Search

Many scientific studies often assumed that the most reliable methods for assessing sperm motility are those that give the highest values, and this leads to misinterpretation of the results. This study aims to propose an objective method to validate sperm motility reliability. Bovine and porcine semen samples were split into two equal fractions. Fraction A was kept alive with a motile population considered at maximum proportion, while fraction B was killed with 0% motile population. A range of motile/non motile sperm was performed by mixing both fractions. The Motility Ratio method, comparing measured and theoretical motility, was validated using LEJA slide and IVOS II and applied to other slides. All slides showed strong Concordance Correlation Coefficient between measured and theoretical motility. However, with IVOS II, LEJA slide showed the lowest bias (< 1) while MAKLER or coverslip showed higher bias (> 2 and > 7 respectively) between measured and theoretical motility. This study shows that the best sperm motility is not always the true motility and highlights the importance of implementing a gold standard methodology for motility reliability such as The Motility Ratio method., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Abstract: In vitro semen purification techniques have been developed that seek to mimic the in vivo selection process in order to generate the highest possible chance of oocyte fertilization following artificial insemination. Numerous methods have been developed to isolate functional spermatozoa for artificial insemination, yet only one method, single-layer centrifugation using commercial preparations like EquiPure, has been widely employed. In this study, we have introduced a novel approach for isolating spermatozoa and compared their quality to those isolated using EquiPure. The AI port system (Memphasys, Ltd. in Sydney, Australia) features a disposable cartridge with an inoculation chamber for depositing extended semen and a harvest chamber for extracting isolated spermatozoa. These chambers are separated by a 5 µm polyethylene terephthalate (PETE) membrane, allowing highly motile spermatozoa to migrate from the inoculation chamber to the harvest chamber over a 20-minute period. This migration effectively leaves behind seminal plasma and other cell types, such as leukocytes. Comparative analyses between spermatozoa isolated with the AI port and EquiPure demonstrated that, across all measured sperm parameters, including yield, vitality, motility, morphology, DNA fragmentation, and mitochondrial superoxide generation, the AI port-isolated cells exhibited comparable or superior performance, particularly in terms of DNA fragmentation. In summary, the AI port system demonstrates the potential to efficiently isolate high-quality spermatozoa, possibly offering a cost-effective and user-friendly alternative that may enhance the success rates of artificial insemination in breeding programs., Lay Summary: This study aimed to create a new method for refining stallion semen to increase the likelihood of a successful pregnancy through artificial insemination. While there are existing techniques for isolating high-quality sperm, the most common involves a complicated process using a centrifuge, which spins the semen to separate it. This research introduces a new approach called the AI port system that uses a disposable cartridge with two separate chambers for putting in semen and getting out isolated sperm. A membrane between the chambers acts like a filter, letting highly motile sperm swim across, leaving behind unwanted substances like bacteria and blood cells. Compared to the centrifugation method, the AI port system effectively produces sperm with comparable or better quality in various aspects, including vitality, movement, shape, DNA integrity, and energy production. In summary, the AI port system is an easy-to-use alternative with the potential to improve the success of artificial insemination in horse breeding programs.

The objective of this study was to assess the ability of camel spermatozoa to bind in the Hyaluronan Binding Assay (HBA), to determine if conventional sperm quality parameters, in vitro fertilization capacity, and precursor of A-Kinase Anchoring Protein 4 (proAKAP4) values correlate with HBA results. The potential to predict post-thaw fertilization performance from HBA for fresh dromedary camel sperm was also evaluated. Semen samples were collected and assessed both fresh and post thawing, at 0 h and 1.5 h. Conventional semen analysis, HBA, and a proAKAP4 biomarker-test were used to validate sperm quality. A heterologous sperm penetration assay using zona pellucida-free goat oocytes was used to assess in vitro sperm fertilizing capacity. The results showed that dromedary camel spermatozoa bound to hyaluronan with no correlation between results from fresh samples and after thawing. Furthermore, the proAKAP4 test results showed a negative correlation with HBA at 0 h after thawing (r = - 0.62; P = 0.03). In the conventional analysis, only progressive motility (r = 0.65; P = 0.02) and straightness correlated with HBA for fresh semen (r = 0.69; P = 0.01). In the sperm penetration assay, a moderate but non-significant correlation was identified between fresh sperm HBA and penetration (r = 0.52; P = 0.07). In conclusion, results suggested that HBA can be used to assess camel sperm properties, but further investigation is needed to understand its correlation with other sperm quality parameters. The HBA score from fresh dromedary camel sperm was unable to predict post-thaw fertilization performance., Competing Interests: Declaration of Competing Interest No conflict of interest to declare., (Copyright © 2024. Published by Elsevier B.V.)

Characterization of long non-coding telomeric repeat-containing RNAs in sperm of normozoospermic and oligoasthenozoospermic men as new biomarker of idiopathic male infertility. We conducted an observational prospective study with two groups of men with normal or orligoasthenozoospermic spermiogram, aged 40 and above. Fertility parameters were analyzed in men undergoing intracytoplasmic sperm injection with donor oocytes, to avoid the female factor. Telomeric RNAs and telomere length were measured by quantitative fluorescent in situ hybridization. Data from seminal parameters and in-vitro fertilization were assessed according to IVIRMA protocols. Patients with oligoasthenozoospermia, who had worse seminal parameters, also obtained embryos with lower inner-cell-mass quality (p = 0.04), despite using donor oocytes. While mean levels of telomeric RNAs were similar for both groups, the percentage of spermatozoa with more than 3 foci was higher in oligoasthenozoospermic men (p = 0.02). Regarding telomere length, oligoasthenozoospermic men had shorter mean, a higher accumulation of short telomeres (15th percentile; p = 0.03) and a lower percentage of very-long telomeres (85th percentile; p = 0.01). Finally, a positive correlation was found between telomeric-RNAs intensity and total progressive motility in the spermatozoa of normozoospermic patients (r = 0.5; p = 0.03). Telomeric parameters were altered in the spermatozoa of the oligoasthenozoospermic group, which also showed lower quality embryos. Interestingly, in the normozoospermic group, a correlation was found between progressive motility and telomeric RNA levels, suggesting that they could be a good biomarker of sperm quality. Further studies are required to confirm these results and translate them into the clinical practice.Trial registration number: 1711-MAD-109-CB, 07/07/2021., (© 2024. The Author(s), under exclusive licence to Society for Reproductive Investigation.)

This study aimed to investigate the influence of various sperm quality characteristics, including morphology, motility, and count, on the success rates of clinical pregnancy achieved through assisted reproductive technologies (ART) such as in-vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI), and intrauterine insemination (IUI). The secondary objective was to assess the impact of these sperm parameters on the clinical pregnancy rate that resulted in the detection of a fetal heartbeat during the 11th week of gestation, a crucial milestone in successful ART-derived pregnancies. The researchers employed a retrospective analysis, evaluating data from 734 couples undergoing IVF/ICSI and 1197 couples undergoing IUI across two infertility centers. Exclusion criteria included cases involving donated eggs or sperm, surrogate uteri, and infertile couples with combined male and female factors. Five ensemble machine-learning models were utilized to predict the clinical pregnancy success rates. The Random Forest (RF) model achieved the highest mean accuracy (0.72) and area under the curve (AUC) (0.80), outperforming the other models for both IVF/ICSI and IUI procedures. The Shapley Additive Explanations (SHAP) value analysis revealed that for IUI cycles, all three sperm parameters (morphology, motility, and count) had significant negative impacts on the prediction of clinical pregnancy success. In contrast, for IVF/ICSI cycles, sperm motility had a positive effect, while sperm morphology and count were negative factors. In cycles with 1 to 5 retrieved eggs, sperm motility, and count, they positively affected the clinical pregnancy rate. The study also identified cut-off values for sperm count, with 54 and 35 being the respective thresholds for IVF/ICSI and IUI. Additionally, a significant cut-off point 30 was found for the sperm morphology parameter across all procedures. This study underscores the immense potential of leveraging ensemble machine learning models with traditional sperm quality assessments. This integrated approach can elevate the precision and personalization of clinical decision-making in the field of assisted reproductive technologies, ultimately offering more hope and better outcomes for couples struggling with infertility., (© 2024. The Author(s).)

Our research focuses on semen collection from 42 African penguin males, grouped by age, habituation levels, and reproductive season timing. We assess behavioral and physiological responses to dorso-abdominal massage, evaluating species-specific seminal traits using conventional and advanced methods. Positive behavioral responses corresponded with successful semen collection. Young and human-reared males exhibited more favorable behaviors, and samples containing spermatozoa were more likely collected during the reproductive season. Age did not influence sample collectability or spermatozoa presence, though mature males produced higher semen volumes. Young males exhibited more sperm morphological abnormalities, especially outside the breeding season. Sperm concentration and number per ejaculate showed no significant differences across age or seasonal groups. Young males had higher motile and progressive sperm percentages, while mature males had more static sperm. Additionally, percentages of live sperm and intact acrosomes were higher in mature males. Young males and samples from non-reproductive season presented more dead sperm with damaged acrosomes. Plasma membrane integrity positively correlated with age for live sperm and negatively for dying sperm. This research demonstrates the feasibility of semen collection from African penguins regardless of habituation level, fills the knowledge gap by describing sperm morphological abnormalities, and provides insights into using flow cytometry in Sphenisciformes., (© 2024. The Author(s).)

The reproductive efficiency of livestock is crucial for agricultural productivity and economic sustainability. One critical factor in successful fertilization and the viability of offspring is the quality of semen. Poor semen quality, especially in frozen-thawed semen used in artificial insemination (AI) have been shown to influence conception outcomes, resulting a negative impact on livestock production. Recent advancements in genetic research have identified specific markers linked to semen quality traits in various livestock species, such as cattle, sheep, goats, pigs, buffalo, and equines. These genetic markers are essential in screening males for breeding suitability, which in turn enhances selective breeding programs. Understanding these markers is crucial for improving reproductive performance and increasing productivity in livestock populations. This review offers a comprehensive overview of the genetic markers associated with semen quality in key livestock. It explores the underlying genetic mechanisms and their practical implications in animal breeding and management. The review underscores the importance of integrating genetic insights into breeding strategies to optimize reproductive efficiency and ensure the sustainable development of livestock industries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Khan, Chen, Naz, Liu, Liang, Chen, Kou, Liu, Ashraf, Han, Peng, Wang and Zahoor.)

Objectives: To determine if histological evaluation of the vasa is useful when post-vasectomy semen analysis (PVSA) compliance is low and to determine whether compliance could be predicted., Methods: A retrospective evaluation of patients undergoing vasectomy between 2018 and 2022 was undertaken. A comparison of the PVSA between three vasa histological categorisations was made: complete divisions, incomplete division(s), absent vas(a). A multivariate model was constructed to predict PVSA compliance., Results: From 388 patients, 191 (49.2%) undertook PVSA. Four patients had a revision of vasectomy. On 3 occasions this was due to the histology findings and once from semen analysis with normal histology. There was no significant difference in the number of azoospermic samples (95.4% vs 91.2%, ns), of samples with presence of Rare Non-Motile Sperm (RNMS) (2.6% vs 8.8%, ns) and those with sperm present (2.0 vs 0%, ns), between patients with complete division of the vasa on both sides and those with incomplete division on one side respectively. There was no difference in patient characteristics between those who complied with PVSA and those who did not., Conclusions: This paper suggests that there is a role for histological evaluation of the vasa when PVSA compliance is poor. Incompletely divided vasa on histology are not associated with an adverse PVSA.

Objective assessment of sperm morphology is an essential component for assessing ejaculate quality. Due to economic limitations, investigators often divert to conducting observational studies instead of experimental ones, which provide the strongest statistical power, yielding more heterogeneous data regardless of the number of data sources (barns/farms). Using such data inevitably leads to higher variances of estimates, which negatively impacts the statistical power of a study. In this article, we describe a statistical methodology called finite mixture modeling (FMM), which, based on the supplied data and assumed number of sub-classes, classifies the data into two or more homogeneous types of distributions and determines their fractional size relative to the entire cohort. The goal is to use statistical methods that will confound the variance of the sample. A figure from a previous publication was used to generate simulated data (n=1559) on the cytoplasmic droplet rate. We identified that a bi-modal distribution with two latent classes best described the simulated data. Post-hoc estimation showed that about 80 % of observations belonged to latent class 1, with 20 % in latent class 2. The FMM methodology identified a cutoff point of 8.7 %. Finally, when estimating the standard error for the total cohort, the FMM methodology yielded a 40 % reduction in the standard error compared to standard methodologies. In conclusion, here we show that FMM successfully confounded the variance of the data and, as such, yielded lower estimates of the variance than standard methodologies, increasing the statistical power of the cohort., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024. Published by Elsevier B.V.)

Among the many factors with a proven relation to semen quality and male fertility, the determination of seminal plasma cytokines provides a promising direction for research into the identification of factors connected with male infertility. The interleukins: IL-1α, -1β, -2, -4, -6, -8, -10, -12p40, -12p70, -18, IFNγ, and GM-CSF, total oxidant (TOS) and antioxidant (TAS) status, were simultaneously examined in seminal plasmas and blood sera in terato- (n = 32), asthenoterato- (n = 33), and oligoasthenoteratozoospermic (n = 29) infertile men and in normozoospermic fertile men (n = 20). Our research shows different cytokine composition of the sera and seminal plasmas in all studied groups, along with much higher concentrations of seminal plasma GM-CSF, IFNγ, IL-1α, IL-4, IL-6, and IL-8 and lower IL-18 and TOS in the comparison to their sera levels. The seminal plasma concentrations of GM-CSF, IFNγ, IL-1α, -4, and -6 differ significantly between fertile and infertile as well as between teratozoospermic, asthenoteratozoospermic, and oligoasthenoteratozoospermic groups. The indication of the cause of different concentrations of cytokines in seminal plasmas of infertile men, and their associations with semen parameters and oxidative status, may be a promising direction for the search for new therapeutic targets that would directly affect the cells and tissues of male reproductive organs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Purpose: Chromosomal abnormalities play an important role in male infertility, which is becoming a significant issue in human fertility. Aim of this study was to evaluate the incidence of spermatic aneuploidies and diploidies in human sperm, according to semen parameters., Methods: We performed semen analysis according to the 6th edition of WHO criteria in 50 male subjects; samples were divided into normozoospermic (n = 23) or those with altered seminal parameters (n = 27). To assess chromosomal numerical alterations of sperm, fluorescence in situ hybridization (FISH) was used., Result: A significant increase in aneuploidies and diploidies was observed in samples with altered seminal parameters. Furthermore, stratifying this group, we observed a significant increase in aneuploidies and total abnormalities in oligozoospermic, asthenoteratozoospermic (AT), and oligoteratoasthenozoospermic (OAT) samples compared to normozoospermic., Conclusion: Our results showed the correlation between altered seminal parameters and numerical chromosomal abnormalities, confirming that sperm FISH analysis could be an additional clinical tool to assess reproductive potential in infertile males. Moreover, our results point to the importance of updating the normality ranges for detecting chromosomal aneuploidies using FISH., (© 2024. The Author(s).)

Objective: To investigate the levels of 12 kinds of cytokines in seminal plasma and their correlations with routine semen parameters., Methods: The remaining seminal plasma samples of 134 patients undergoing routine semen examination were collected for detecting cytokines. The parameters for sperm concentration, percentage of progressively motile sperm (PR), and motility were analyzed by a computer-assisted sperm analysis (CASA) system. According to the results of sperm concentration, PR and motility, 134 patients were divided into the normal routine semen parameters group, oligoasthenospermia group and azoospermia group. The levels of 12 kinds of cytokines in seminal plasma, including interleukin (IL)-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12P70, IL-17, interferin (IFN)-α, IFN-γ, and tumor necrosis factor (TNF)-α, were detected by flow cytometry. Two seminal plasma samples were detected for 10 times, respectively, to calculate the coefficients of variation (CV) of each cytokine. The linear range of each cytokine was measured using the standard, and the correlation coefficient (r) was calculated., Results: The r 2 of 12 kinds of cytokines detected by flow cytometry were all greater than 0.99. The reproducibility of 2 seminal plasma samples showed that the CVs of all cytokines were lower than 15 % except for TNF-α in sample 1 (15.15 %). Seminal plasma IL-6 levels were negatively correlated with semen volume (P < 0.01). Seminal plasma IL-5 levels were positively correlated with sperm concentration (P < 0.01). Seminal plasma IL-8 levels were negatively correlated with sperm motility (P < 0.01). Seminal plasma IL-8, IL-17 and IL-12P70 levels were negatively correlated with sperm PR (P < 0.05). In addition to the significant negative correlation between IL-5 and IL-17 (P < 0.05), there was a significant positive correlation between the majority of other cytokines. The levels of seminal plasma IL-17 and IL-12P70 in the oligoasthenospermia group and IL-1β and IL-12P70 in the azoospermia group were significantly higher than those in the normal routine semen parameters group (P ≤ 0.05), while the levels of IL-10 in the azoospermia group were significantly lower than that in the normal routine semen parameters group (P < 0.05)., Conclusion: There are certain correlations between seminal plasma cytokines and routine semen parameters and strong correlations between different seminal plasma cytokines, suggesting that the imbalance between seminal plasma cytokines may affect sperm quality. However, it still needs to be further confirmed by large samples and multi-center clinical studies and related basic researches., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Xia Zhao reports administrative support and article publishing charges were provided by Special Fund Project for Health Science and Technology Development of Nanjing Health Construction Commission. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Male factors may be present in up to 50-70% of infertile couples and the prevalence of male infertility accounts for 20-30% of infertility cases. Understanding the mechanisms and causes behind male infertility remains a challenge, but new diagnostic tools such as DNA fragmentation might aid in cases where the routine semen analysis is insufficient. DNA fragmentation, which refers to damages or breaks of the genetic material of the spermatozoa, is considered one of the main causes of male infertility due to impaired functional capability of sperm. The aim of the present narrative review is to investigate and enlighten the potential correlation between DNA fragmentation and male infertility parameters such as the seminal profile and the reproductive outcomes. Comprehensive research in PubMed/Medline and Scopus databases was conducted and 28 studies were included in the present review. Fourteen studies provided data regarding the impact of DNA fragmentation and seminal parameters and showed a correlation of significantly lower sperm count, lower concentration, motility, and abnormal morphology with an increased DNA fragmentation index (DFI). Similarly, 15 studies provided data regarding the impact of DFI on reproductive outcomes. Two studies showed higher aneuploidy rates with higher DFI values, and seven studies showed significantly lower pregnancy rates and live birth rates with higher DFI values. Ultimately, the studies included in this review highlight, collectively, the importance of measuring sperm DFI in the assessment of male infertility. Further studies are needed to explore the effectiveness of interventions aiming to reduce DFI levels.

Infertility is a global health issue, and more and more people are hoping to have babies by means of assisted reproductive technology. However, there are still many challenges in fertilization and pregnancy outcomes. Sperm quality is a key factor affecting the success rate of assisted reproduction. Therefore, sperm quality screening is crucial for achieving breakthroughs in assisted reproduction technology. At present, with its capabilities in the field of image recognition, artificial intelligence (AI) is providing new ideas and methods for sperm screening. Various attempts have been made with AI-based models to evaluate indicators such as sperm morphology, DNA quality, and motility level, and some results have been achieved. Herein, we reviewed the application of AI in sperm quality analysis and selection, providing support for the future development of AI and the improvement in the fertilization rate and outcomes of assisted reproductive technology., Competing Interests: 利益冲突　所有作者均声明不存在利益冲突, (© 2024《四川大学学报（医学版）》编辑部 版权所有Copyright ©2024 Editorial Office of Journal of Sichuan University (Medical Sciences).)

Mature, vital, and motile spermatozoa are essential for reaching the oocyte and binding to hyaluronic acid (HA) in the cumulus oophorus matrix. This study aims to determine the relationship between sperm-migration ability and HA-binding potential, as well as the relationship between sperm concentration and motility. Semen samples were collected from 702 men aged 20-56 years (median 34.8). We evaluated the sperm concentration and motility from basic semen analysis, the swim-up test (expressed as millions per mL and the migration efficiency percentage), and the hyaluronan-binding assay (HBA). A moderate positive correlation was found between the migration test results and HBA (R = 0.48). The highest correlation was observed between the concentration of motile spermatozoa and the migration test results (R = 0.85) and HBA (R = 0.4). The sperm migration efficiency strongly correlated with progressive motility (R = 0.6). Although significantly higher sperm migration was observed in patients with normal HBA results, the results of the functional tests were found to differ in some cases. For infertility treatment, the current diagnostic algorithm should be enhanced with more comprehensive seminological methods that assess the sperm-migration ability and HA-binding potential. We also recommend incorporating the swim-up method into the diagnostic protocol before planning assisted reproductive technology (ART) treatment.

Fourier harmonic analysis (FHA) is a robust method for identification of minute changes in sperm nuclear shape that are indicative of reduced fertility. The current study was designed to develop a fertility prediction model for Nili-Ravi buffalo bulls through FHA of sperm. In experiment I, FHA technique was standardized, average sperm nuclear perimeter was measured and sperm nuclear shape plot of buffalo bull was constructed. Sperm of buffalo bulls (n = 10) were stained with YOYO-1 and Hoechst-33342 to differentiate live and dead, and digital images were captured using phase contrast and fluorescent microscopy. The images were analyzed by ImageJ software and 100 sperm/bull were evaluated. The results are described as mean ± SEM values of mean harmonic amplitude (mharm), skewness harmonic amplitude (skharm), kurtosis harmonic amplitude (kurharm) and variance harmonic amplitude (varharm) at Fourier frequencies 0-5 along with the cartesian and polar coordinate plots of buffalo bull sperm. In experiment II, a fertility prediction model was developed based on FHA of buffalo bull sperm. Semen samples of low (n = 6), medium (n = 3) and high (n = 8) fertility bulls were investigated for FHA of sperm and harmonic amplitudes (HA) were generated. Firstly, to determine if live and dead sperm population have unique nuclear shape distribution; the mean, skewness, kurtosis and variance HA 0-5 of 1700 live and 1294 dead spermatozoa of 17 bulls were evaluated. T-test signified a difference in the mharm0 (2.363 ± 0.01 vs. 2.439 ± 0.02), skharm0 (-0.0002 ± 0.07 vs. -0.266 ± 0.09), kurharm0 (-0.156 ± 0.07 vs. 0.260 ± 0.18), kurharm2 (0.142 ± 0.11 vs. 1.031 ± 0.32) and varharm4 (0.109 ± 0.00 vs. 0.082 ± 0.00) of live vs. dead sperm population (p < 0.05). Therefore, 100 live sperm/bull were further evaluated for mean, skewness, kurtosis and variance HA 0-5 values among high (n = 6) and low-fertility (n = 6) groups. Results of T-test showed higher values of mharm2 (0.739 ± 0.01 vs. 0.686 ± 0.00), mharm4 (0.105 ± 0.001 vs. 0.007 ± 0.001), and skharm0 (0.214 ± 0.109 vs. -0.244 ± 0.097) in high vs. low-fertility group (p < 0.05). In next step, five significantly different combinations of discriminant measures between high and low-fertility groups were obtained by discriminant analysis. In conclusion, mharm4, skharm0 and varharm2 correctly identified 91.7 % of bulls into their respective fertility groups, and upon cross validation the value of the canonical correlation was 0.928., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The objective of this study was to compare the results of semen analysis using the manual method and the SQA-Vision sperm analyser after four years of practice and with a large cohort of patients. This was a comparative study of 1130 cases collected for semen analysis between October 2019 and October 2023, which were analysed simultaneously and independently by different operators using the manual microscopic method and an SQA-V automated analyser. For each sample, sperm concentration, progressive motility, motility, normal morphology, and round cells count were performed. There was no significant difference between the SQA-V method and manual assessment for all sperm parameters (Mann-Whitney test p  > 0.05). According to the parameter studied, there was a strong correlation (rho = 0.81) and a very high correlation (rho = 0.98) between manual assessment and the SQA-V method. In the analysis of sperm concentration, the sensitivity and specificity were 0.90 and 0.99, respectively. The sensitivity and specificity for the analysis of sperm progressive motility were 0.98 and 0.99, respectively, while the sensitivity and specificity for the analysis of sperm motility were 0.87 and 0.99, respectively. The sensitivity and specificity for the analysis of normal morphology were 0.88 and 0.99, respectively. Regarding the analysis of round cells, the sensitivity and specificity were 0.98 and 0.99, respectively. The results of this retrospective study indicate that the SQA-V system offers satisfactory performance for routine sperm analysis.

Objectives: This study aimed to investigate the correlation between testicular shear wave elastography (SWE) values and semen analysis results in men with infertility., Methods: This was a retrospective case-control study. Patients were categorized as normal, abnormal, or azoospermic based on sperm analysis results. Testicular volume was measured using B-mode ultrasonography using the Lambert formula. Subsequently, 40-80 regions of interest measuring 1.5 × 1.5 mm were manually positioned in both testicles based on their size, and two-dimensional SWE was applied through virtual touch imaging quantification software., Results: The patients had a mean age of 33.79 ± 6.3 years, with semen analysis revealing normal results in 15 patients (22.4%), pathological findings in 35 patients (52.2%), and azoospermia in 17 patients (25.4%). Right, left, total, and mean testicular volumes were significantly lower in patients with azoospermia compared to those in both normal and impaired semen parameters (P < .05). Conversely, testicular elastography scores were higher in patients with azoospermia than in the other groups (P < .05). The significant negative correlation between volume and elastographic findings remained independent of age (r = 0.4, P < .001). The accuracy rates for detecting impaired semen parameters and azoospermia were 94.3% and 94.1%, respectively, after considering factors such as age, testicular volume (right/left/total), and elastography (right/left/total). Notably, the total mean elastography score ranked first, with 100% in the independent normalized importance distribution of these variables., Conclusion: SWE can be used effectively alone or in combination with other diagnostic tools to evaluate histopathological changes in the testicles of male patients with infertility., (© 2024 American Institute of Ultrasound in Medicine.)

Reproductive efficiency is crucial for animal agriculture. This economically important aspect can be influenced by environmental burdens, nutritional imbalance, and gonadal or gametic malformations of genetic origin. Successful implementation of genomic-driven selective breeding in cattle depends on the reproductive performance of artificial insemination (AI) sires with valuable genomic production traits. Reproduction is traditionally viewed as a complex set of polygenic traits that are negatively impacted by using a small number of often closely related sires selected for AI due to their superior genetics. Despite recent progress, it remains difficult to define relationships between sire genome and variation in sperm phenotypes, even though several types of heritable, non-compensable sperm defects have been identified. In this review, we discuss the concept of sperm quality biomarker discovery and genomics of male fertility. We also outline a multidisciplinary genome-to-phenome approach for investigating heritable mutations and their impacts on bull fertility, sperm phenotypes and paternal contributions to early pregnancy. High-precision phenotyping requires novel, state-of-the-art instrumentation for sperm quality evaluation and development of new biomarkers of sperm quality in farm animals, with potential for incorporation into andrology-specific machine learning protocols and translation to human andrology. We conclude that reproduction is a complex phenotype that can be deciphered and explored for more precise male fertility evaluation and higher reproductive efficiency., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Sperm metabolism consists of a sophisticated network of biochemical reactions and varies between species, resulting in different metabolic strategies for ATP production to maintain sperm functionality. ATP can be produced through glycolysis or in the mitochondria by oxidative phosphorylation (OXPHOS). Since OXPHOS is the predominant metabolic pathway in horses spermatozoa, various assessments of mitochondrial activity are used to evaluate fertility, utilizing techniques such as fluorescent probes analysed via microscopy or flow cytometry, and polarographic electrode assays to measure current flow in response to an applied voltage. Though, these methods are limited by low throughput, as they assess mitochondrial activity at a single time point under a specific treatment condition. This study explores, for the first time, the application of the Agilent Seahorse XFp Technology to evaluate metabolism in stallion spermatozoa. This method enables real-time measurement of cellular metabolism across multiple samples or experimental conditions simultaneously. Ejaculates from eight different stallions were collected, and pools were prepared from three of them. Sperm viability and mitochondrial activity were evaluated by fluorescence microscopy, sperm motility by a computer-assisted sperm analysis system, and sperm metabolism was analysed via the Seahorse XFp analyser. Results confirmed a preference for OXPHOS over glycolysis in ATP production in stallion sperm, with mitochondria contributing significantly to total ATP generation. The Seahorse XFp Technology proved effective in evaluating equine sperm bioenergetics, offering insights into metabolic pathways critical for sperm function. In conclusion, this technology grants a new method for high-throughput analysis of sperm metabolism and quality, which could be applied to future reproductive studies in male equine fertility., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The objectives of this study were to validate diagnostic tests to detect polymorphonuclear cells (PMNs) in bull semen, and to determine the prevalence of leucospermia in beef bulls with varying semen quality. We hypothesized that all tests have comparable diagnostic value, and that leucospermia is more prevalent in unsatisfactory breeders in association with poor semen quality. For the analytical validation, one ejaculate was obtained from five bulls. Aliquots of 50 × 10 6 purified sperm were incubated in triplicate with six concentrations of purified bovine PMNs: 1) no PMNs, 2) 0.25 × 10 6 PMN/ml, 3) 0.5 × 10 6 PMN/ml, 4) 2.5 × 10 6 PMN/ml, 5) 5 × 10 6 PMN/ml, 6) 10 × 10 6 PMN/ml. The PMNs were quantified using a hemacytometer, cytology, a leucocyte esterase dipstick test (LEDT), a peroxidase test, and CD45 immunolabeling. The number of leucocytes detected with the LEDT differed among treatments (P < 0.0001). The quantitative tests detected differences with the control treatment at a PMN concentration of ≥2.5 × 10 6 PMN/ml (P < 0.0001). Sperm motion parameters after 4 h of incubation at 38 °C were lower in samples with ≥5 × 10 6 PMN/ml (P < 0.05). For the clinical validation, semen samples from 305 beef bulls were evaluated. Unsatisfactory breeders (n = 83) had more CD45-positive cells (P = 0.016) and positive LEDT results (P = 0.008) than satisfactory breeders (n = 222). With CD45 immunostaining as the gold standard, the hemacytometer count had the highest clinical sensitivity (64.3 %) but the lowest specificity (73.3 %). A higher specificity was obtained with the peroxidase test (95.1 %) or semen cytology (98.8 %). In conclusion, the presence of ≥5 × 10 6 PMN/ml was associated with decreased semen quality in beef bulls. The hemacytometer count was the most sensitive bull-side test. But due to the low specificity, positive hemacytometer counts should be confirmed with the identification of peroxidase-positive cells or morphological identification of leucocytes on semen cytology. The CD45 immunostaining is the gold standard for the diagnosis of leucospermia in bulls., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: To find the machine learning (ML) method that has the highest accuracy in predicting the semen quality of men based on basic questionnaire data about lifestyle behavior., Methods: The medical records of men whose semen was analyzed for any reason were collected. Those who had data about their lifestyle behaviors were included in the study. All semen analyses of the men included were evaluated according to the WHO 2021 guideline. All semen analyses were categorized as normozoospermia, oligozoospermia, teratozoospermia, and asthenozoospermia. The Extra Trees Classifier, Average (AVG) Blender, Light Gradient Boosting Machine (LGBM) Classifier, eXtreme Gradient Boosting (XGB) Classifier, Logistic Regression, and Random Forest Classifier techniques were used as ML algorithms., Results: Seven hundred thirty-four men who met the inclusion criteria and had data about lifestyle behavior were included in the study. 356 men (48.5%) had abnormal semen results, 204 (27.7%) showed the presence of oligozoospermia, 193 (26.2%) asthenozoospermia, and 265 (36.1%) teratozoospermia according to the WHO 2021. The AVG Blender model had the highest accuracy and AUC for predicting normozoospermia and teratozoospermia. The Extra Trees Classifier and Random Forest Classifier models achieved the best performance for predicting oligozoospermia and asthenozoospermia, respectively., Conclusion: The ML models have the potential to predict semen quality based on lifestyles., (© 2024. The Author(s).)

Objective: To demonstrate nanoscale motion tracing of spermatozoa and present analysis of the motion traces to characterize the consistency of motion of spermatozoa as a complement to progressive motility analysis., Design: Anonymized sperm samples were videographed under a quantitative phase microscope, followed by generating and analyzing superresolution motion traces of individual spermatozoa., Setting: Not applicable., Patient(s): Centrifuged human sperm samples., Intervention(s): Not applicable., Main Outcome Measure(s): Precision of motion trace of individual sperms, presence of a helical pattern in the motion trace, mean and standard deviations of helical periods and radii of sperm motion traces, speed of progression., Result(s): Spatially sensitive quantitative phase imaging with a superresolution computational technique MUltiple SIgnal Classification ALgorithm allowed achieving motion precision of 340 nm using ×10, 0.25 numerical aperture lens whereas the diffraction-limited resolution at this setting was 1,320 nm. The motion traces thus derived facilitated new kinematic features of sperm, namely the statistics of helix period and radii per sperm. Through the analysis, 47 sperms with a speed >25 μm/s were randomly selected from the same healthy donor semen sample, it is seen that the kinematic features did not correlate with the speed of the sperms. In addition, it is noted that spermatozoa may experience changes in the periodicity and radius of the helical path over time. Further, some very fast sperms (e.g., >70 μm/s) may demonstrate irregular motion and need further investigation. Presented computational analysis can be used directly for sperm samples from both fertility patients with normal and abnormal sperm cell conditions. We note that MUltiple SIgnal Classification ALgorithm is an image analysis technique that may vaguely fall under the machine learning category, but the conventional metrics for reporting found in Enhancing the QUAlity and Transparency Of health Research network do not apply. Alternative suitable metrics are reported, and bias is avoided through random selection of regions for analysis. Detailed methods are included for reproducibility., Conclusion(s): Kinematic features derived from nanoscale motion traces of spermatozoa contain information complementary to the speed of the sperms, allowing further distinction among the progressively motile sperms. Some highly progressive spermatozoa may have irregular motion patterns, and whether irregularity of motion indicates poor quality regarding artificial insemination needs further investigation. The presented technique can be generalized for sperm analysis for a variety of fertility conditions., Competing Interests: Declaration of Interests S.B. has nothing to disclose. A patent application containing the idea and application of this work as a subset of the claims has been submitted, where A.B. and K.A. are coinventors. They have not received any monetary gains pertaining this. They have received funding from European Research Council and Forskningsradet for commercialization of the patented technology; University hospital of North Norway UNN’s fertility clinic provided anonymized and centrifuged sperm samples. S.A. has nothing to disclose. D.H.H. has nothing to disclose. J.-C.T. has nothing to disclose. M.N. has nothing to disclose. D.S.M. has nothing to disclose. K.A. reports European Research Council Starting grant 804233 and European Research Council PoC 101123485 that funded the study., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Objective: To assess the accuracy of the YO home sperm test by real-world/amateur users., Methods: This multisite study investigated whether amateur users could use the FDA-cleared YO Home Sperm Test to obtain accurate motile sperm concentration (MSC) results when compared to trained laboratory technicians. The qualitative MSC results of amateur and professional YO users were compared to each other as well as to the results of an established automated sperm quality analyzer (SQA-V) above and below a 6 m/mL MSC cut-off., Results: This was a blinded, prospective study of 316 amateur users and 3 professional laboratory technicians across 3 study sites. Amateur vs Professional YO users demonstrated an accuracy of >97%. Qualitative results of amateurs and professionals vs SQA-V results showed a >95.7% accuracy., Conclusion: Amateur users with no prior training were able to follow the YO test directions to receive highly accurate qualitative motile sperm concentration (MSC) results. The YO test is user-friendly and can be used as an effective initial home screening tool to gain preliminary insight into the fertility status of the male in a real-world setting. Furthermore, the YO test results correlated with those obtained by the FDA-cleared SQA-V laboratory analyzer, confirming that the YO test delivers accurate MSC results in the hands of amateur users., Competing Interests: Declaration of Competing Interest No conflict., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

This study aims to compare the efficacy of computer-assisted sperm analysis (CASA) and smartphone-applied sperm analysis (SASA) in assessing the quality of frozen-thawed bull semen. A total of 75 straws (n = 75) semen samples were used from different production batches of five Holstein bulls. The semen analyses were conducted in three groups: Group I (CASA-37°C), semen samples were evaluated using the CASA system at 37°C (n = 25); Group II (SASA-25°C), semen samples were assessed using the SASA system at a temperature of room heat (25°C) (n = 25); and Group III (SASA-37°C), semen samples were evaluated using the SASA system at 37°C (n = 25). The frozen-thawed bull semen samples were analysed in terms of total motility (TM), progressive motility (PM), immotile, velocity average path (VAP), velocity curve linear (VCL), velocity straight line (VSL) and sperm concentration. There was no significant difference between the groups in terms of spermatozoa concentration (p > .05). However, significant differences among the groups were observed for total motile spermatozoa values (p < .001). Values of progressive motile spermatozoa were lower in Group I and Group II compared to Group III (p < .001). The immotile spermatozoa values were significant between the groups (p < .001) and were found to be proportional to total motile spermatozoa values. Additionally, the VAP, VCL and VSL values were comparable between Group II and Group III, but lower when compared to Group I. In conclusion, the results of the study demonstrate that the Sperm Cell™ system can accurately analyse the concentration of frozen-thawed bull semen. The analyses performed at room temperature indicate a parallelism between the PM value and CASA results. However, it is thought that SASA devices require a series of standardization studies in different semen extenders, different sample concentrations and different animal species, analogous to the standardization evolution process of CASA devices in semen analysis., (© 2024 The Author(s). Reproduction in Domestic Animals published by Wiley‐VCH GmbH.)

Competing Interests: Declaration of Competing Interest Lev Rabinovitch is a paid consultant to Medical Electronic Systems (MES) and the study is funded by MES; Stanton Honig and Natan Bar-Chama have no conflict as authors and the study is funded by MES.

Purpose: To identify the sperm preparation procedure that selects the best sperm population for medically assisted reproduction., Methods: Prospective observational study comparing the effect of four different sperm selection procedures on various semen parameters. Unused raw semen after routine diagnostic analysis was split in four fractions and processed by four different methods: (1) density gradient centrifugation (DGC), (2) sperm wash (SW), (3) DGC followed by magnetic activated cell sorting (MACS), and (4) using a sperm separation device (SSD). Each fraction was analyzed for progressive motility, morphology, acrosome index (AI), and DNA fragmentation index (DFI)., Results: With DGC as standard of care in intraclass correlation coefficient analysis, only SSD was in strong disagreement regarding progressive motility and DFI [0.26, 95%CI (- 0.2, 0.58), and 0.17, 95%CI (- 0.19, 0.45), respectively]. When controlling for abstinence duration, DFI was significantly lower after both MACS and SSD compared to DGC [- 0.27%, 95%CI (- 0.47, - 0.06), p = 0.01, and - 0.6%, 95%CI (- 0.80, - 0.41), p < 0.001, respectively]. Further comparisons between SSD and MACS indicate significantly less apoptotic cells [Median (IQR) 4 (5), 95%CI (4.1, - 6.8) vs Median (IQR) 5 (8), 95%CI (4.9, - 9.2), p < 0.001, respectively] and dead cells [Median (IQR) 9.5 (23.3), 95%CI (13.2, - 22.4) vs Median (IQR) 22 (28), 95%CI (23.1, - 36.8), p < 0.001, respectively] in the SSD group., Conclusion: The selection of a population of highly motile spermatozoa with less damaged DNA from unprocessed semen is ideally performed with SSD. Question remains whether this method improves the embryological outcomes in the IVF laboratory., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Objectives: Infertility is a disease of the male or female reproductive systems. Male reproductive workup is based on routine semen analysis, although of limited value. The 2021 WHO Manual incorporated Sperm DNA Fragmentation (SDF) assessment, and highlighted the need for individual laboratories to define suitable thresholds. This study aimed to present an alternative to address this issue, determine an SDF cut-off value with fertile donors, and characterize SDF in a patient cohort and their relationship with semen parameters., Study Design: A service unit was established to remotely perform TUNEL assay in a 2 step-process. Semen samples were received at andrology laboratories, subjected to routine semen analysis (WHO, 2010), partially processed and transported to the service unit for SDF evaluation. Using this setting, studies were done in fertile donors (n = 15) to define the cut-off value, and in men undergoing infertility workup (n = 318)., Results: A cut-off value of 9.17 % was determined with the fertile donor cohort. With this cut-off, a 64.46 % abnormal SDF incidence was determined in the patient cohort. SDF negatively correlated with sperm number, vitality and motility, and positively with abnormal morphology and male age (P < 0.05). TUNEL-positive cases depicted lower sperm quality and higher male age (P < 0.05). A similar abnormal SDF incidence was determined among patients with semen abnormalities. Asthenozoospermic and ≥40 years patient samples depicted higher (P < 0.05) SDF than those of the general population. SDF incidence was also high in normozoospermic patients., Conclusions: Using a 2-step remote approach with a standardized procedure and an SDF cut-off value established with fertile donors, high SDF incidence in semen samples depicting normal and abnormal quality were identified in men consulting for infertility, highlighting the relevance of its evaluation as part of the male fertility workup., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

We investigated a screening method using only serum hormone levels and AI (artificial intelligence) predictive analysis. Among 3662 patients, numbers for NOA (non-obstructive azoospermia), OA (obstructive azoospermia), cryptozoospermia, oligozoospermia and/or asthenozoospermia, normal, and ejaculation disorder were 448, 210, 46, 1619, 1333, and 6, respectively. "Normal" was defined as semen findings normal according to the WHO (World Health Organization) Manual for Human Semen Testing of 2021. We extracted age, LH (luteinizing hormone), FSH (follicle stimulating hormone), PRL (prolactin), testosterone, E2 (estradiol), and T (testosterone)/E2 from medical records. A total motility sperm count of 9.408 × 10 6 (1.4 ml × 16 × 10 6 /ml × 42%) was defined as the lower limit of normal. The Prediction One-based AI model had an AUC (area under the curve) of 74.42%. For the AutoML Tables-based model, AUC ROC (receiver operating characteristic) was 74.2% and AUC PR (precision-recall) 77.2%. In a ranking of feature importance from 1st to 3rd, FSH came a clear 1st. T/E2 and LH ranked 2nd and 3rd for both Prediction One and AutoML Tables. Using data from 2021 and 2022 to verify the Prediction One-based AI model, the predicted and actual results for NOA were 100% matched in both years., (© 2024. The Author(s).)

Background: In the field of male infertility, when sperm is normal/subnormal, a few "add-on" routine tests can complete the basic semen examination., Objectives: The aim of this study was to develop and evaluate a faster, simplified motile sperm organelle morphology examination (MSOME) technique for selected infertile patients with apparently normal/subnormal sperm and, in their background: failure of two or three intrauterine insemination (IUI) cycles, repeatedly fragmented embryos, embryonic development to blastocyst-stage failures, repeated miscarriages, a long period of infertility or 2 or more IVF attempts without pregnancy. Our test results were correlated with IUI, conventional in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and intracytoplasmic morphologically selected sperm injection (IMSI) outcomes., Materials and Methods: We validated an adapted version of the MSOME analysis called the pre-IMSI test (PIT), based on vacuole evaluation alone. 248 infertile patients from our assisted reproductive technology (ART) Center were retrospectively selected and split into three PIT score subgroups (patients with ≤8% (score I), 9 to 15% (score II) and ≥16% normal spermatozoa (score III)) based on the correlation between PIT results and each ART technique outcome. The choice of one or another of these ART techniques had been made according to the usual clinico-biological criteria., Results: Clinical outcomes for each of the three PIT subgroups were compared individually for the different ART techniques. For ICSI, the effect of the PIT score subgroup was significant for clinical pregnancies (p = 0.0054) and presented a trend for live births (p = 0.0614). Miscarriage rates of IVF attempts were statistically different depending on the PIT score (p = 0.0348). Furthermore, the odds ratios of clinical pregnancy rates were significantly different according to PIT score subgroup when comparing ICSI vs. IMSI or IVF vs. ICSI attempts., Discussion: IMSI appears to be recommended when sperm belongs to PIT score I, ICSI when it belongs to PIT score II and IVF or IUI when sperm is of PIT score III quality in selected infertile couples. The lack of statistical power in these PIT subgroups means that we must remain cautious in interpreting results., Conclusion: Our results support the interest of this simplified test for certain couples with normal/subnormal sperm to help choose the most efficient ART technique, even as first-line treatment., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sigala et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

To investigate the mechanisms underlying the differences in the freezability of boar semen, Yorkshire boars with freezing-tolerant semen (YT, n = 3), Yorkshire boars with freezing-sensitive semen (YS, n = 3), Landrace boars with freezing-tolerant semen (LT, n = 3), and Landrace boars with freezing-sensitive semen (LS, n = 3) were selected for this study. Their sperm was subjected to protein extraction, followed by data-independent acquisition proteomics and functional bioinformatics analysis. A total of 3042 proteins were identified, of which 2810 were quantified. Some key KEGG pathways were enriched, such as starch and sucrose metabolism, carbohydrate digestion and absorption, mineral absorption, the HIF-1 signaling pathway, and the necroptosis pathways. Through PRM verification, we found that several proteins, such as α-amylase and epididymal sperm-binding protein 1, can be used as molecular markers of the freezing resistance of boar semen. Furthermore, we found that the addition of α-amylase to cryoprotective extender could significantly improve the post-thaw motility and quality of boar semen. In summary, this study revealed some molecular markers and potential molecular pathways contributing to the high or low freezability of boar sperm, identifying α-amylase as a key protein. This study is valuable for optimizing boar semen cryopreservation technology.

This comprehensive review delves into the evolving landscape of assisted reproductive technologies (ARTs) in bovine species, particularly focusing on the pivotal roles of semen additives in the cryopreservation of buffalo and cattle semen. In developing nations, where ARTs are still emerging, these techniques significantly influence bovine reproductive strategies. In contrast, developed regions have embraced them as primary approaches for dairy buffalo and cattle breeding. Semen cryopreservation, while offering advantages like extended storage and genetic propagation, also presents challenges. These include diminished sperm quality due to reactive oxygen species (ROS) production, alterations in sperm structure, and temperature fluctuations. Further, the effect of cryopreservation differs between cattle and buffaloes, with the latter exhibiting poorer semen viability and fertility due to inherent lipid composition susceptibilities. The generation and implications of ROS, especially hydrogen peroxide, contribute significantly to sperm DNA damage and functional impairments. To counteract these challenges, research has intensified on semen additives, aiming to bolster semen quality and protect against oxidative stress-induced damage. As the field advances, the review emphasizes the need for optimized cryopreservation techniques and tailored antioxidant strategies to harness the full potential of ARTs in bovine breeding programs. Doi.org/10.54680/fr24410110112.

In this cross-sectional prospective study, advanced next-generation sequencing technology was used to compare the molecular karyotyping of individual human sperm cells in infertile couples with severe oligoteratozoospermia (i.e., low sperm count and motility) to those of infertile couples with normal semen. Fourteen infertile couples who were patients at Ramathibodi Hospital in Bangkok, Thailand, were recruited from January to November 2023, and they were categorized into two groups based on semen analysis results. The study group comprised couples with severe oligoteratozoospermia, whereas the control group exhibited normal semen. Individual sperm cells from the semen samples were isolated by the micromanipulation technique for subsequent whole-genome amplification and next-generation sequencing, where the primary outcome was the aneuploidy rate. Seventy individual sperm cells were isolated with a 90% success rate for amplification. The next-generation sequencing results showed that the aneuploidy rate was 25%-75%, with a mean of 48.28% in the study group. In contrast, the control group exhibited aneuploidy rates of 0-75%, with a mean of 15.15%. The difference between the two groups was statistically significant (odds ratio: 5.8, 95% confidence interval: 1.30-26.03). Sperm cells of the study group showed a threefold higher aneuploidy rate than those in the control group, even though the sperm cells were selected by micromanipulation for their normal morphology. Comprehensive counseling is recommended to address elevated aneuploidy rates that potentially surpass those of the general infertile population. Guidance on preimplantation genetic testing is also recommended to ensure the transfer of embryos with normal chromosomes., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Sassanarakkit et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad," defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions, and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington and Alzheimer disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, deoxyribonucleic acid, and accessory structures of the sperm head and tail. Introduction of next-generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high-throughput phenotyping of spermatozoa in humans and economically important livestock animals., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction.)

The process-of-male reproduction is intricate, and various medical conditions-have the potential to disrupt spermatogenesis. Moreover, infertility in males can serve as an indicator of-potential future health issue. Numerous conditions with systemic implications have been identified, encompassing genetic factors (such as Klinefelter Syndrome), obesity, psychological stress, environmental factors, and others. Consequently, infertility assessment-presents an opportunity for comprehensive health counseling, extending-beyond discussions about reproductive goals. Furthermore, male infertility has been suggested as a harbinger of future health problems, as poor semen quality and a diagnosis of-male infertility are associated with an increased risk of hypogonadism, cardiometabolic disorders, cancer, and even mortality. This review explores the existing-literature on the relationship between systemic illnesses and male fertility, impacting both clinical-outcomes and semen parameters. The majority of the literature analyzed, which compared gonadal function with genetic, chronic, infectious or tumoral diseases, confirm the association between overall male health and infertility., (© 2024 the author(s), published by De Gruyter, Berlin/Boston.)

Background: There is no consensus on tests required to either diagnose unexplained infertility or use for research inclusion criteria. This leads to heterogeneity and bias affecting meta-analysis and best practice advice., Objectives: This systematic review analyses the variability of inclusion criteria applied to couples with unexplained infertility. We propose standardised criteria for use both in future research studies and clinical diagnosis., Search Strategy: CINAHL and MEDLINE online databases were searched up to November 2022 for all published studies recruiting couples with unexplained infertility, available in full text in the English language., Data Collection and Analysis: Data were collected in an Excel spreadsheet. Results were analysed per category and methodology or reference range., Main Results: Of 375 relevant studies, only 258 defined their inclusion criteria. The most commonly applied inclusion criteria were semen analysis, tubal patency and assessment of ovulation in 220 (85%), 232 (90%), 205 (79.5%) respectively. Only 87/220 (39.5%) studies reporting semen analysis used the World Health Organization (WHO) limits. Tubal patency was accepted if bilateral in 145/232 (62.5%) and if unilateral in 24/232 (10.3%). Ovulation was assessed using mid-luteal serum progesterone in 115/205 (56.1%) and by a history of regular cycles in 87/205 (42.4%). Other criteria, including uterine cavity assessment and hormone profile, were applied in less than 50% of included studies., Conclusions: This review highlights the heterogeneity among studied populations with unexplained infertility. Development and application of internationally accepted criteria will improve the quality of research and future clinical care., (© 2023 The Authors. BJOG: An International Journal of Obstetrics and Gynaecology published by John Wiley & Sons Ltd.)

Objective: The Neubauer hemocytometer, as well as the Makler chamber, are devices commonly used in andrology laboratories. The present study aimed to verify if both methods yield comparable results, and whether they can be used interchangeably to determine sperm concentration., Methods: Sperm and latex beads concentration measurements were performed with the Neubauer hemocytometer and the Makler chamber. Fixed and proportional biases were estimated, and the method agreement was determined by assessing sperm concentration results with the Bland and Altman plot. The Coefficient of Variation (CV) and relative bias were calculated as an index of precision and accuracy, respectively, by measuring latex beads target concentrations in both chambers., Results: The Makler chamber systematically overestimated the Neubauer hemocytometer concentration measurements by a mean of -7.99%, with limits of agreement (LOA) between -41% to 25.61% (p<0.001). The fixed bias was found for concentration values inferior to 40 x 106/ml range (p<0.001), but not higher concentration results (p>0.05). Measurements with the Neubauer hemocytometer showed the greatest consistency in the study with the CV ranging from 3.01% to 6.67%; while the CV with the Makler chamber ranged from 8.46% to 25.64%. The relative bias for the Neubauer hemocytometer determinations varied from 0.12% to 8.40%, while for the Makler chamber varied from 7.6% to an overestimation of 38.0%., Conclusions: Measurements made with the Makler chamber demonstrated more variability and a higher degree of overestimation. The Makler chamber is a poor substitute to the Neubauer hemocytometer for evaluation of oligozoospermic samples, although both chambers render similar results for highly concentrated samples.

In biotechnological processes such as chromosomal manipulation studies, semen has become a reference in the ploidy verification of the evaluated material. However, the use of fresh samples is limited to the use at field conditions because the analysis is performed under laboratory conditions. Thus, this study aimed to develop a simpler procedure for storing dry semen at 28 °C to reduce cold storage costs. For this, semen samples were evaluated according to established quality semen parameters, a protocol for dry, and 3 sterilization treatments of dry semen were applied to the store. The integrity of the DNA was evaluated every two months, using fresh semen, dry semen (untreated), and particles 3C to compare the peaks by flow cytometry. The results indicated that all samples evaluated before and after drying showed no significant difference in the DNA content. UV-treated semen showed a 1C peak in the histogram up to 180 days of storage and a non-significant difference (P > 0.05) from fresh control in the number of DNA particles up to 120 days and untreated only showed a 1C peak up to 120 days. The developed method may become an interesting procedure to serve as a reference peak for practical flow cytometric analysis, not only in the field of fish biology but also in biomedical and agricultural sciences. Furthermore, dried semen can become a tool for the preservation of genetic material and is a promising low-cost storage technique for biobanking., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Society for Cryobiology. Published by Elsevier Inc. All rights reserved.)

Male infertility is a global health issue, with 40-50% attributed to sperm abnormalities. The subjectivity and irreproducibility of existing detection methods pose challenges to sperm assessment, making the design of automated semen analysis algorithms crucial for enhancing the reliability of sperm evaluations. This paper proposes a comprehensive sperm tracking algorithm (Sperm YOLOv8E-TrackEVD) that combines an enhanced YOLOv8 small object detection algorithm (SpermYOLOv8-E) with an improved DeepOCSORT tracking algorithm (SpermTrack-EVD) to detect human sperm in a microscopic field of view and track healthy sperm in a sample in a short period effectively. Firstly, we trained the improved YOLOv8 model on the VISEM-Tracking dataset for accurate sperm detection. To enhance the detection of small sperm objects, we introduced an attention mechanism, added a small object detection layer, and integrated the SPDConv and Detect&#95;DyHead modules. Furthermore, we used a new distance metric method and chose IoU loss calculation. Ultimately, we achieved a 1.3% increase in precision, a 1.4% increase in recall rate, and a 2.0% improvement in mAP@0.5:0.95. We applied SpermYOLOv8-E combined with SpermTrack-EVD for sperm tracking. On the VISEM-Tracking dataset, we achieved 74.303% HOTA and 71.167% MOTA. These results show the effectiveness of the designed Sperm YOLOv8E-TrackEVD approach in sperm tracking scenarios., Competing Interests: The authors declare no conflicts of interest.

Background: Deep learning has been increasingly investigated for assisting clinical in vitro fertilization (IVF). The first technical step in many tasks is to visually detect and locate sperm, oocytes, and embryos in images. For clinical deployment of such deep learning models, different clinics use different image acquisition hardware and different sample preprocessing protocols, raising the concern over whether the reported accuracy of a deep learning model by one clinic could be reproduced in another clinic. Here we aim to investigate the effect of each imaging factor on the generalizability of object detection models, using sperm analysis as a pilot example., Methods: Ablation studies were performed using state-of-the-art models for detecting human sperm to quantitatively assess how model precision (false-positive detection) and recall (missed detection) were affected by imaging magnification, imaging mode, and sample preprocessing protocols. The results led to the hypothesis that the richness of image acquisition conditions in a training dataset deterministically affects model generalizability. The hypothesis was tested by first enriching the training dataset with a wide range of imaging conditions, then validated through internal blind tests on new samples and external multi-center clinical validations., Results: Ablation experiments revealed that removing subsets of data from the training dataset significantly reduced model precision. Removing raw sample images from the training dataset caused the largest drop in model precision, whereas removing 20x images caused the largest drop in model recall. by incorporating different imaging and sample preprocessing conditions into a rich training dataset, the model achieved an intraclass correlation coefficient (ICC) of 0.97 (95% CI: 0.94-0.99) for precision, and an ICC of 0.97 (95% CI: 0.93-0.99) for recall. Multi-center clinical validation showed no significant differences in model precision or recall across different clinics and applications., Conclusions: The results validated the hypothesis that the richness of data in the training dataset is a key factor impacting model generalizability. These findings highlight the importance of diversity in a training dataset for model evaluation and suggest that future deep learning models in andrology and reproductive medicine should incorporate comprehensive feature sets for enhanced generalizability across clinics., (© 2024. The Author(s).)

Study Question: Is a microfluidic sperm sorter (MSS) able to select higher quality sperm compared to conventional methods?, Summary Answer: The MSS selects sperm with improved parameters, lower DNA fragmentation, and higher fertilizing potential., What Is Known Already: To date, the few studies that have compared microfluidics sperm selection with conventional methods have used heterogeneous study population and have lacked molecular investigations., Study Design, Size, Duration: The efficiency of a newly designed MSS in isolating high-quality sperm was compared to the density-gradient centrifugation (DGC) and swim-up (SU) methods, using 100 semen samples in two groups, during 2023-2024., Participants/materials, Setting, Methods: Semen specimens from 50 normozoospermic and 50 non-normozoospermic men were sorted using MSS, DGC, and SU methods to compare parameters related to the quality and fertilizing potential of sperm. The fertilizing potential of sperm was determined by measurement of phospholipase C zeta (PLCζ) and post-acrosomal sheath WW domain-binding protein (PAWP) expression using flow cytometry, and the chromatin dispersion test was used to assess sperm DNA damage., Main Results and the Role of Chance: In both normozoospermic and non-normozoospermic groups, the MSS-selected sperm with the highest progressive motility, PLCζ positive expression and PLCζ and PAWP fluorescence intensity the lowest non-progressive motility, and minimal DNA fragmentation, compared to sperm selected by DGC and SU methods (P < 0.05)., Limitation, Reasons for Caution: The major limitations of our study were the low yield of sperm in the MSS chips and intentional exclusion of severe male factor infertility to yield a sufficient sperm count for molecular experiments; thus testing with severe oligozoospermic semen and samples with low count and motility is still required. In addition, due to ethical considerations, at present, it was impossible to use the sperm achieved from MSS in the clinic to assess the fertilization rate and further outcomes., Wider Implications of the Findings: Our research presents new evidence that microfluidic sperm sorting may result in the selection of high-quality sperm from raw semen. This novel technology might be a key to improving clinical outcomes of assisted reproduction in infertile patients., Study Funding/competing Interest(s): The study is funded by the Iran University of Medical Sciences and no competing interest exists., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Context Conventional sperm quality tests may not be sufficient to predict the fertilising ability of a given ejaculate; thus, rapid, reliable and sensitive tests are necessary to measure sperm function. Aims This study sought to address whether a cluster analysis approach based on flow cytometry variables could provide more information about sperm function. Methods Spermatozoa were exposed to either isotonic (300mOsm/kg) or hypotonic (180mOsm/kg) media for 5 and 20min, and were then stained with SYBR14 and propidium iodide (PI). Based on flow cytometry dot plots, spermatozoa were classified as either viable (SYBR14+ /PI- ) or with different degrees of plasma membrane alteration (SYBR14+ /PI+ and SYBR14- /PI+ ). Moreover, individual values of electronic volume (EV), side scattering (SS), green (FL1) and red (FL3) fluorescence were recorded and used to classify sperm cells through cluster analysis. Two strategies of this approach were run. The first one was based on EV and the FL3/FL1 quotient, and the second was based on EV, SS and the FL3/FL1 quotient. Key results The two strategies led to the identification of more than three sperm populations. In the first strategy, EV did not differ between membrane-intact and membrane-damaged sperm, but it was significantly (P P P Conclusions Cluster analysis based on flow cytometry variables provides more information about sperm function than conventional assessment does. Implications Combining flow cytometry with cluster analysis is a more robust approach for sperm evaluation.

Sperm head morphology is crucial for male factor infertility diagnosis and assessment of male reproductive potential. Several criteria are available to analyze sperm head morphology, but they are limited by poor methodology comparability and population applicability. This study aimed to explore comprehensive and new normal morphometric reference values for spermatozoa heads in fertile Asian males. An automated sperm morphology analysis system captured 23 152 stained spermatozoa from confirmed fertile males. Of these samples, 1856 sperm head images were annotated by three experienced laboratory technicians as "normal". We employed 14 novel morphometric features to describe sperm head size (head length, head width, length/width ratio, and girth), shape (ellipse intersection over union, girth intersection over union, short-axis symmetry, and long-axis symmetry), area (head, acrosome, postacrosomal areas, and acrosome area ratio), and degrees of acrosome and nuclear uniformity. This straight-forward method for the morphometric analysis of sperm by accurate visual measurements is clinically applicable. The measured parameters present valuable information to establish morphometric reference intervals for normal sperm heads in fertile Asian males. The presented detailed measurement data will be valuable for interlaboratory comparisons and technician training. In vitro fertilization and andrology laboratory technicians can use these parameters to perform objective morphology evaluation when assessing male fertilization potential., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Objective: To explore the taxonomic and predicted functional relationship between the urine microbiome and alterations of semen analysis (SA) parameters., Design: Cross-sectional study., Setting: Academic medical center., Patient(s): Men presenting for fertility evaluation or men presenting for vasectomy consultation with proven biological paternity were recruited and stratified on the basis of alterations, or lack thereof, in SA parameters., Main Outcome Measure: Changes in the functional and taxonomic urine microbiome profiles of participants with or without alterations in SA parameters., Results: Seventy-three participants were included in our study. Men with abnormal sperm motility (N = 27) showed a nearly 50-fold higher abundance of Dialister micraerophilus compared with those with normal sperm motility (N = 46). This relationship persisted on canonical correlational analysis (r = 0.439). Men with abnormal sperm concentration (N = 20) showed a lower abundance of Enterococcus faecalis and Staphylococcus aureus, compared with those with normal sperm concentration (N = 53). The urine of participants with impaired sperm motility demonstrated dramatic differences in predictive functional profiles in pathways involved in oxidation-reduction balance and cell longevity., Conclusions: Our findings underscore differences in the urinary microbiome and abnormalities in semen parameters, especially sperm motility. By incorporating predictive functional profiling, we also highlight possible mechanisms that may drive the observed differences in sperm parameters., Competing Interests: Declaration of Interests V.O. has nothing to disclose. A.B. has nothing to disclose. R.K. has nothing to disclose. J.T.S. has nothing to disclose. T.P.F. has nothing to disclose. J.S.A. has nothing to disclose. T.K. has nothing to disclose. S.F.M. has nothing to disclose. C.D.T. has nothing to disclose. T.-C.M.H. has nothing to disclose. J.N.M. is a consultant for Halozyme Pharma, Boston Scientific, and Endo Pharmaceuticals. S.V.E. has nothing to disclose., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background/aim: Intrauterine insemination (IUI) is the most common assisted-reproduction treatment. However, it has lower success rate in comparison to other treatments. Therefore, determining factors that contribute to IUI success is of particular interest and this was the purpose of this prospective study., Patients and Methods: In this study, only homologous inseminations with fresh semen samples were included. All women received mild ovarian stimulation with clomiphene citrate and gonadotropins. Before IUI, basic semen analysis, evaluation of DNA fragmentation index (DFI), as well as measurement of sperm redox potential, were performed on each semen sample. Semen was processed with density-gradient centrifugation and 500 μl of processed sperm was used for insemination., Results: In 200 cycles, there were 36 pregnancies, six of them ectopic. Cycles with ongoing pregnancies were characterized by younger male and female age and higher number of follicles. Multivariate logistic regression analysis showed that only female age was significantly associated with ongoing pregnancy. DFI was positively correlated with male age and negatively correlated with sperm concentration and progressive motility. Semen redox potential showed a strong negative correlation with sperm concentration and positive correlation with DFI., Conclusion: Female age seems to be the most important determinant factor for the achievement of an ongoing pregnancy in homologous IUI cycles with fresh semen., (Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)