Phase angle at bioelectric impedance analysis is associated with detrimental sperm quality in idiopathic male infertility: a preliminary clinical study.

Background: In 2020, 38% of adults were affected by obesity, while infertility globally affected 1 in 6 people at some stage of their lives.Body mass index (BMI) provides an easy but occasionally inaccurate estimation of body composition. To achieve a more precise assessment, bioelectric impedance analysis serves as a validated tool that administers electrical energy through surface electrodes. Phase angle as a function of the relationship between tissues resistance and reactance, is a trustworthy predictor of body composition and cell membrane integrity. Objectives: We aim to assess whether there is an association between phase angle and seminal parameters, as well as sperm DNA fragmentation percentage. Design: Semen samples of 520 idiopathic infertile patients were analyzed according to 2021 World Health Organization guidelines and evaluated for sperm DNA fragmentation rate. Each participants underwent bioelectric impedance analysis. Results: Median age was 40 years old, median BMI was 26.3 kg/m2, median phase angle was 6.2°. In the logistic regression analysis adjusted for age and total intracorporeal water, phase angle (continuous) was significantly associated with oligozoospermia (odds ratio [OR]:0.4; p<0.01) and sperm morphology (OR: 0.65; p=0.05) and slightly with sperm DNA fragmentation (OR: 0.98; p=0.07). In subgroup analysis, the logistic regression analysis adjusted for the mentioned parameters showed that a phase angle between 6.2 and 7 (°) (OR: 0.63; p=0.02) and >7 (°) (OR: 0.12; p<0.01) were associated with a reduced risk of oligozoospermia compared to values <6.2 (°). Similarly, a phase angle between 6.2 and 7 (°) (OR: 0.57; p< 0.01 and OR: 0.58; p= 0.01) and PA > 7 (°) (OR: 0.12; p= 0.03 and OR: 0.21; p< 0.01) were associated with a reduced risk of lower sperm concentration and lower total sperm count, respectively, compared to a phase angle < 6.2 (°). Conclusion: Our study suggests a negative association between phase angle and detrimental sperm parameters in male idiopathic infertility.

Clustering of spermatozoa examined through flow cytometry provides more information than the conventional assessment: a resilience to osmotic stress example.

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.

Testing the generalizability and effectiveness of deep learning models among clinics: sperm detection as a pilot study.

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.

Effects of Slow Freezing and Vitrification of Human Semen on Post-Thaw Semen Quality and miRNA Expression.

Semen cryopreservation has played an important role in medically assisted reproduction for decades. In addition to preserving male fertility, it is sometimes used for overcoming logistical issues. Despite its proven clinical usability and safety, there is a lack of knowledge of how it affects spermatozoa at the molecular level, especially in terms of non-coding RNAs. Therefore, we conducted this study, where we compared slow freezing and vitrification of good- and poor-quality human semen samples by analyzing conventional sperm quality parameters, performing functional tests and analyzing the expression of miRNAs. The results revealed that cryopreservation of normozoospermic samples does not alter the maturity of spermatozoa (protamine staining, hyaluronan binding), although cryopreservation can increase sperm DNA fragmentation and lower motility. On a molecular level, we revealed that in both types of cryopreservation, miRNAs from spermatozoa are significantly overexpressed compared to those in the native semen of normozoospermic patients, but in oligozoospermic samples, this effect is observed only after vitrification. Moreover, we show that expression of selected miRNAs is mostly overexpressed in native oligozoospermic samples compared to normozoospermic samples. Conversely, when vitrified normozoospermic and oligozoospermic samples were compared, we determined that only miR-99b-5p was significantly overexpressed in oligozoospermic sperm samples, and when comparing slow freezing, only miR-15b-5p and miR-34b-3p were significantly under-expressed in oligozoospermic sperm samples. Therefore, our results imply that cryopreservation of normozoospermic sperm samples can modulate miRNA expression profiles in spermatozoa to become comparable to those in oligozoospermic samples.

Sperm Chromatin Dispersion Test Detects Sperm DNA Fragmentation Mainly Associated with Unviable Spermatozoa and Underestimates the Values with Respect to TUNEL Assay.

Several clinical laboratories assess sperm DNA fragmentation (sDF) in addition to semen analysis in male infertility diagnosis. Among tests evaluating sDF, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) and SCD (Sperm Chromatin Dispersion) are widely used. Our lab developed a modified version of TUNEL (TUNEL/PI) able to distinguish two sperm populations (PI Brighter and PI Dimmer) differently associated with sperm viability and reproductive outcomes. The aim of this study was to compare sDF levels detected by SCD and TUNEL/PI in the semen samples from 71 male subjects attending our Andrology Laboratory. Our results demonstrate that SCD is less sensitive in determining sDF compared to TUNEL/PI. The statistically significant positive correlation found between sDF evaluated by SCD and PI Dimmer (consisting of all dead spermatozoa) suggests that SCD mainly detects sDF in unviable spermatozoa. We confirmed that most spermatozoa detected by SCD are unviable by performing SCD after incubation in hypo-osmotic medium to discriminate viable and unviable cells in 52 samples. Such results might explain the lower ability of this test in discriminating couples having successful ART outcomes demonstrated in published metanalyses. Overall, our results indicate that SCD is less sensitive in evaluating sDF for diagnostic purposes.

Determinant Factors for Ongoing Pregnancy in Homologous Intrauterine Insemination Cycles - A Prospective Study.

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.

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.

The correlation between human seminal plasma sialoproteins and ejaculate parameters.

Sialic acids are negatively charged carbohydrates that are components of saccharide chains covalently linked to macromolecules. Sialylated glycoproteins are important for most biological processes, including reproduction, where they are associated with spermatogenesis, sperm motility, immune responses, and fertilization. Changes in the glycoprotein profile or sialylation in glycoproteins are likely to affect the quality of ejaculate. The aim of this study was to determine differences in the degree of sialylation between normozoospermic ejaculates and ejaculates with a pathological spermiogram using two lectins, Sambucus nigra (SNA) and Maackia amurensis (MAL II/MAA) recognizing α-2,6 or α-2,3 linkage of Sia to galactosyl residues. Our results show a close relationship between seminal plasma (SP) sialoproteins and the presence of anti-sperm antibodies in the ejaculate, apoptotic spermatozoa, and ejaculate quality. Using mass spectrometry, we identified SP sialoproteins such as, semenogelins, glycodelin, prolactin-inducible protein, lactotransferrin, and clusterin that are associated with spermatozoa and contribute to the modulation of the immune response and sperm apoptosis. Our findings suggest a correlation between the degree of SP glycoprotein sialylation and the existence of possible pathological states of spermatozoa and reproductive organs. Glycoproteins sialylation represents a potential parameter reflecting the overall quality of ejaculate and could potentially be utilised in diagnostics.

Effect of the technical variability of counting chambers upon the interpretation of sperm concentration results.

RESEARCH QUESTION: Does the choice of sperm-counting chamber affect the proportion of samples generating results with an erroneous interpretation? DESIGN: Laboratories in an external quality assurance programme were sent 141 semen samples over a 12-year period and asked to return the sperm concentration and whether or not the result was abnormal. Only those using 5th edition of the World Health Organization manual (WHO5) interpretation criteria were included. Submissions from specialist fertility laboratories were used to calculate assigned values for each sample. Laboratory50 values determined the sperm concentration at which the laboratories reported a majority transition from abnormal to normal interpretations, i.e. the tipping point, which should coincide with the lower reference limit. RESULTS: The median and range of bias from the assigned values of each sample were determined for the Makler (-3.3%; -88.6% to +332.8%), haemocytometer (10.6%; -93.3% to +645.5%), Kova (+65.3%; -71.7% to +581.8%) and Vetriplast (+72.4%; -100.0% to +709.1) chambers. Laboratory50 values for the Makler (17.3  ×  106/ml), haemocytometer (13.6  ×  106/ml), Kova (10.0  ×  106/ml) and Vetriplast chambers (8.8  ×  106/ml) reflected the under- and overestimation of the chambers and confirmed a shift in the adjusted lower reference limit then used. The proportion of laboratories reporting erroneous interpretations of the four chambers for oligozoospermic samples were 10.9%, 15.1.%, 40.1% and 44.0%, respectively, and rose as the adjusted lower reference limit decreased. CONCLUSIONS: The between-laboratory and within-sample variation for all the chambers was high and remains a concern. The main impact of an increasing bias of the chambers was a lowering of the laboratory50 tipping point, resulting in an under-reporting of abnormal semen samples.

Agreement and internal quality assurance of the Neubauer hemocytometer and Makler chamber for human sperm concentration determination.

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.

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.

Microfluidic sperm sorting selects a subpopulation of high-quality sperm with a higher potential for fertilization.

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.

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.

Urine microbes and predictive metagenomic profiles associate with abnormalities in sperm parameters: implications for male subfertility.

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.

Unraveling the Impact of Sperm DNA Fragmentation on Reproductive Outcomes.

In recent years, there has been a growing interest in identifying subcellular causes of male infertility, and sperm DNA fragmentation (SDF) research has been at the forefront of this focus. DNA damage can occur during spermatogenesis due to faulty chromatin compaction or excessive abortive apoptosis. It can also happen as sperm transit through the genital tract, often induced by oxidative stress. There are several methods for SDF testing, with the sperm chromatin structure assay, terminal deoxynucleotidyl transferase d-UTI nick end labeling (TUNEL) assay, comet assay, and sperm chromatin dispersion test being the most commonly used. Numerous studies strongly support the negative impact of SDF on male fertility potential. DNA damage has been linked to various morphological and functional sperm abnormalities, ultimately affecting natural conception and assisted reproductive technology outcomes. This evidence-based review aims to explore how SDF influences male reproduction and provide insights into available therapeutic options to minimize its detrimental impact.

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.

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.

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.

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.