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This comprehensive review explores the evolving landscape of sperm selection techniques within the realm of Assisted Reproductive Technology (ART). Our analysis delves into a range of methods from traditional approaches like density gradient centrifugation to advanced techniques such as Magnetic-Activated Cell Sorting (MACS) and Intracytoplasmic Morphologically Selected Sperm Injection (IMSI). We critically assess the efficacy of these methods in terms of sperm motility, morphology, DNA integrity, and other functional attributes, providing a detailed comparison of their clinical outcomes. We highlight the transition from conventional sperm selection methods, which primarily focus on physical characteristics, to more sophisticated techniques that offer a comprehensive evaluation of sperm molecular properties. This shift not only promises enhanced prediction of fertilization success but also has significant implications for improving embryo quality and increasing the chances of live birth. By synthesizing various studies and research papers, we present an in-depth analysis of the predictability of different sperm selection procedures in ART. The review also discusses the clinical applicability of these methods, emphasizing their potential in shaping the future of assisted reproduction. Our findings suggest that the integration of advanced sperm selection strategies in ART could lead to more cost-effective treatments with reduced duration and higher success rates. This review aims to provide clinicians and researchers in reproductive medicine with comprehensive insights into the current state and future prospects of sperm selection technologies in ART.

Sperm characterization is an important part of reproductive biology and is particularly important for the conservation of endangered species in the wild. However, research on this aspect of the Chinese pangolin, an endangered species from mainland China, was extremely limited. Therefore, the present study fills the gap in this research area by first systematically providing the sperm collection methods and the characteristics of the spermatozoa of Chinese pangolin. Abstract The Chinese pangolin (Manis pentadactyla) is a critically endangered species. However, there is a paucity of research on the male reproductive gamete biology of this species. The present study was the first to systematically analyse the sperm characterization of the Chinese pangolin, including semen collection, sperm morphometry and ultrastructure. The semen of five male Chinese pangolins was successfully collected using the electroejaculation method. CASA (computer-assisted sperm analysis) was used to assess semen quality and take images for sperm morphometric analysis. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used for sperm ultrastructure observation. The results showed that the semen of the Chinese pangolin was yellow to pale yellow in colour, viscous, with a fishy odour, and a slightly alkaline pH of between 7.7 and 7.9. The head defects were the main sperm defects; there were 13 kinds of head defects counted in this study. The total sperm length, head length, head width and tail length were 67.62 ± 0.21 μm, 10.47 ± 0.06 μm, 1.33 ± 0.006 μm and 57.16 ± 0.20 μm, respectively. SEM observed that the spermatozoa had a rod-shaped head with a distinct apical ridge, which was different from most mammals and similar to that in avians and reptiles. Interestingly, TEM found that the acrosome membrane of the Chinese pangolin had a double membrane structure rather than a multiple bi-lamellar membrane structure as reported by the previous study. Collectively, this study contributes to the development of artificial breeding efforts and assisted reproductive techniques for the Chinese pangolin, as well as providing technical support for research on germplasm conservation of this species.

This study investigated the effects of pure and methyl-β-cyclodextrin loaded forms of resveratrol (10 µg/mL, 20 µg/mL, and 40 µg/mL) on ram sperm functions post-thawing. Semen samples were pooled and divided into ten groups: Control, RES10, RES20, RES40, CD10, CD20, CD40, RLC10, RLC20, and RLC40. The groups were pre-diluted with media containing the group-specific chemicals, followed by 15 min of incubation, dilution, and freezing. To assess the effects of the chemicals, a post-thaw sperm quality assessment was conducted. Motility and other velocity parameters were evaluated using computer-assisted semen analysis. The functional integrity of spermatozoa membranes was assessed with the hypo-osmotic swelling test, and the capacitation status of spermatozoa was determined through fluorescent microscopic evaluation. Additionally, flow cytometry was used to evaluate mitochondrial activity, oxidative stress, and the integrity of the sperm membrane and acrosome. The results indicated that cyclodextrin adversely affected sperm functions following freezing–thawing, notably increasing the rate of spermatozoa exhibiting pre-capacitation and mitochondrial activity by approximately 34% and 16%, respectively (p < 0.05). It was found that 20 µg/mL resveratrol prevented pre-capacitation (p < 0.05). Both resveratrol and resveratrol-loaded cyclodextrin groups improved post-thaw sperm qualities overall, demonstrating their utility for freezing ram semen. However, higher concentrations of resveratrol were found to negatively impact sperm functions.

In wildlife conservation, breeding programmes focused on reintroduction are critical to recovering endangered species. Assisted reproductive technologies (ARTs) and biobanking play pivotal roles in these efforts but depend on high‐quality gametes. Spermatozoa, due to their structural simplicity and well‐documented quality assessments, are often considered the preferred cell type for cryopreservation and ARTs, especially in rare or exotic species. However, inconsistent retrieval of sperm samples with sufficient volume and concentration, especially for critically endangered species, can pose major limitations to assessing overall sperm quality. To address this information gap, we have developed the Wildlife Sperm Index (WSI), a standardized scoring system that integrates key sperm quality metrics—such as motility, viability and DNA integrity—into a weighted composite score. We designed the WSI as both a quality assessment and decision‐making tool, enabling users to evaluate sample suitability for cryopreservation, ARTs or downstream applications. While this study applies the WSI to sperm, the framework is adaptable to other cell types used in biobanking and reproductive technologies, broadening its potential utility. Unlike traditional sperm assessments that rely solely on microscopy, the WSI provides a scalable and quantitative framework for evaluating samples across species, including those that yield low volume or concentration sperm samples. By enabling standardized quality tracking, the WSI provides a foundation for more informed reproductive decisions to support global conservation efforts.

Background Semen quality, a predictor of male fertility, has been suggested declining worldwide. Among other life style factors, male coffee/caffeine consumption was hypothesized to influence semen parameters, but also sperm DNA integrity. To summarize available evidence, we performed a systematic review of observational studies on the relation between coffee/caffeine intake and parameters of male fertility including sperm ploidy, sperm DNA integrity, semen quality and time to pregnancy. Methods A systematic literature search was performed up to November 2016 (MEDLINE and EMBASE). We included all observational papers that reported the relation between male coffee/caffeine intake and reproductive outcomes: 1. semen parameters, 2. sperm DNA characteristics, 3. fecundability. All pertinent reports were retrieved and the relative reference lists were systematically searched in order to identify any potential additional studies that could be included. Results We retrieved 28 papers reporting observational information on coffee/caffeine intake and reproductive outcomes. Overall, they included 19,967 men. 1. Semen parameters did not seem affected by caffeine intake, at least caffeine from coffee, tea and cocoa drinks, in most studies. Conversely, other contributions suggested a negative effect of cola-containing beverages and caffeine-containing soft drinks on semen volume, count and concentration. 2. As regards sperm DNA defects, caffeine intake seemed associated with aneuploidy and DNA breaks, but not with other markers of DNA damage. 3. Finally, male coffee drinking was associated to prolonged time to pregnancy in some, but not all, studies. Conclusions The literature suggests that caffeine intake, possibly through sperm DNA damage, may negatively affect male reproductive function. Evidence from epidemiological studies on semen parameters and fertility is however inconsistent and inconclusive. Well-designed studies with predefined criteria for semen analysis, subject selection, and life style habits definition, are essential to reach a consistent evidence on the effect of caffeine on semen parameters and male fertility.

In the field of assisted reproductive techniques (ART), computer-assisted sperm analysis (CASA) systems have proved their utility and potential for assessing sperm quality, improving the prediction of the fertility potential of a seminal dose. Although most laboratories and scientific centers use commercial systems, in the recent years certain free and open-source alternatives have emerged that can reduce the costs that research groups have to face. However, these open-source alternatives cannot analyze sperm kinetic responses to different stimuli, such as chemotaxis, thermotaxis or rheotaxis. In addition, the programs released to date have not usually been designed to encourage the scalability and the continuity of software development. We have developed an open-source CASA software, called OpenCASA, which allows users to study three classical sperm quality parameters: motility, morphometry and membrane integrity (viability) and offers the possibility of analyzing the guided movement response of spermatozoa to different stimuli (useful for chemotaxis, thermotaxis or rheotaxis studies) or different motile cells such as bacteria, using a single software. This software has been released in a Version Control System at Github. This platform will allow researchers not only to download the software but also to be involved in and contribute to further developments. Additionally, a Google group has been created to allow the research community to interact and discuss OpenCASA. For validation of the OpenCASA software, we analysed different simulated sperm populations (for chemotaxis module) and evaluated 36 ejaculates obtained from 12 fertile rams using other sperm analysis systems (for motility, membrane integrity and morphology modules). The results were compared with those obtained by Open-CASA using the Pearson's correlation and Bland-Altman tests, obtaining a high level of correlation in all parameters and a good agreement between the different used methods and the OpenCASA. With this work, we propose an open-source project oriented to the development of a new software application for sperm quality analysis. This proposed software will use a minimally centralized infrastructure to allow the continued development of its modules by the research community.

ObjectivesEnvironmental exposure to chemicals has been considered a potential factor contributing to deteriorated semen quality. However, previous literature on exposure to air pollution and semen quality is inconsistent. We therefore investigated the health effects of short-term and long-term exposure to fine particulate matter (PM2.5) on semen quality in Taiwanese men from the general population.MethodsA cross-sectional study was conducted among 6475 male participants aged 15–49 years who participated in a standard medical examination programme in Taiwan between 2001 and 2014. Semen quality was assessed according to the WHO 1999 guidelines, including sperm concentration, total motility, progressive motility and morphology. Three-month and 2-year average PM2.5 concentrations were estimated at each participant’s address using a spatiotemporal model based on satellite-derived aerosol optical depth data. Multivariable linear and logistic regressions were used to examine the associations between PM2.5 and semen quality.ResultsA robust association was observed between exposure to PM2.5 and decreased normal morphology. Every increment of 5 µg/m3 in 2-year average PM2.5 was significantly associated with a decrease of 1.29% in sperm normal morphology and a 26% increased risk of having the bottom 10% of sperm normal morphology, after adjusting for a wide range of potential confounders (p<0.001). On the other hand, an increment of 5 µg/m3 in 2-year average PM2.5 was associated with an increase of 1.03×106/mL in sperm concentration and a 10% decreased risk of being the bottom 10% of sperm concentration (both p<0.001). Similar results were found for 3-month PM2.5.ConclusionsExposure to ambient PM2.5 air pollution is associated with a lower level of sperm normal morphology and a higher level of sperm concentration.

The purpose of this study was to examine sperm quality after cryopreservation of ejaculates collected as a bulk sample, which is routinely part of semen collection, and to compare this quality with the sperm-rich fraction in boars. Ejaculates were collected as sperm-rich fractions (SRF) and bulk samples (BE) using a gloved-hand technique. Fresh semen quality in terms of semen volume, sperm concentration, total sperm motility and pH were conventionally evaluated. Then, semen was cryopreserved using the liquid nitrogen vapour method. The post-thaw sperm quality was evaluated by assessing sperm motility, live sperm with normal apical ridge and high mitochondrial energy status, lipid peroxidation was evaluated using CASA and fluorescent multiple staining and MDA levels were determined using a spectrophotometer, respectively. In terms of fresh semen quality, sperm motility in fresh semen did not differ significantly between the two groups. The treatment with the greater mean volume (BE; P < 0.05) had a lower mean sperm concentration (P < 0.05); meanwhile, the mean ejaculate pH collected as BE was more basic compared with SRF (P < 0.05). However, there were no significant post-thaw quality changes between sperm-rich fractions and bulk samples of semen. In conclusion, ejaculates can be collected as bulk samples without the need to classify fractions for boar semen cryopreservation.

Epidemiological evidence remains equivocal on the associations between environmentally relevant levels of per-/polyfluoroalkyl substances (PFASs) and human semen quality. We aimed to test whether the potential effects on semen quality could be better observed when seminal PFAS levels were used as an exposure marker compared with serum PFAS levels. Matched semen and serum samples from 664 adult men were collected from a cross-sectional population in China from 2015 to 2016. Multiple semen parameters were assessed, along with measurement of 16 target PFASs in semen and serum. Partitioning between semen and serum was evaluated by the ratio of matrix-specific PFAS concentrations. Regression model results were expressed as the difference in each semen parameter associated with the per unit increase in the ln-transformed PFAS level after adjusting for confounders. Perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS), and emerging chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) were detected at their highest concentrations in both semen and serum, with median concentrations of 0.23, 0.10, and in semen, respectively, and a semen-to-serum ratio of 1.3:3.1. The between-matrix correlations of these PFAS concentrations were high ( ). Seminal PFOA, PFOS, and 6:2 Cl-PFESA levels were significantly associated with a lower percentage of progressive sperm and higher percentage of DNA fragmentation (false discovery rate-adjusted ). Associations between serum PFAS levels and semen parameters were generally statistically weaker, except for DNA stainability, which was more strongly associated with serum-based PFASs than with semen-based PFASs. Our results suggest the potential for deleterious effects following exposure to 6:2 Cl-PFESA and other PFASs. Compared with serum PFAS levels, the much clearer association of seminal PFAS levels with semen parameters suggests its advantage in hazard assessment on semen quality, although the potential for confounding might be higher. Exposure measurements in target tissue may be critical in clarifying effects related to PFAS exposure. https://doi.org/10.1289/EHP4431.

Atrazine (ATZ), as a widely used triazine herbicide, is an environmental endocrine disruptor (EDC) that can cause many health problems. Therefore, we conducted this study based on the evidence of rats and mice to figure out the characteristics of ATZ damage to the reproductive system and further evaluate its health effects on the human. PRISMA’s guidelines were followed according to the principles recommended by the Cochrane Handbook for Systematic Review. Health assessment was performed using the OHAT approach. Our new data were obtained from randomized controlled trials in rats designed in accordance with toxicological guidelines. Exposure to ATZ was significantly associated with decreased testosterone production (SMD = − 0.90, 95% CI − 1.27 to − 0.53), and reduced absolute weights of testis (SMD = − 0.41, 95% CI − 0.61 to − 0.22) and other reproductive organs. The damaging effect of sperm quality was also observed clearly, which included reduction of sperm count both in epididymis (SMD = − 2.32, 95% CI − 2.83 to − 1.81) and testis (SMD = − 1.01, 95% CI − 1.37 to − 0.64), decrease in sperm motility (SMD = − 8.86, 95% CI − 10.88 to − 6.83), and increase in sperm abnormality. Subgroup analysis revealed consistency across different species, life stage, and dosage. In addition, we found that ATZ exposure at a daily dose of 120 mg/kg during adolescence could cause decrease in weight gain and histological damage to the testis. The gene expression levels of Nrf2/HO-1 and Bcl-2/caspase signaling pathways in testis tissues were changed significantly. The results of this SR indicated that exposure to ATZ was associated with impairment of male reproductive system in rodents regardless of species, exposure life stage, and dosage. It is believed that ATZ exposure may have similar effects on male reproductive system of human beings. Pathways related to oxidative stress and apoptosis may be the mechanism leading to testicular damage in rats treated with ATZ.