Health risk and germ cell toxicity of five commercially available sachet waters in Nigeria: a public health concern.

Background: Sachet water is the most common form of portable water commercially available in Nigeria. Methodology: Using the murine sperm count and sperm abnormality assay, the germ cell toxicity of five common commercially available sachet waters in Nigeria was assessed in this study. The levels of hormones such as Follicle Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Total Testosterone (TT); and activities of catalase (CAT), alanine aminotransferase (ALT), superoxide dismutase (SOD), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were evaluated. The heavy metal and physicochemical parameters of the sachet waters were also analyzed. Healthy male mice were allowed to freely drink the sachet waters for 35 days after which they were sacrificed. Results: The findings indicated that the concentrations of some heavy metals (As, Cr, and Cd) in the sachet waters exceeded the limit by regulatory organizations. The data of the total carcinogenic risk (TCR) and total non-carcinogenic risk (THQ) of some heavy metals associated with the ingestion of sachet water for adults and children showed that the values exceeded the acceptable threshold, and thus, is indicative of a high non-carcinogenic and carcinogenic risks. The data of the sperm abnormality assay showed that in the exposed mice, the five sachet waters induced a statistically significant (P < 0.05) increase in abnormal sperm cells and a significantly lower mean sperm count. Additionally noted were changes in the serum activities of TT, FSH, ALP, AST, ALT, and LH. Conclusion: Thus, the sachet waters studied contained agents that can induce reproductive toxicity in exposed humans. This is of public health importance and calls for immediate action by regulatory bodies.

Optimization of spermatozoa analysis in mice: A comprehensive protocol.

Sperm quality is critical to predict reproductive alterations caused by immunological factors or toxicant agents. Yet, no detailed protocol has been published focusing on analyses of sperm parameters in mice. Our aim was to evaluate the most efficient diluent for mice sperm analyses and to optimize the sperm morphology classification, through the comparison of different staining methods. The diluents assessed were PBS (baseline), HTF, DMEM, 1 % BSA in PBS and 9 % skimmed powdered milk diluted in PBS. Spermatozoa were evaluated for vitality, motility, and morphology, smears were stained with Papanicolaou, HE, Giemsa, and Rapid staining. Sperm vitality and total motility reached better scores in milk based and DMEM diluents. HE raised up as an effective option since its combination with any of the diluents we tested, resulted in a fair staining, which was appropriated to evaluate mice spermatozoa. Finally, based on WHO manual, we have updated the current morphological classification for mice sperm, since we have detailed the head defects as well as included midpiece and tail defects on it. Taken together, we presented a useful, low cost, and reliable method to assess sperm morphology that could be employed worldwide by laboratories dedicated to study reproductive biology on mice model.

Selection based on the Breeding Soundness Evaluation is associated with the improvement of the reproductive quality of young Nellore bulls.

Breeding soundness evaluation (BSE) is the best methodology to estimate the fertility potential of future bulls and performing indirect selection for their fertility. However, the outcome of the BSE is influenced by several factors, including genetics, environment, and BSE guidelines. Herein, in this retrospective study, our first aim was to characterize the reasons for failure in 46,566 BSE from 2-year-old beef Bos indicus bulls (Nellore) born from 1997 to 2018. Our second aim was to determine whether or not BSE was associated with reproductive potential improvement of the bulls over the years. Due to changes in the BSE criteria, we used the same dataset, but only bulls born from 2002 to 2018 were included resulting in 35,856 BSE. For the second aim, the effect of the year and farm were included in the model of the multivariate logistic regression. We also determined if the main reasons for BSE failure decreased over time. Bulls were classified as approved (satisfactory potential breeders and qualified for natural breeding service) and not approved (deferred and unsatisfactory potential breeders). The reasons for BSE failure in Nellore bulls were poor semen quality (53.1 %) and physical defects (46.9 %), with the main physical defect being testis abnormalities (19.7 %). The overall percentage of bulls approved each year was 87.1 %, with no improvement over the years of study. However, the percentage of approved bulls at the first BSE increased over the years (P < 0.05). This increase was evident by a reduction in the difference between the overall percentage of the bulls approved vs the percentage of bulls approved at the first BSE. Furthermore, there was an increase in the percentage of bulls classified as satisfactory potential breeders in the BSE and an evident decrease in the percentage of bulls qualified only for natural breeding service (P < 0.05). In addition, an increase of the scrotal circumference (SC) of the herd was found (P < 0.05). These results indicate the overall quality of the bulls improved over the years. To associate and identify the main sperm abnormalities, 3461 not approved bulls were clustered. The most frequent defects were strongly coiled tail spermatozoa, proximal droplets, and acrosomal defects. Overall, there was no change in the frequency of bulls not approved by the sperm morphology nor the frequency of the main sperm abnormalities over the years. Nevertheless, the frequency of the defects remained very low, implying they were controlled. Additionally, abnormalities in the testis decreased over the years and was associated with the increase in the SC of the herd and a decrease of culled bulls due to low SC. In conclusion, this study demonstrates that there is an association between implementation and use of BSE with improvements in the reproductive quality of future generation bulls.

High rate of isolated teratospermia in a population of fertile men and the questionable clinical utility of sperm morphology.

Objective: To better understand the impact of sperm morphology on fertility by assessing sperm morphology in a population of known fertile men. Design: A prospective cohort study. Setting: Fertility center associated with the university. Patients: Healthy men >18 years of age were recruited to provide one semen sample before a vasectomy appointment scheduled between March 2020 and November 2022. Patients were included in the study when they had at least one biologic child and no history of difficulty achieving pregnancy or fertility procedures. Interventions: None. Main Outcome Measures: Sperm morphology. Results: A total of 68 patients (mean age 36.7 years) were included. Thirty-eight (55.9%) patients had 3% or lower normal sperm morphology, including two patients who had 0 normal morphology. The most common morphologic abnormalities were head-shaped defects (n = 59, 84.3%), followed by coiled tails (n = 14, 20.3%). Count, concentration, motility, and progressive motility were normal in >90% of patients. Conclusions: More than half (55.9%) of fertile male patients had lower than normal sperm morphology in our study. The results of our study further question the clinical relevance of sperm morphology on fertility outcomes and when the current approach in assessing morphology is too strict.

What sperm parameters effect blastocyst formation and quality during ICSI with severe male infertility.

Sperm-derived genetic material contributes half of the genome to the embryo, hence it's crucial to investigate which sperm parameter influences blastocyst formation in the intracytoplasmic sperm injection (ICSI) cycles with severe male infertility. The retrospective study analyzed 296 ICSI cycles with severe oligoasthenoteratozoospermia (OAT) and 99 ICSI cycles with preimplantation genetic testing for aneuploidy (PGT-A). Following the correlation analysis, data stratifications were performed in the OAT ICSI subgroup. The results showed that the matching blastocyst in the OAT ICSI cycles had inferior sperm parameters. DFI and sperm morphology had an influence on the blastocyst formation rate and the high-quality blastocysts formation rate on Day6, but no significant effect on the blastocyst development on Day 5. The high-quality blastocysts formation rate and ratio of high-quality blastocyst on Day 6 were demonstrably better in the subgroup of the teratozoospermic morphology when DFI was within the normal range. In the case of the normal sperm morphology, no statistically significant difference was found in blastocyst development, although there were numerical differences within different DFI subgroups. It was concluded that the blastocyst quality and development declined with the decreased sperm qualities.

Chronic unpredictable stress (CUS) reduced phoenixin expression, induced abnormal sperm and testis morphology in male rats.

Chronic stress caused by prolonged emotional pressure can lead to various physiological issues, including reproductive dysfunction. Although reproductive problems can also induce chronic stress, the impact of chronic stress-induced reproductive dysfunction remains contentious. This study investigates the effects of chronic unpredictable stress (CUS) on reproductive neuropeptides, sperm quality, and testicular morphology. Sixteen twelve-week-old Sprague Dawley rats were divided into two groups: a non-stress control group and a CUS-induced group. The CUS regimen involved various stressors over 28 days, with both groups undergoing behavioural assessments through sucrose-preference and forced-swim tests. Hypothalamic gene expression levels of CRH, PNX, GPR173, kisspeptin, GnRH, GnIH, and spexin neuropeptides were measured via qPCR, while plasma cortisol, luteinizing hormone (LH), and testosterone concentrations were quantified using ELISA. Seminal fluid and testis samples were collected for sperm analysis and histopathological evaluation, respectively. Results showed altered behaviours in CUS-induced rats, reflecting stress impacts. Hypothalamic corticotropin-releasing hormone (CRH) expression and plasma cortisol levels were significantly higher in CUS-induced rats compared to controls (p < 0.05). Conversely, phoenixin (PNX) expression decreased in the CUS group (p < 0.05), while kisspeptin, spexin, and gonadotropin-inhibitory hormone (GnIH) levels showed no significant differences between groups. Despite a significant increase in GnRH expression (p < 0.05), plasma LH and testosterone concentrations were significantly lower (p < 0.05) in CUS-induced rats. Histopathological analysis revealed abnormal testis morphology in CUS-induced rats, including disrupted architecture, visible interstitial spaces between seminiferous tubules, and absence of spermatogenesis. In conclusion, CUS affects reproductive function by modulating PNX and GnRH expression, influencing cortisol levels, and subsequently reducing plasma LH and testosterone concentrations. This study highlights the complex interplay between chronic stress and reproductive health, emphasizing the significant impact of stress on reproductive functions.

The Effect of Neutral Alpha-Glucosidase on Semen Parameters.

INTRODUCTION: The objective of this study was to investigate the relationship between the activity of neutral α-glucosidase in seminal plasma and semen quality and to explore the effect of secretory capability of the epididymis on male fertility. METHODS: A retrospective analysis of 542 men treated in the Center for Reproductive Medicine and Infertility from February to December 2022, the semen parameters and neutral α-glucosidase were tested and compared among different groups. These 542 men included normozoospermia, oligospermia, asthenospermia, and teratozoospermia. RESULTS: There was statistical difference in neutral alpha-glucosidase (NAG) level among different groups with different sperm concentration, motility, and morphology (p < 0.001). The NAG activity in seminal plasma was positively correlated with ejaculate volume and sperm concentration; meanwhile, a very weak positive correlation was found between NAG level and sperm motility, sperm morphology, respectively. CONCLUSIONS: Our results indicated that the secretion of NAG affected the volume, concentration, motility, and morphology of sperm to a certain extent. Given that NAG is a specific and marker enzyme in epididymis, where is the site of sperm maturation, we can conclude that there is a close relationship between NAG and sperm quality. Therefore, seminal plasma NAG has a definite clinical value in helping diagnosis of male infertility.

An association weight matrix identified biological pathways associated with bull fertility traits in a multi-breed population.

Using seven indicator traits, we investigated the genetic basis of bull fertility and predicted gene interactions from SNP associations. We used percent normal sperm as the key phenotype for the association weight matrix-partial correlation information theory (AWM-PCIT) approach. Beyond a simple list of candidate genes, AWM-PCIT predicts significant gene interactions and associations for the selected traits. These interactions formed a network of 537 genes: 38 genes were transcription cofactors, and 41 genes were transcription factors. The network displayed two distinct clusters, one with 294 genes and another with 243 genes. The network is enriched in fertility-associated pathways: steroid biosynthesis, p53 signalling, and the pentose phosphate pathway. Enrichment analysis also highlighted gene ontology terms associated with 'regulation of neurotransmitter secretion' and 'chromatin formation'. Our network recapitulates some genes previously implicated in another network built with lower-density genotypes. Sequence-level data also highlights additional candidate genes relevant to bull fertility, such as FOXO4, FOXP3, GATA1, CYP27B1, and EBP. A trio of regulatory genes-KDM5C, LRRK2, and PME-was deemed core to the network because of their overarching connections. This trio probably influences bull fertility through their interaction with genes, both known and unknown as to their role in male fertility. Future studies may target the trio and their target genes to enrich our understanding of male fertility further.

High percentage of midpiece defects in Brangus bull sperm with no reduction in sperm kinematics.

The study investigated midpiece defects in sperm from a 5-year-old Brangus bull with a high rate of semen batch rejection, due to morphologically abnormal sperm, with no reduction in sperm kinematics. A comprehensive evaluation was conducted over a 16-month period, involving 28 ejaculates. Notably, despite the high proportion of midpiece defects (average 37.73%, from 3% to 58%), the study revealed stable sperm production, with no discernible differences in the kinematic data before and after cryopreservation. Electron microscopy identified discontinuities in the mitochondrial sheath, characteristic of midpiece aplasia (MPA). The anomalies were attributed to be of genetic origin, as other predisposing factors were absent. Additionally, the electron microscopy unveiled plasma membrane defects, vacuoles and chromatin decondensation, consistent with previous findings linking acrosome abnormalities with midpiece defects. The findings underscored the necessity of conducting thorough laboratory evaluations before releasing cryopreserved semen for commercialization. Despite substantial morphological alterations, the initial semen evaluation data indicated acceptable levels of sperm kinematics, emphasizing the resilience of sperm production to severe morphological changes. This case report serves as a contribution to the understanding of midpiece defects in bull sperm, emphasizing the need for meticulous evaluation and quality control in semen processing and commercialization.

Age, sexual abstinence duration, sperm morphology, and motility are predictors of sperm DNA fragmentation.

Purpose: Sperm DNA fragmentation (SDF) has recently received attention as a cause of male infertility. However, SDF cannot be fully assessed using conventional semen parameter evaluations alone. Therefore, the authors aimed to elucidate the relationship between SDF and sperm parameters via computer-assisted sperm analysis (CASA) to improve treatment strategies in reproductive medicine. Methods: This retrospective observational study analyzed the relationship between sperm parameters assessed by CASA and SDF values determined by the TUNEL assay in 359 patients who visited the Mie University Hospital for infertility treatment. The methodology involved semen analyses covering concentration, motility, and morphology, followed by SDF quantification using the flow cytometry. Results: Statistical analysis revealed significant correlations between SDF and various factors, including age, sexual abstinence period, and specific CASA-measured parameters. Notably, lower sperm motility rates and abnormal head dimensions were associated with higher SDF values, indicating that these parameters were predictive of SDF. Conclusions: This study highlights the importance of sperm motility and head morphology as indicators of SDF, suggesting their usefulness in assessing male fertility. These findings demonstrate the efficacy of detailed sperm analysis, potentially increasing the success rate of assisted reproductive technologies by improving sperm selection criteria.

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.

Ascorbic acid attenuates gasoline-induced testicular toxicity, sperm quality deterioration, and testosterone imbalance in rats.

The present study evaluated the protective effect of ascorbic acid (ASCB) against gasoline fumes (PET) induced testicular oxidative stress, sperm toxicity, and testosterone imbalance in Wistar rats. Twenty-four (24) male albino rats (75 ± 16 g) were randomized into three experimental groups (N = 8). The control group: received normal saline, PET group: exposed to PET 6 h daily by inhalation in an exposure chamber and PET + 200 mg ASCB/kg body weight group: exposed to PET 6 h daily by inhalation and administered ASCB per os. Treatment of ASCB and PET exposure was done thrice and five times weekly for a period of 10 weeks respectively. ASCB co-treatment prevented PET-induced increases in the oxidative stress markers (glutathione, glutathione S-transferase, superoxide dismutase, catalase, hydrogen peroxide generation, nitric oxide, and lipid peroxidation) and serum testosterone concentration (p < .05). Sperm quality was low and those with damaged heads and tails increased alongside histological injuries in the PET-exposed rats, which were also minimized with ASCB administration. ASCB protected against PET-induced oxidative stress, sperm, and testis damage in rats.

Genome-Wide Association Analysis of Semen Characteristics in Piétrain Boars.

Since artificial insemination is common practice in pig breeding, the quality and persistence of the semen are decisive for the usability of individual boars. In the current study, genome-wide association analyses were performed to investigate the genetic variability underlying phenotypic variations in semen characteristics. These traits comprise sperm morphology and sperm motility under different temporal and thermal storage conditions, in addition to standard semen quality parameters. Two consecutive samples of the fourth and fifth ejaculates from the same boar were comprehensively analyzed in a genotyped Piétrain boar population. A total of 13 genomic regions on different chromosomes were identified that contain single-nucleotide polymorphisms significantly associated with these traits. Subsequent analysis of the genomic regions revealed candidate genes described to be involved in spermatogenesis, such as FOXL3, GPER1, PDGFA, PRKAR1B, SNRK, SUN1, and TSPO, and sperm motility, including ARRDC4, CEP78, DNAAF5, and GPER1. Some of these genes were also associated with male fertility or infertility in mammals (e.g., CEP78, GPER1). The analyses based on these laboriously determined and valuable phenotypes contribute to a better understanding of the genetic background of male fertility traits in pigs and could prospectively contribute to the improvement of sperm quality through breeding approaches.

Relationship between male aging and semen quality: a retrospective study on over 2500 men.

PURPOSE: We aimed to evaluate whether and to what extent an association exists between male aging and worsening of semen parameters and to determine whether a threshold age can be identified above which the decline in semen quality becomes statistically significant. METHODS: 2612 men (age: 16-56 years) attending an andrology outpatient clinic for semen analysis and clinical evaluation were studied. Semen analyses were performed according to the ongoing WHO-recommended procedures. Total motile count (TMC) and total progressive motile count (TPMC) were calculated by multiplying total sperm number by total motility and progressive motility, respectively. RESULTS: Significant negative correlations were found between age and total motility (r = - 0.131, p < 0.0001), progressive motility (r = - 0.112, p < 0.0001), TPMC (r = - 0.042, p = 0.037), and normal sperm morphology (r = - 0.053, p = 0.007). All these associations persisted in multivariate regression models adjusted for abstinence time, smoking, history of male accessory gland infections, varicocele and the year in which semen analysis was performed. When comparisons were performed among quartiles of increasing age, the fourth quartile, corresponding to the age group > 40 years, was associated with a significant decrease in total and progressive motility. An earlier decline in the TPMC and percentage of normal forms was also observed. CONCLUSION: Advancing male age exhibits an independent association with a decrease in the percentage of motile and morphologically normal spermatozoa, with greater evidence from the age of > 40 years. Further studies are warranted to elucidate the mechanisms and clinical reflections of these associations.

Associations between Sperm Epigenetic Age and Semen Parameters: An Evaluation of Clinical and Non-Clinical Cohorts.

The well-documented relationship between chronological age and the sperm methylome has allowed for the construction of epigenetic clocks that estimate the biological age of sperm based on DNA methylation, which we previously termed sperm epigenetic age (SEA). Our lab demonstrated that SEA is positively associated with the time taken to achieve pregnancy; however, its relationship with semen parameters is unknown. A total of 379 men from the Longitudinal Investigation of Fertility and Environment (LIFE) study, a non-clinical cohort, and 192 men seeking fertility treatment from the Sperm Environmental Epigenetics and Development Study (SEEDS) were included in the study. Semen analyses were conducted for both cohorts, and SEA was previously generated using a machine learning algorithm and DNA methylation array data. Association analyses were conducted via multivariable linear regression models adjusting for BMI and smoking status. We found that SEA was not associated with standard semen characteristics in SEEDS and LIFE cohorts. However, SEA was significantly associated with higher sperm head length and perimeter, the presence of pyriform and tapered sperm, and lower sperm elongation factor in the LIFE study (p < 0.05). Based on our results, SEA is mostly associated with defects in sperm head morphological factors that are less commonly evaluated during male infertility assessments. SEA shows promise to be an independent biomarker of sperm quality to assess male fecundity.

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.

The Bull Breeding Soundness Examination and Its Application in the Production Setting.

breeding soundness for several reasons and at several times during their life span. These include before sale for the reason of affirming their sale ability or before a breeding season to determine their readiness for breeding. Bulls may also be evaluated for diagnostic purposes. The breeding soundness examination (BSE) is universally promoted as an important management tool, but there continues to be a level of inconsistency in its performance. A complete bull BSE consists of a thorough physical examination including internal and external reproductive tract, measurement of the circumference of the scrotum and evaluation of individual sperm motility and morphology.

Cryopreservation-Induced Morphological Changes in Freshwater Fish Sperm: A Systematic Review.

A systematic review was performed to summarize the scientific evidence and critically evaluate the effects of cryopreservation on sperm morphology in freshwater fish, and to assess the methodologies for sperm morphology classification. The search strategy was applied to four electronic databases (CAB Direct, Pub Med, Scopus, and ISI Web of Science). The main inclusion criteria involved studies on semen from freshwater fish subjected to the cryopreservation process and evaluation of sperm quality through morphology. The risk of bias was assessed with respect to randomization, allocation concealment, blinding, incomplete outcome data, and selective reporting. A total of 6 publications reporting sperm cryopreservation from 4 species with a total 74 fish individuals were included in this review. A high methodological variability among the results of the studies was observed due to the species-specific protocols and diversity of freshwater fish species studied. All included studies reported negative effects of cryopreservation on sperm quality, especially morphology, highlighting the increase in incidence of sperm abnormalities. However, only five studies statistically compared abnormalities between groups (fresh and cryopreserved sperm). Our results suggest the need to elaborate on a new morphological classification of fish spermatozoa, by considering the structure and physiology of fish sperm. This classification should be developed based on the sperm characterization and observing damage caused by different cryopreservation protocols.

A variant in sperm-specific glycolytic enzyme enolase 4 (ENO4) causes human male infertility.

BACKGROUND: Although defects in sperm morphology and physiology lead to male infertility, in many instances, the exact disruption of molecular pathways in a given patient is often unknown. The glycolytic pathway is an essential process to supply energy in sperm cell motility. Enolase 4 (ENO4) is crucial for the glycolytic process, which provides the energy for sperm cells in motility. ENO4 is located in the sperm principal piece and is essential for the motility and organization of the sperm flagellum. In the present study, we characterized a family with asthenozoospermia and abnormal sperm morphology as a result of a variant in the enolase 4 (ENO4) gene. METHODS: Computer-assisted semen analysis, papanicolaou smear staining and scanning electron microscopy were used to examine sperm motility and morphology for semen analysis in patients. For genetic analysis, whole-exome sequencing followed by Sanger sequencing was performed. RESULTS: Two brothers in a consanguineous family were being clinically investigated for sperm motility and morphology issues. Genetic analysis by whole-exome sequencing revealed a homozygous variant [c.293A>G, p.(Lys98Arg)] in the ENO4 gene that segregated with infertility in the family, shared by affected but not controls. CONCLUSIONS: In view of the association of asthenozoospermia and abnormal sperm morphology in Eno4 knockout mice, we consider this to be the first report describing the involvement of ENO4 gene in human male infertility. We also explore the possible involvement of another variant in explaining other phenotypic features in this family.

Ancillary Methods for Semen Evaluation.

The cause of subfertility or poor fertility in naturally mated bulls should be differentiated from impotentia coeundi, generandi, or erigendi prior to ancillary semen evaluation. Bulls used for artificial insemination may undergo ancillary semen evaluation following low fertility rates as judged by poor conception or low pregnancy rates. Morphologically abnormal sperm have long been associated with bull subfertility and infertility. Some morphological defects such as improper sperm chromatin condensation are not visible using traditional light microscopy and require specialized staining. Ancillary semen evaluation is useful in cases where the reason for low or absence of fertility needs to be identified. As compared to SEM, TEM can be extremely useful for identifying minuscule acrosome defects, issues with chromatin, and centrosome defects and is considered the gold standard method for the identification of midpiece and tail defects.