, USA). Data were expressed as mean ± standard deviation. Parametric or non-parametric tests were employed for comparisons according to the normality of data distribution and the available data set. P value of 0.05 was considered statistically significant. Results: Vitrification was found to be associated with a decrease in the percentage of sperm motility (P ≤ 0.0001), vitality (P ≤ 0.0001), spermatozoa exhibiting normal morphology (P &gt; 0.05), and PLC ζ protein (P &gt; 0.05), however, the latter two, only, insignificantly. There was increased dominance in the post-acrosomal localization of PLC ζ after vitrification (P ≤ 0.001). Conclusions: The post-acrosomal localization of PLC ζ has been reported to have the highest positive correlation with oocyte fertilization and the present study showed the predominant pattern of the same. The implications for quality maintenance for long storage periods can be suggested as better sperm quality was observed at 3 months of storage during this study. This raises the hypothesis that the vitrification method of sperm cryopreservation may be the method of choice for routine clinical use in the assisted reproductive technology settings.

Effects of an Anti-Fertility Product on Reproductive Structures of Common Vole Males and Residues of Compounds.

Some rodent species cause significant damage to agriculture and forestry, and some can transmit pathogens to humans and livestock. The common vole (Microtus arvalis) is widespread in Europe, and its population outbreaks have resulted in massive crop loss. Bait-based fertility control could contribute to rodent pest management. Bait containing 4-vinylcyclohexene diepoxide (VCD) and triptolide (TP), registered as ContraPest®, was delivered to male common voles for 14 or 28 consecutive days. The effects on reproductive structures and residues in the liver and testes were assessed. There was no effect on testis weight, sperm viability, sperm motility and oxidative stress in sperm cells. Results regarding the mitochondrial membrane potential of sperm, DNA fragmentation and progressively motile sperm cells were inconclusive. However, there was an increase in morphological sperm defects in voles treated for 14/28 days and fewer normal sperm cells in voles treated for 28 days. There were no TP residues in the testes, few and low TP residues and no VCD residues in liver tissues, making considerable secondary exposure to non-target species unlikely. Treatments with VCD + TP seemed to have minor effects on the reproductive organs of males. Further studies should evaluate the effect of VCD + TP on females and on the reproductive success of common voles and other pest rodent species.

Exogenous Oxidative Stress in Human Spermatozoa Induces Opening of the Mitochondrial Permeability Transition Pore: Effect on Mitochondrial Function, Sperm Motility and Induction of Cell Death.

Oxidative stress (OS) and disrupted antioxidant defense mechanisms play a pivotal role in the etiology of male infertility. The alterations in reactive oxygen species (ROS) production and calcium (Ca2+) homeostasis are the main activators for the mitochondrial permeability transition pore (mPTP) opening. The mPTP opening is one of the main mechanisms involved in mitochondrial dysfunction in spermatozoa. This alteration in mitochondrial function adversely affects energy supply, sperm motility, and fertilizing capacity and contributes to the development of male infertility. In human spermatozoa, the mPTP opening has been associated with ionomycin-induced endogenous oxidative stress and peroxynitrite-induced nitrosative stress; however, the effect of exogenous oxidative stress on mPTP opening in sperm has not been evaluated. The aim of this study was to determine the effect of exogenous oxidative stress induced by hydrogen peroxide (H2O2) on mPTP opening, mitochondrial function, motility, and cell death markers in human spermatozoa. Human spermatozoa were incubated with 3 mmol/L of H2O2 for 60 min, and intracellular Ca2+ concentration, mPTP opening, mitochondrial membrane potential (ΔΨm), ATP levels, mitochondrial reactive oxygen species (mROS) production, phosphatidylserine (PS) externalization, DNA fragmentation, viability, and sperm motility were evaluated. H2O2-induced exogenous oxidative stress caused increased intracellular Ca2+, leading to subsequent mPTP opening and alteration of mitochondrial function, characterized by ΔΨm dissipation, decreased ATP levels, increased mROS production, and the subsequent alteration of sperm motility. Furthermore, H2O2-induced opening of mPTP was associated with the expression of apoptotic cell death markers including PS externalization and DNA fragmentation. These results highlight the role of exogenous oxidative stress in causing mitochondrial dysfunction, deterioration of sperm motility, and an increase in apoptotic cell death markers, including PS externalization and DNA fragmentation, through the mPTP opening. This study yielded new knowledge regarding the effects of this type of stress on mitochondrial function and specifically on mPTP opening, factors that can contribute to the development of male infertility, considering that the role of mPTP in mitochondrial dysfunction in human sperm is not completely elucidated. Therefore, these findings are relevant to understanding male infertility and may provide an in vitro model for further research aimed at improving human sperm quality.

Haplotype-resolved assembly of a pig genome using single-sperm sequencing

Single gamete cell sequencing together with long-read sequencing can reliably produce chromosome-level phased genomes. In this study, we employed PacBio HiFi and Hi-C sequencing on a male Landrace pig, coupled with single-sperm sequencing of its 102 sperm cells. A haplotype assembly method was developed based on long-read sequencing and sperm-phased markers. The chromosome-level phased assembly showed higher phasing accuracy than methods that rely only on HiFi reads. The use of single-sperm sequencing data enabled the construction of a genetic map, successfully mapping the sperm motility trait to a specific region on chromosome 1 (105.40–110.70 Mb). Furthermore, with the assistance of Y chromosome-bearing sperm data, 26.16 Mb Y chromosome sequences were assembled. We report a reliable approach for assembling chromosome-level phased genomes and reveal the potential of sperm population in basic biology research and sperm phenotype research.

Cytotoxic Oxidative Stress Effects of Neutrophil Extracellular Traps' Components on Cattle Spermatozoa.

Bovine spermatozoa are highly susceptible to oxidative stress (OS), and it is known to affect their cellular functions. The main leukocyte producers of reactive oxygen species (ROS) in mammalian semen are polymorphonuclear neutrophils (PMN). PMN activation can result in the formation of neutrophil extracellular traps (NETs), which have been shown to affect the motility and function of spermatozoa. However, OS effects on bull spermatozoa derived from individual NETs components have not been investigated. The hypothesis of this study was that specific NETs components might generate OS on bull spermatozoa. Bovine sperm cells were incubated with five NETs-associated molecules, including 30 μg/mL histone 2A (H2A), neutrophil elastase (NE), 1 μg/mL myeloperoxidase (MPO), cathepsin G (Cat-G), and cathelicidin LL37 (LL-37), for a time course ranging from 15 to 240 min. Fluorescence microscopy was used to evaluate the coincubation of bovine PMN and sperm cells. Within 15 min, H2A, NE, and LL-37 caused membrane disruption, while MPO and Cat-G caused OS on bull spermatozoa after 1 h of coincubation. NET formation was observed within 15 min of coincubation in co-cultures of bovine PMN/sperm cells. This study is the first to report on the role of cytotoxic OS effects caused by NETs-derived components in bovine sperm in vitro.

DNA methylation patterns in patients with asthenospermia and oligoasthenospermia

BackgroundSpermatogenesis is a highly regulated and complex process in which DNA methylation plays a crucial role. This study aimed to explore the differential methylation profiles in sperm DNA between patients with asthenospermia (AS) and healthy controls (HCs), those with oligoasthenospermia (OAS) and HCs, and patients with AS and those with OAS.ResultsSemen samples and clinical data were collected from five patients with AS, five patients with OAS, and six age-matched HCs. Reduced representation bisulfite sequencing (RRBS) was performed to identify differentially methylated regions (DMRs) in sperm cells among the different types of patients and HCs. A total of 6520, 28,019, and 16,432 DMRs were detected between AS and HC, OAS and HC, and AS and OAS groups, respectively. These DMRs were predominantly located within gene bodies and mapped to 2868, 9296, and 9090 genes in the respective groups. Of note, 12, 9, and 8 DMRs in each group were closely associated with spermatogenesis and male infertility. Furthermore, BDNF, SMARCB1, PIK3CA, and DDX27; RBMX and SPATA17; ASZ1, CDH1, and CHDH were identified as strong differentially methylated candidate genes in each group, respectively. Meanwhile, the GO analysis of DMR-associated genes in the AS vs. HC groups revealed that protein binding, cytoplasm, and transcription (DNA-templated) were the most enriched terms in the biological process (BP), cellular component (CC), and molecular function (MF), respectively. Likewise, in both the OAS vs. HC and AS vs. OAS groups, GO analysis revealed protein binding, nucleus, and transcription (DNA-templated) as the most enriched terms in BP, CC, and MF, respectively. Finally, the KEGG analysis of DMR-annotated genes and these genes at promoters suggested that metabolic pathways were the most significantly associated across all three groups.ConclusionsThe current study results revealed distinctive sperm DNA methylation patterns in the AS vs. HC and OAS vs. HC groups, particularly between patients with AS and those with OAS. The identification of key genes associated with spermatogenesis and male infertility in addition to the differentially enriched metabolic pathways may contribute to uncovering the potential pathogenesis in different types of abnormal sperm parameters.

Variation of sperm quality and circular RNA content in men exposed to environmental contamination with heavy metals in 'Land of Fires', Italy.

Can illegal discharge of toxic waste into the environment induce a new condition of morpho-epigenetic pathozoospermia in normozoospermic young men? Toxic environmental contaminants promote the onset of a new pathozoospermic condition in young normozoospermic men, consisting of morpho-functional defects and a sperm increase of low-quality circular RNA (circRNA) cargo, tightly linked to contaminant bioaccumulation in seminal plasma. Epidemiological findings have reported several reproductive anomalies depending on exposure to contaminants discharged into the environment, such as germ cell apoptosis, steroidogenesis defects, oxidative stress induction, blood-testis barrier dysfunctions, and poor sperm quality onset. In this scenario, a vast geographical area located in Campania, Italy, called the 'Land of Fires', has been associated with an excessive illegal discharge of toxic waste into the environment, negatively impacting human health, including male reproductive functions. Semen samples were obtained from healthy normozoospermic men divided into two experimental groups, consisting of men living in the 'Land of Fires' (LF; n = 80) or not (CTRL; n = 80), with age ranging from 25 to 40 years. The study was carried out following World Health Organization guidelines. Quality parameters of semen from CTRL- and LF-normozoospermic men were evaluated by computer-assisted semen analysis; high-quality spermatozoa from CTRL and LF groups (n = 80 for each experimental group) were obtained using a 80-40% discontinuous centrifugation gradient. Seminal plasma was collected following centrifugation and used for the dosage of chemical elements, dioxins and steroid hormones by liquid chromatography with tandem mass spectrometry. Sperm morpho-functional investigations (cellular morphology, acrosome maturation, IZUMO1 fertility marker analysis, plasma membrane lipid state, oxidative stress) were assessed on the purified high-quality spermatozoa fraction by immunochemistry/immunofluorescence and western blot analyses. Sperm circRNA cargo was evaluated by quantitative RT-PCR, and the physical interaction among circRNAs and fused in sarcoma (FUS) protein was detected using an RNA-binding protein immunoprecipitation assay. Protein immunoprecipitation experiments were carried out to demonstrate FUS/p-300 protein interaction in sperm cells. Lastly, in vitro lead (Pb) treatment of high-quality spermatozoa collected from normozoospermic controls was used to investigate a correlation between Pb accumulation and onset of the morpho-epigenetic pathozoospermic phenotype. Several morphological defects were identified in LF-spermatozoa, including: a significant increase (P < 0.05 versus CTRL) in the percentage of spermatozoa characterized by structural defects in sperm head and tail; and a high percentage (P < 0.01) of peanut agglutinin and IZUMO1 null signal cells. In agreement with these data, abnormal steroid hormone levels in LF seminal plasma suggest a premature acrosome reaction onset in LF-spermatozoa. The abnormal immunofluorescence signals of plasma membrane cholesterol complexes/lipid rafts organization (Filipin III and Flotillin-1) and of oxidative stress markers [3-nitrotyrosine and 3-nitrotyrosine and 4-hydroxy-2-nonenal] observed in LF-spermatozoa and associated with a sperm motility reduction (P < 0.01), demonstrated an affected membrane fluidity, potentially impacting sperm motility. Bioaccumulation of heavy metals and dioxins occurring in LF seminal plasma and a direct correlation between Pb and deregulated circRNAs related to high- and low-sperm quality was also revealed. In molecular terms, we demonstrated that Pb bioaccumulation promoted FUS hyperacetylation via physical interaction with p-300 and, in turn, its shuttling from sperm head to tail, significantly enhancing (P < 0.01 versus CTRL) the endogenous backsplicing of sperm low-quality circRNAs in LF-spermatozoa. Participants were interviewed to better understand their area of origin, their eating habits as well as their lifestyles, however any information incorrectly communicated or voluntarily omitted that could potentially compromise experimental group determination cannot be excluded. A possible association between seminal Pb content and other heavy metals in modulating sperm quality should be explored further. Future investigations will be performed in order to identify potential synergistic or anti-synergistic effects of heavy metals on male reproduction. Our study provides new findings regarding the effects of environmental contaminants on male reproduction, highlighting how a sperm phenotype classified as normozoospermic may potentially not match with a healthy morpho-functional and epigenetic one. Overall, our results improve the knowledge to allow a proper assessment of sperm quality through circRNAs as biomarkers to select spermatozoa with high morpho-epigenetic quality to use for ART. This study was supported by 'Convenzione Azienda Sanitaria Locale (ASL) Caserta, Regione Campania' (ASL CE Prot. N. 1217885/DIR. GE). The authors have no conflict of interest to declare. N/A.

Contextual analysis strategy leads to rise in conviction rate for sexual assault cases

Sexual assault cases typically involve intense physical contact between victim and perpetrator, resulting in a high chance of trace transfer. In addition the victim’s, and sometimes the suspect’s, declaration is available, and can help direct the forensic investigation. However, many cases are still closed because of insufficient proof or an unknown perpetrator.Previously in Belgium, the forensic analysis strategy was often limited to the detection of human sperm fluid and if not found, the forensic part of the case was closed.Therefore, the Forensic Advice service from the Belgian National Institute for Criminalistics and Criminology (NICC) together with the public prosecutor’s office of the Judicial district Antwerp, department Antwerp started “Project code 37”. In Belgium every type of crime receives a code and sexual assault cases are categorized under code 37. Because a forensic advisor focuses on the potential of forensic examination on physical objects, the project focused on cases with samples taken on the victim or clothing available.The goal of the project was to perform a contextual analysis in order to go beyond the standard sperm fluid-orientated approach and evaluate if this had a positive impact on the conviction rate. The project also aimed to evaluate the impact of the consistent use of toxicological analyses on conviction rates. Through this systematic contextual analysis, it became clear that four clear categories could be defined: (1) cases with an unknown perpetrator, (2) cases where the suspect denies all contact, (3) cases where the suspect denies (part of) the sexual contact, and (4) cases where only the consent of the victim is disputed. These categories all assumed one female victim and one male perpetrator. Together with the NICC’s DNA, trace evidence and toxicology lab, an analysis strategy was drafted for the four categories. All cases that do not fit this description were gathered in a fifth category (custom-made cases), as the victim’s/perpetrator’s sex has a significant effect on the forensic analysis strategy. At the end of the project, a comparison was made with the same set of cases from another judicial year where the standard analysis strategy was used. For all the categories for which data was available, there was a rise of the conviction rate and a decrease in the number of dismissed cases.

Environmental Influences on Frozen Epididymal Sperm Cell Viability in White-Tailed Deer

The objective of this study was to evaluate the association between Temperature and Humidity Index (THI), age, and post-collection time with post-thawing sperm cell parameters such as progressive motility, viability, morphology, and cytoplasmic droplets. Samples were collected from 4 male white-tailed deer after regulated hunting. THI was calculated on collection days. Two THI categories were determined, ≥70 and &lt;70. Thawed semen was placed on a slide and observed under a phase-contrast microscope to evaluate the progressively motile sperm cells. Morphology, viability, and cytoplasmic droplets were assessed on a smear stained with eosin-nigrosine and classified as normal or abnormal, viable or not viable, and present or absent, respectively. When THI was ≥70, the progressive motility (51.0 vs 30.67%), sperm viability (51.0 vs 36.33%), and the proportion of sperm cells with normal morphology (65.0 vs 37.67%) were higher than to days with a THI &lt;70, without differences in the presence of cytoplasmic droplets. The 4-years bucks had a higher proportion of sperm cells with normal morphology (81.0 vs 35.5 and 26.0%) and cytoplasmic droplets (27.0 vs. 12.0 and 7.5%) compared to 2- and 3-year-old animals. Samples processed after 4 hours (51.0%) had a higher progressive motility than samples processed after 6 and 8 hours (32.0 and 25.0%, respectively). Spermatozoa processed after 8 hours (30.0%) had a lower viability than spermatozoa processed after 4 hours (51.0%). Compared to samples processed after 6 and 8 hours, those processed after 2 and 4 hours had a higher proportion of sperm cells with normal morphology (26.0 and 6.0 vs 81.0 and 65.0%, respectively). Samples processed after 2 hours had a higher (27.0 vs. 12.0%) proportion of sperm cells with cytoplasmic droplets than to those processed after 6 hours. High THI favors progressive motility and sperm viability. The older bucks had a greater proportion of sperm cells with normal morphology and cytoplasmic droplets than to the young ones. The greater progressive motility, viability, morphology, and presence of cytoplasmic droplets were found when the samples were processed within the first 4 hours.

Impacts of Acrylamide on testis and spermatozoa.

Acrylamide (ACR) is an industrial chemical used to produce polyacrylamide, a synthetic polymer with a wide range of applications. Depending on the dosage, its presence in occupational and environmental sources poses potential health risks to humans and animals. ACR can be formed in starchy foods cooked at high temperatures. Its effects on human sperm are not well understood. Animal studies indicate that ACR induces toxicity in the male reproductive system through oxidative stress mechanisms. Exposure to ACR alters the normal structure of testicular tubules, leading to congestion, interstitial edema, degeneration of spermatogenic cells, formation of abnormal spermatid giant cells, and necrosis and apoptosis. It also disrupts the balance of important biomarkers such as malondialdehyde, nitric oxide, superoxide dismutase, catalase, and glutathione. ACR has a negative impact on mitochondrial function, antioxidant enzymes, ATP production, and sperm membrane integrity, resulting in decreased sperm quality. Furthermore, it interferes with the expression of steroidogenic genes associated with testosterone biosynthesis. This review explores the detrimental effects of ACR on sperm and testicular function and discusses the potential role of antioxidants in mitigating the adverse effects of ACR on male reproduction.

Assessing the Impact of the Novel Sperm Selection Technique 'Annexin-V Coated Polystyrene Bead Technique' on Mouse Assisted Reproductive Techniques Outcomes: Preliminary Findings

ICSI is one of the most commonly used techniques to treat infertility. The sperm selection for the procedure is done ‘randomly’ by the embryologist according to the motility and morphology parameters which is known not to reflect the potential of a sperm for fertilization, pregnancy and a healthy childbearing. Since the apoptosis rate is higher in sperm cells of infertile patients, it is more likely to choose an apoptotic sperm by the 'random selection method'. We recently introduced a novel sperm selection technique namely ‘Annexin-V coated polystrene bead technique’(APB-Tech), for the selection of non-apoptotic sperm cells. The principal of the technique is based on the binding affinity of an apoptotic sperm to ‘Annexin-V covered beads’ enabling to distinguish a viable and a healthy sperm by light microscopy. The aim of the present study was to observe the effects of this technique on ICSI outcomes in mice. Sibling-oocyte trial was conducted and the outcome measures were compared with the results of traditional sperm selection method. Embryo and blastocyst qualities and blastocyst development rates were significantly increased in APB-Tech group, while the other parameters were not affected. Promising results obtained from the technique reflect its promising potential as a new and powerful tool for sperm selection and thus infertility techniques.

The effects of saluang belum (Luvunga sarmentosa) root infusion on sperm motility, viability, and testicle histology in mice (Mus musculus)

Background: Men's infertility is frequently caused by a decrease in sperm quality. Herbal medicine, which is relatively cheap and easy to obtain, can be used to treat this condition. People in Central Borneo frequently consume boiled Saluang Belum root (Luvunga sarmentosa). L. sarmentosa root has the potential to improve sperm quality. Objective: To investigate the effects of Saluang Belum root infusion on sperm cell survival and testicle histology in mice (Mus musculus). Method: L. sarmentosa root was extracted using an infusion method, analysed and administered to mice at doses of 200 mg/kgBW, 400 mg/kgBW, and 600 mg/kgBW. Aquadest was used as a negative control for 15 days. On the 16th day, sperm motility and viability were assessed. The right testicles were removed and examined under a microscope at 100x and 400x magnification for five fields of view to examine mice testicle histology. Result: Infusion of L. sarmentosa contained triterpenoids, flavonoids, steroids, alkaloids, saponins, and phenols. Statistical tests of infused L. sarmentosa root on the motility, viability sperm and spermatogenic potentials of mice indicated that the doses of 200mg/kgBW, 400mg/kgBW, and 600mg/kgBW are significantly increased than control. Conclusion: L. sarmentosa root infusion increased sperm motility and viability, and the spermatogenic cells, as well as Leydig cells in mice.

Unraveling the Impact of the PROCA1 Mutation in Male Infertility: Incorporating Whole Exome Sequencing in Teratozoospermia Patients and Analyzing Proca1 Knockout Mice.

In the world, about 15% of couples are infertile, and nearly half of all infertility was caused by men. A large number of genetic mutations are thought to affect spermatogenesis by regulating acrosome formation. Here, we identified three patients harbouring the protein interacting with cyclin A1 (PROCA1) mutation by whole exome sequencing (WES) and Sanger sequencing among patients with predominantly acrosome-deficient teratozoospermia. However, the expression and roles of PROCA1 in infertile men remain unclear. We found that PROCA1 is predominantly expressed in the testis, where it is specifically localized to the acrosome of normal human sperm. Proca1 knockout (KO) mice were subsequently generated using CRISPR-Cas9 technology. However, Proca1 KO adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type (WT) mice. Testicular tissue or sperm morphology were not significantly different in Proca1 KO mice compared to WT mice. Expression of the acrosome markers PNA and SP56 in the acrosome was comparable between Proca1 KO and WT mice. In summary, these findings suggested that the PROCA1 mutation identified in humans does not affect acrosome biogenesis in mice.

Scriptaid is a prospective agent for improving human asthenozoospermic sample quality and fertilization rate in vitro.

Male infertility is a global issue caused by poor sperm quality, particularly motility. Enhancement of the sperm quality may improve the fertilization rate in assisted reproductive technology (ART) treatment. Scriptaid, with a novel human sperm motility-stimulating activity, has been investigated as a prospective agent for improving sperm quality and fertilization rate in ART. We evaluated the effects of Scriptaid on asthenozoospermic (AZS) semen, including its impact on motility stimulation and protective effects on cryopreservation and duration of motility, by computer-aided sperm analysis (CASA). Sperm quality improvement by Scriptaid was characterized by increased hyaluronan-binding activity, tyrosine phosphorylation, adenosine triphosphate (ATP) concentration, mitochondrial membrane potential, and an ameliorated AZS fertilization rate in clinical intracytoplasmic sperm injection (ICSI) experiments. Furthermore, our identification of active Scriptaid analogs and different metabolites induced by Scriptaid in spermatozoa lays a solid foundation for the future biomechanical exploration of sperm function. In summary, Scriptaid is a potential candidate for the treatment of male infertility in vitro as it improves sperm quality, prolongs sperm viability, and increases the fertilization rate.

Hamster spermatozoa incorporate hypotaurine via TauT for self-protection.

Mammalian spermatozoa actively generate reactive oxygen species (ROS) during capacitation, a maturational process necessary for fertilization in vivo. This study shows that hypotaurine, a precursor of taurine present in the oviduct, is incorporated and concentrated in hamster sperm cells via the taurine transporter, TauT, for cytoprotection against self-produced ROS. To achieve fertilization competence, mammalian spermatozoa undergo capacitation, during which they actively generate reactive oxygen species (ROS). Therefore, mammalian spermatozoa must protect themselves from these self-generated ROS. The mammalian oviductal fluid is rich in hypotaurine, a taurine precursor, which reportedly protects mammalian spermatozoa, including those of hamsters, from ROS; however, its precise mechanism remains unknown. This study aimed to elucidate the mechanism underlying hypotaurine-mediated protection of spermatozoa from ROS using hamsters, particularly focusing on the taurine/hypotaurine transporter TauT. The effect of hypotaurine on sperm motility and ROS levels was tested using sperm motility analysis and the CellROX dye and luminol assays. RNA sequencing analysis was performed to verify TauT expression. We found that hypotaurine was necessary for maintaining sperm motility and hyperactivated motility. Hypotaurine did not scavenge extracellular ROS but lowered intracellular ROS levels and was incorporated and concentrated in hamster spermatozoa. TauT was detected at both mRNA and protein levels. β-Alanine blocked hypotaurine transport, increased intracellular ROS levels, and inhibited hyperactivation. Elimination of Na+ or Cl- ions inhibited hypotaurine transport and increased intracellular ROS levels. Thus, these results indicated that hamster spermatozoa incorporated and concentrated hypotaurine in sperm cells via TauT to protect themselves from self-generated ROS.