Establishment and validation of a 5-factor diagnostic model for obstructive and non-obstructive azoospermia based on routine clinical parameters.

Azoospermia is a serious leading male-factor cause of infertility in couples of childbearing age. The two main azoospermia types, obstructive (OA) and non-obstructive (NOA) azoospermia, differ in their treatment approaches. Therefore, their clinical diagnosis is extremely important, requiring an accurate, efficient, and easy-to-use diagnostic model. This retrospective observational study included 707 patients with azoospermia treated between 2017 and 2021, 498 with OA, and 209 with NOA. Hematological and seminal plasma parameters, hormone levels, and testicular volume were used in logistic regression analysis to evaluate and compare their diagnostic performance, results showed that the optimal diagnostic model is constructed by five variables including semen volume, semen pH, seminal plasma neutral α-glucosidase activity, follicle-stimulating hormone in the serum, and testicular volume, compared with follicle-stimulating hormone-based and testicular volume-based models. The 5-factor diagnostic model had an accuracy of 90.4%, sensitivity of 96.4%, positive predictive value of 90.6%, negative predictive value of 89.8%, and area under the curve of 0.931, all higher than in the other two models. However, its specificity (76.1%) was slightly lower than in the other models. Meantime, the internal 5-fold cross-validation results indicated that the 5-factor diagnostic model had a good clinical application value. This study established an accurate, efficient, and relatively accessible 5-factor diagnostic model for OA and NOA, providing a reference for clinical decision-making when selecting an appropriate treatment.

Impact of Season Variation on Semen Quality: A Comprehensive Retrospective Analysis of Data From Patients at an Eastern Iranian Tertiary Care Fertility Center Over a Decade.

Seasonal changes are assumed to affect various sperm characteristics based on photoperiods, temperature, and air pollution. According to the literature, most studies were performed on populations of Western countries, and there are limited studies performed in the Middle East with variable results. This study evaluated the seasonality of sperm characteristics among men of reproductive age in an andrology center in Kerman, Iran, where the seasonal temperature varies significantly, with average temperatures ranging from 50 °F (10 °C) to 75.2 °F (24 °C). We retrospectively evaluated the sperm analysis test record. Sperm samples were obtained from 2,948 men during 10 years, excluding those with azoospermia. Samples were assessed for volume, concentration, motility, and morphology according to the World Health Organization (WHO) criteria. We performed a comprehensive comparative literature review of the studies investigating the association between seasonal variation and sperm quality. The mean semen volume was higher in the summer compared with other seasons (p = .04). The mean percentage of sperm motility was higher in the spring and less in winter (p = .03). Sperm morphology-related parameters, measured by the percent of normal morphology, were significantly better in winter (p = .03). Our findings suggest seasonality of sperm characteristics among men of fertility age. Semen volume, motility, and morphology were affected by the photoperiod of reproductive seasons. Results might support the influential role of seasonal variations in the possibility of fertility, especially among those using assisted reproductive technologies and those with oligospermia.

APHRODITE criteria: addressing male patients with hypogonadism and/or infertility owing to altered idiopathic testicular function.

RESEARCH QUESTION: Can a novel classification system of the infertile male - 'APHRODITE' (Addressing male Patients with Hypogonadism and/or infeRtility Owing to altereD, Idiopathic TEsticular function) - stratify different subgroups of male infertility to help scientists to design clinical trials on the hormonal treatment of male infertility, and clinicians to counsel and treat the endocrinological imbalances in men and, ultimately, increase the chances of natural and assisted conception? DESIGN: A collaboration between andrologists, reproductive urologists and gynaecologists, with specialization in reproductive medicine and expertise in male infertility, led to the development of the APHRODITE criteria through an iterative consensus process based on clinical patient descriptions and the results of routine laboratory tests, including semen analysis and hormonal testing. RESULTS: Five patient groups were delineated according to the APHRODITE criteria; (1) Hypogonadotrophic hypogonadism (acquired and congenital); (2) Idiopathic male infertility with lowered semen analysis parameters, normal serum FSH and normal serum total testosterone concentrations; (3) A hypogonadal state with lowered semen analysis parameters, normal FSH and reduced total testosterone concentrations; (4) Lowered semen analysis parameters, elevated FSH concentrations and reduced or normal total testosterone concentrations; and (5) Unexplained male infertility in the context of unexplained couple infertility. CONCLUSION: The APHRODITE criteria offer a novel and standardized patient stratification system for male infertility independent of aetiology and/or altered spermatogenesis, facilitating communication among clinicians, researchers and patients to improve reproductive outcomes following hormonal therapy. APHRODITE is proposed as a basis for future trials of the hormonal treatment of male infertility.

Performance evaluation of sperm concentration, motility, and morphological analysis for GSA-810 series of sperm quality analysis system.

BACKGROUND: The performance evaluation of each computer-assisted sperm analysis (CASA) system may provide a basis for the interpretation of clinical results and further improvement of the CASA system. METHODS: The accuracy of the GSA-810 CASA system was evaluated by detecting latex bead quality control products. The precision of sperm concentration, morphology, and percentages of progressively motile sperm (PR) were evaluated by coefficient of variation (CV). Three samples with sperm concentration of about 100 × 106 /mL were diluted to evaluate the linear range. RESULTS: The detection values of latex beads were within the range of target values. The CVs of sperm concentration and PR were significantly and negatively correlated with sperm concentration (r = -0.561, p = 0.001) and PR value (r = -0.621, p < 0.001), respectively. The R2 values of the linear range of sperm concentration were ≥0.99. There was no significant difference in sperm motility and PR within 1-10 min at 36.5°C ± 0.5°C. The coincidence rates of sperm morphology and sperm head morphology for 36 semen samples analyzed by the GSA-810 system and manual method were 99.40% and 99.67%, respectively. The CVs of the percentage of sperm with abnormal morphology and percentage of sperm with abnormal head morphology were less than 5%. CONCLUSION: The GSA-810 system can accurately analyze normal semen samples, but the repeatability of the results is poor for oligozoospermia and asthenozoospermia samples. The future CASA system for analyzing sperm morphology should focus on recognizing the middle and tail segments of a spermatozoon.

Improving standard practices in studies using results from basic human semen examination.

The purpose of this article is to provide an explanation of the background behind a checklist that declares the laboratory methods used in a scientific study. It focuses primarily on implementing laboratory procedures to yield reliable results in basic semen examinations. While the World Health Organization (WHO) and international standards provide recommendations for basic semen examination, manuscripts submitted to Andrology frequently lack transparency regarding the specific techniques used. In addition, the terminology used for semen examination results often fails to provide a clear definition of the groups under study. Furthermore, the WHO's reference limits are often misinterpreted as strict boundaries between fertility and infertility. It is important to note that valid clinical andrological diagnoses and treatments cannot rely solely on semen examination results; they require proper laboratory procedures as a foundation for diagnosing and treating male patients. Therefore, scientific journals should promote the adoption of robust laboratory practices and an accurate definition of patient groups. A checklist can facilitate the design of high-quality studies and the creation of informative publications. Further, it can help journals assess submitted manuscripts and improve the overall quality of their publications.

Impact of semen oxidative stress on sperm quality: initial results from Vietnam.

OBJECTIVES: This study aimed to determine the relationship between oxidative stress (OS) measured by the oxidation-reduction potential (ORP) and the results of semen analysis among men from infertile couples. METHODS: This cross-sectional study included 166 men from infertile couples, determined according to the World Health Organization guidelines. The general characteristics, semen analysis, sperm chromatin dispersion assay, and ORP of all subjects were evaluated and analyzed statistically. RESULTS: Among 166 men from infertile couples, individuals with OS had a significantly higher DNA fragmentation index (DFI) than men without OS (22.37% ± 11.67% vs. 17.98% ± 8.98%). The sperm concentration, total sperm count, motility rate, and normal morphology were negatively correlated, while and an abnormal head and neck-tail were positively correlated with ORP. There was also a positive association between the DFI and OS level. The optimal ORP threshold for determining sperm quality was 0.77 mV/106 sperm/mL (sensitivity, 50.4%; specificity, 93.5%; positive predictive value, 52.9%; negative predictive value, 32.3%). CONCLUSIONS: Determining the ORP suggests that OS has an adverse effect on the total sperm count, sperm motility, sperm concentration, morphology, vitality, and DNA fragmentation index.

Oxido-Reduction Potential as a Method to Determine Oxidative Stress in Semen Samples.

There are different estimates for the incidence of infertility. Its occurrence may vary from area to area, but on average, it affects 15% of couples and 10-12% of men worldwide. Many aspects of infertility can be linked to reactive oxygen species (ROS) and the process of oxidative stress (OS). The association between poor semen quality and OS is well known. Unfortunately, there is no accepted protocol for the diagnosis and treatment of OS in andrology. Oxido-reduction potential (ORP) measurement is a new method for determining the ratio between oxidant and antioxidant molecules. Currently, ORP measurement is one of the fastest and most user-friendly methods of andrological OS determination and our goals were to confirm published correlations between ORP values and sperm parameters, examine how sperm concentration influences these results, and investigate whether intracellular ROS formations are also manifested in the ORP values or not after artificial ROS induction. Intracellular ROS formations were induced by menadione (superoxide anion inducer), hydrogen peroxide, and tert-butyl hydroperoxide (lipid peroxidation inducer) treatments; sperm parameters like motility and viability were determined with an SCA Scope system, and ORP changes were recorded by the Mioxsys system. Significant correlations were noticed among the ORP, spermatozoa concentration, motility, progressive motility, and viability. Nevertheless, only the ORP value after normalization with the sperm count correlated with these parameters. Due to normalization, very low and very high sperm concentrations can give misleading results. The means of the non-normalized ORP values were almost the same. All of the applied treatments resulted in decreases in the viability, motility, and progressive motility, and interestingly, altered ORP levels were detected. In addition, it was determined that seminal plasma had a significant protective effect on spermatozoa. The elimination of seminal plasma caused higher sensitivity of spermatozoa against used OS inducers, and higher ORP levels and decreased viabilities and motilities were measured. The ORP level could be a good indicator of male OS; however, in cases of low and high sperm counts, its result can be misleading. Overall, the conclusion can be drawn that ORP determination is a suitable method for detecting intracellular ROS accumulation, but it has limitations that still need to be clarified.

Evaluation of Sperm DNA Fragmentation Using Two Different Methods: TUNEL via Fluorescence Microscopy, and Flow Cytometry.

Background and Objectives: Sperm DNA fragmentation refers to any break in one or both of the strands of DNA in the head of a sperm. The most widely used methodologies for assessing sperm DNA fragmentation are the sperm chromatin structure assay (SCSA), the sperm chromatin dispersion assay (SCD), the single-cell gel electrophoresis assay (SCGE-comet), and the terminal-deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay. The aim of this study was to compare the efficiency and sensitivity of the analysis of sperm DNA fragmentation using TUNEL via fluorescence microscopy, and flow cytometry. Materials and Methods: Semen samples were collected and analyzed for standard characteristics using light microscopy, and for sperm DNA fragmentation using both TUNEL via fluorescence microscopy, and flow cytometry. Results: There were no significant differences in the values of the sperm DNA fragmentation index (DFI) obtained when the analysis was performed using TUNEL or flow cytometry (p = 0.543). Spearman's correlation analysis revealed a significant negative correlation between sperm motility (%) and sperm DNA fragmentation (p < 0.01), as well as between sperm concentration and sperm DNA fragmentation (p < 0.05). The Mann-Whitney U test showed no significant difference in the DFI among couples with repeated implantation failure (RIF) and miscarriages (p = 0.352). Conclusions: Both methods (TUNEL via fluorescence microscopy, and flow cytometry) have a high efficiency and sensitivity in accurately detecting sperm DNA fragmentation, and can be effectively used to assess male fertility.

High-throughput sperm DNA analysis at the single-cell and population levels.

Clinical semen quality assessment is critical to the treatment of infertility. Sperm DNA integrity testing provides critical information that can steer treatment and influence outcomes and offspring health. Flow cytometry is the gold standard approach to assess DNA integrity, but it is not commonly applied at the clinical level. The sperm chromatin dispersion (SCD) assay provides a simpler and cheaper alternative. However, SCD is low-throughput and non-quantitative - sperm assessment is serial, manual and suffers inter- and intra-observer variations. Here, an automated SCD analysis method is presented that enables quantitative sperm DNA quality assessment at the single-cell and population levels. Levering automated optical microscopy and a chromatin diffusion-based analysis, a sample of thousands of sperm that would otherwise require 5 hours is assessed in under 10 minutes - a clinically viable workflow. The sperm DNA diffusion coefficient (DDNA) measurement correlates (R2 = 0.96) with DNA fragmentation index (DFI) from the cytometry-based sperm chromatin structure assay (SCSA). The automated measurement of population-level sperm DNA fragmentation (% sDF) prevents inter-observer variations and shows a good agreement with the SCSA % DFI (R2 = 0.98). This automated approach standardizes and accelerates SCD-based sperm DNA analysis, enabling the clinical application of sperm DNA integrity assessment.

Effects of supplementation of cryopreservation media with cysteine on the post-thaw quality and fertility of brown-marbled grouper (Epinephelus fuscoguttatus) spermatozoa.

The cryopreservation process is associated with the generation of excessive reactive oxygen species, which causes a series of cellular damage to spermatozoa. The objective of the current study was to investigate the effect of different concentrations of cysteine on post-thaw sperm quality of brown-marbled grouper sperm. Semen samples were frozen with cysteine supplemented at 0.5, 1, 2, 5, 10 mM and the control group (no additive). After thawing, sperm quality parameters were analyzed. In comparison to the control, cysteine treatment groups yielded relatively higher sperm total motility, progressive motility, and curvilinear velocity. Different concentrations of cysteine had no effect on average path velocity, straight linear velocity and viability (P > 0.05), while an increase in the concentration of cysteine resulted in a significant improvement in the mitochondrial membrane potential, SOD activity, and ATP content (P < 0.05). As for lipid peroxidation, the extent of which in cysteine treated spermatozoa was less than the control, although the differences were not statistically significant (P > 0.05). In terms of fertilizing capacity, a greater hatching rate (91.7 ± 1.2%) was obtained in thawed sperm treated with 2 mM cysteine, compared to the control (84.3 ± 4.2%; P < 0.05). Overall, it is concluded that the addition of cysteine is helpful in maintaining the function of frozen-thawed brown-marbled grouper sperm, which can be recommended as an effective antioxidant to improve the semen cryopreservation efficiency.

Human Sperm Head Vacuoles Are Related to Nuclear-Envelope Invaginations.

Nuclear vacuoles are specific structures present on the head of the human sperm of fertile and non-fertile men. Human sperm head vacuoles have been previously studied using motile sperm organelle morphology examination (MSOME) and their origin related to abnormal morphology, abnormal chromatin condensation and DNA fragmentation. However, other studies argued that human sperm vacuoles are physiological structures and consequently, to date, the nature and origin of the nuclear vacuoles remains to be elucidated. Here, we aim to define the incidence, position, morphology and molecular content of the human sperm vacuoles using transmission electron microscopy (TEM) and immunocytochemistry techniques. The results showed that ~50% of the analyzed human sperm cells (n = 1908; 17 normozoospermic human donors) contained vacuoles mainly located (80%) in the tip head region. A significant positive correlation was found between the sperm vacuole and nucleus areas. Furthermore, it was confirmed that nuclear vacuoles were invaginations of the nuclear envelope from the perinuclear theca and containing cytoskeletal proteins and cytoplasmic enzyme, discarding a nuclear or acrosomal origin. According to our findings, these human sperm head vacuoles are cellular structures originating from nuclear invaginations and contain perinuclear theca (PT) components, allowing us to define a new term of 'nuclear invaginations' rather than 'nuclear vacuoles'.

SARS-CoV-2 infection reduces quality of sperm parameters: prospective one year follow-up study in 93 patients.

BACKGROUND: Short- and long-term implications of SARS-CoV-2 on the quality of the sperm and the results of this on fertility remain largely unknown due to lack of longitudinal studies. In this longitudinal observational cohort study, we aimed to analyse the differential effect and the impact of SARS-CoV-2 infection on different semen quality parameters. METHODS: Sperm quality was assessed using the World Health Organization criteria, DNA damage to sperm cells by quantifying the DNA fragmentation index (DFI) and the high-density stainability (HDS), IgA- and IgG-anti-sperm antibodies (ASA) were assessed with light microscopy. FINDINGS: SARS-CoV-2 infection was associated with sperm parameters that were independent of spermatogenic cycle like progressive motility, morphology, DFI and HDS, as well as spermatogenic cycle dependent parameters such as sperm concentration. Detection of IgA- and IgG-ASA allowed classification of patients in three different groups according to its sequence of appearance in sperm during post-COVID-19 follow-up. The maximum progressive motility was lowest during follow-up in patients without ASA (41.9%), intermediate in patients with only IgA-ASA (46.2%) and highest inpatients who had both IgA- and IgG-ASA (54.9%). INTERPRETATION: SARS-CoV-2 infection was associated with changes of all analysed sperm parameters to a different degree which is also observed in their return to normality and is suggestive of individual variations in the patient's immune system performance. Firstly, sperm production is decreased through temporal immune mediated arrest of active meiosis, and secondly immune induced sperm DNA damage prevents fertilization if transferred to the oocyte. Both mechanisms are temporal, and most sperm parameters return to baseline after infection. FUNDING: AML (R20-014), Femicare.

Effect of incubation and analysis temperatures on sperm kinematics and morphometrics during human semen analysis / Efecto de las temperaturas de incubación y análisis sobre la cinemática y la morfometría de los espermatozoides durante el análisis de semen humano

Introduction: Human semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed. Methods: Seminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria. Results: The data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT Conclusions: Our results showed that temperature control during both incubation and analysis is needed for accurate semen analysis, recommending the use of 37°C during the entire process. (AU)

Introducción: El análisis de semen humano debe realizarse después de la licuefacción del eyaculado. Esto ocurre aproximadamente a los 30minutos después de la eyaculación. Las temperaturas para esta incubación y el análisis final de la motilidad son cruciales, pero rara vez se tienen en cuenta. Este estudio tiene como objetivo examinar el efecto de estas temperaturas en varios parámetros de los espermatozoides tanto de forma manual (recuento de espermatozoides, motilidad, morfología, viabilidad, condensación y maduración de la cromatina y fragmentación del ADN) como CASA (cinemática y morfometría, utilizando un CASA-Mot ISAS®v1 y Sistemas CASA-Morph, respectivamente) analizados. Métodos: Las muestras seminales de 13 donantes se incubaron durante 10minutos a 37°C, seguidas de 20minutos adicionales a temperatura ambiente (TA, 23°C) o a 37°C y luego se examinaron siguiendo los criterios de la OMS 2010. Resultados: Los datos obtenidos muestran que no hubo diferencias significativas (p>0,05) en los parámetros subjetivos de calidad del esperma con la temperatura de incubación. Por otro lado, los parámetros morfométricos de la cabeza de los espermatozoides fueron significativamente más altos después de la incubación a temperatura ambiente, mostrando, además, una elipticidad más baja (p<0,05). Además, los parámetros cinemáticos se evaluaron tanto a temperatura ambiente como a 37°C para las dos temperaturas de incubación. En general, las cuatro combinaciones de temperatura mostraron que los parámetros cinemáticos siguieron este orden: RT-RT < RT-37 < 37-37 < 37-RT (temperaturas de incubación y análisis, respectivamente). Conclusiones: Nuestros resultados mostraron que el control de la temperatura durante la incubación y el análisis es necesario para un análisis de semen preciso, recomendando el uso de 37°C durante todo el proceso. (AU)

Computer-assisted semen analysis in large falcons (Falco spp.) and comparison to results of conventional semen analysis

OBJECTIVE: Numerous raptor species including some falcon species are facing continuous population decline in the wild and some are threatened by extinction. To support these species, captive breeding and reintroduction programs are attempted. Besides conservation, some large falcon species are commonly used in falconry and therefore bred commercially. Assisted reproduction is established in falcon breeding since the 1970s and semen analysis is an integral part to enable assessment of breeding males, inclusion or exclusion of semen donors and quality control of semen prior to artificial insemination. Conventional methods for semen analysis are widely used, but are time consuming and depend on the investigator's experience and ability. Computer-assisted semen analysis (CASA) would offer an objective, fast and reproducible alternative, but as they have not been established in large falcon species, this was the aim of this study. MATERIAL AND METHODS: To this end, we examined in 3 breeding seasons 109 semen samples of gyr-saker hybrid falcons (n=2) and peregrine falcons (n=4) in 940 fields of view using the Minitube CASA SpermVision and compared these results to results of conventional methods of semen analysis. We used a preprogrammed setting and adapted two settings of CASA according to specific semen characteristics of falcons. RESULTS: Sperm velocity, motility and viability parameters were recorded successfully using CASA. Correlation of conventional and computer-assisted motility analysis improved during the process of adaptation of CASA settings, but both methods differed significantly due to misinterpretation of round bodies and semen impurities by CASA. Viability values of conventional and computer assisted viability analysis using SYBR-PI correlated significantly while sperm concentration did not at all. CONCLUSION: CASA failed to replace conventional semen analysis regarding sperm motility and sperm concentration using 3 different settings, as a reliable differentiation between spermatozoa, spermatids and round bodies was not achieved. CLINICAL RELEVANCE: Using CASA, sperm velocity parameters were measured in spermatozoa of captive-bred large falcons for the first time and may be used as orientation values.

Freezing Does Not Alter Sperm Telomere Length despite Increasing DNA Oxidation and Fragmentation.

Correlations were reported between sperm telomere length (STL) and male fertility, sperm DNA fragmentation, and oxidation. Sperm freezing is widely used for assisted reproductive techniques, fertility preservation, and sperm donation. However, its impact on STL remains unknown. For this study, semen surplus from patients who underwent routine semen analysis were used. The impact of slow freezing on STL was analyzed by performing qPCR before and after freezing. Sperm populations with different STL were evaluated using Q-FISH. The relationship between sperm DNA oxidation, DNA fragmentation, and STL was assessed in fresh and frozen sperm samples. No significant impact of slow freezing on STL was observed, neither measured by qPCR nor Q-FISH. However, Q-FISH allowed for the distinguishing of sperm populations with different STLs within individual sperm samples. Slow freezing induced different STL distributions for some of the analyzed sperm samples, but no correlation was found between STL and sperm DNA fragmentation or oxidation. Slow freezing does not alter STL despite increasing sperm DNA oxidation and fragmentation. As STL alterations could be transmitted to offspring, the lack of impact of the slow freezing method on STL ensures the safety of this procedure.

Novel sperm chromatin dispersion test with artificial intelligence-aided halo evaluation: A comparison study with existing modalities.

BACKGROUND: Sperm chromatin dispersion test is a common and inexpensive technique to assess sperm DNA fragmentation, but its subjectivity in assessing a small number of spermatozoa is a disadvantage. OBJECTIVES: To study the efficacy of a new sperm chromatin dispersion test kit (R10) combined with an artificial intelligence-aided halo-evaluation platform (X12) and compare the results to those of existing sperm DNA fragmentation testing methods. MATERIALS AND METHODS: Semen samples from normozoospermic donors (n = 10) and infertile men with abnormal semen parameters (n = 10) were enrolled. DNA fragmentation indices were examined by multiple assays, including R10, Halosperm G2 (G2), sperm chromatin structure assay, and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling. In R10 assay, the DNA fragmentation indices were obtained both manually (manual R10) and by X12 (AI-R10). The obtained DNA fragmentation indices were analyzed by agreement analyses. RESULTS: The DNA fragmentation indices obtained by manual R10 and those obtained by AI-R10 showed a strong significant correlation (r = 0.97, p < 0.001) and agreement. The number of spermatozoa evaluated by AI-R10 was 2078 (680-5831). The DNA fragmentation indices obtained by manual R10 and AI-R10 both correlated with those of G2 (r = 0.90, p < 0.001; r = 0.88, p < 0.001). Between the AI-R10 and G2 results, Passing-Bablok regression showed no systematic or proportional difference, and Bland-Altman plots revealed overall agreement and a mean bias of 6.3% with an SD of 6.9% (95% limit of agreement: -7.2% to 19.9%). AI-R10 and sperm chromatin structure assays showed systematic differences with a mean bias of -1.9%, while AI-R10 and terminal deoxynucleotidyl transferase deoxynucleotidyl transferase nick end labeling revealed proportional differences with a mean bias of -10.7%. CONCLUSIONS: The novel sperm chromatin dispersion kit and artificial intelligence-aided platform demonstrated significant correlation and agreement with existing sperm chromatin dispersion methods by assessing greater number of spermatozoa. This technique has the potential to provide a rapid and accurate assessment of sperm DNA fragmentation without technical expertise or flow cytometry.

Tinospora cordifolia modulates the seminal parameters, leakage of intracellular enzymes and seminal antioxidants in equilibrated and cryopreserved semen of Sahiwal bulls.

The present study was undertaken to assess the effects of stem extract of Tinospora cordifolia (Giloy or Guduchi) in the semen extender on seminal parameters, leakage of intracellular enzymes and antioxidants in semen of Sahiwal bull. A total of 48 ejaculates from four bulls were selected for the study. Spermatozoa of 25 × 106 were incubated in 100, 300 and 500 µg of stem extract of Guduchi as Gr II, III and IV, respectively, and pre-freeze and post-thaw semen samples were analysed for seminal parameters [motility, viability, total sperm abnormality (TSA), plasma membrane integrity (PMI) and acrosomal integrity (AcI)], intracellular enzymes [aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)] and seminal antioxidants [superoxide dismutase (SOD) and catalase] in comparison with an untreated control group (Gr I). The results revealed that stem extract-treated semen had significantly (p < .05) higher motility, viability, PMI, AcI, SOD and catalase and had significantly (p < .05) lower TSA, AST and LDH compared to those in untreated control group at pre-freeze and post-thaw stages. Semen treated with 100 µg stem extract/25 × 106 spermatozoa had significantly (p < .05) higher motility, viability, PMI, AcI, SOD and catalase and had significantly (p < .05) lower TSA, AST and LDH compared to those in control, 300- and 500-µg-treated groups at pre-freeze and post-thaw stages. Further, these seminal parameters and antioxidants were showing decreasing trend and TSA and leakage of intracellular enzymes were showing increasing trend from Gr II to Gr IV at pre-freeze and post-thaw stages. Thus, 100 µg/25 × 106 spermatozoa were optimum or suitable dose for cryopreservation of Sahiwal bull semen. The study concluded that T. cordifolia stem extract 100 µg/25 × 106 spermatozoa in the semen extender can be effectively utilized to reduce the oxidative stress and improve the pre-freeze and post-thaw seminal parameters in Sahiwal bull. However, further studies on effects of different concentrations of stem extract on in vitro or in vivo fertility trials are to be conducted to assess the impact of the stem extract supplementation in the semen extender on field pregnancy outcomes in bovine species.

Analytical variability and interpretation of results of a 3-category sperm motility assessment: 5 years' of an Australian external quality assurance programme.

RESEARCH QUESTION: How do laboratories perform when assessing sperm motility with a 3-category system and interpreting results as per the fifth edition of the World Health Organization manual (WHO5), and will the use of a 4-category system as per the sixth edition of the WHO manual improve their performance? DESIGN: Eighty video recordings of sperm samples were sent to over 200 laboratories spanning a 5-year period for the assessment of progressive motility. The results were reviewed relative to the all-laboratory trimmed mean (ALTM) in terms of the minimum and maximum values reported, the coefficient of variation and the proportion of laboratories indicating an abnormal result. A further 20 video recordings were sent over 1 year, with 6-11 laboratories per distribution adjusting to reporting rapid progressive motility using the 4-category system. RESULTS: For the 3-category system, the videos covered a mean assessed progressive motility range of 12.0-81.1%. The mean difference between the minimum and maximum values per sample was 50.3% and the coefficients of variation were negatively correlated with the ALTM (r = -0.87, P < 0.00001). Progressive motility abnormality reporting formed a sigmoid curve, and the inflection point (50% of laboratories identifying an abnormality) gave an ALTM value of 32.01%. Preliminary results for laboratories using the 4-category system showed no performance improvement but the number of laboratories was small. CONCLUSIONS: Analytical variation can result in laboratories crossing the clinical cut-off of the lower reference limit for samples whose motility is close to the WHO5 lower reference limit, but is less important for samples with extreme values. The benefits of a 4-category motility system are yet to be shown.

Effect of incubation and analysis temperatures on sperm kinematics and morphometrics during human semen analysis.

INTRODUCTION: Human semen analysis must be performed after the liquefaction of the ejaculate. This takes place about 30min after ejaculation and samples must be maintained in the lab during this time. The temperatures for this incubation and the final analysis of motility are crucial but seldom taken into account. This study aims to examine the effect of these temperatures on various sperm parameters both manually (sperm count, motility, morphology, viability, chromatin condensation and maturation and DNA fragmentation) and CASA (kinematics and morphometrics, using an ISAS®v1 CASA-Mot and CASA-Morph systems, respectively) analyzed. METHODS: Seminal samples from thirteen donors were incubated for 10min at 37°C followed by additional 20min at either room temperature (RT, 23°C) or 37°C and then examined following WHO 2010 criteria. RESULTS: The data obtained show that there were no significant differences (P>0.05) in the subjective sperm quality parameters with incubation temperature. On the other hand, the head sperm morphometric parameters were significantly higher after room temperature incubation showing, in addition, lower ellipticity (P<0.05). Furthermore, kinematic parameters were evaluated both at RT and 37°C for the two incubation temperatures. In general, the four temperature combinations showed that kinematic parameters followed this order: RT-RT

A matter of agreement: The effect of the technique and evaluator on the analysis of morphologic defects in stallion sperm.

Analysis of sperm morphology is an important part of the stallion breeding soundness evaluation since it provides an objective measure of a stallion's sperm quality and is one of many factors that estimate a stallion's fertility potential. To describe the effect of sperm quality level on the technique (Differential Interference Contrast - DIC; Phase-contrast - PH; Dip-Quick staining - DQ; and eosin-nigrosin staining - EN; semen samples fixed in buffered-formal saline) and evaluator (three evaluators; using only DIC), stallions were categorized based on sperm quality into three categories: High: >57% normal sperm, Moderate: 23-56% normal sperm, or Low: <23% normal sperm (four stallions per category). The data were analyzed using three different statistical methods: Analysis of Variance (ANOVA), correlative analysis, and Bland-Altman method (agreement). A higher level of agreement among techniques was observed between DIC and PH for morphologically normal sperm. The agreement between the alternative methods (EN, DQ, or PH) and the standard method (DIC) varied, depending on the sperm quality level (High, Moderate, or Low). Some morphological defects (e.g., AH, AMP) were constantly underestimated with the staining methods (DQ, EN) compared to DIC and PH, particularly in ejaculates with low sperm morphology. Underestimation of some abnormalities, due to the technique or the evaluator, has the potential to alter the clinical interpretation of stallion fertility.