The use of microfluidic sperm sorting (MFSS) systems in infertility treatment is increasing due to their practicality and ease of use. While often presented as highly effective, their efficacy in patients with varying sperm analysis results remains uncertain. In this study, we evaluated the effectiveness of MFSS compared with the swim-up (SU) technique in terms of oxygen radical levels and spermiogram parameters. Samples from each patient were processed using both methods, followed by assessments of sperm concentration, motility, morphology, DNA integrity, acrosomal status, and mitochondrial membrane potential. Participants were selected based on sperm analysis and categorized as normozoospermic (n = 40) or non-normozoospermic (n = 28). An analysis of separation techniques revealed no significant differences, except for a lower percentage of DNA-fragmented sperm in the MFSS group compared with SU within the non-normozoospermic cohort (SU: 10.0% vs. MFSS: 5.69%, p = 0.027). No differences were observed between SU and MFSS in normozoospermic men. The MFSS method is a simple technique, frequently used in laboratories, that yields good results but does not offer a substantial advantage over SU. The primary benefit of MFSS appears to be a significant reduction in the proportion of sperm with DNA fragmentation compared with SU in patients with abnormal sperm analysis results.

• Klíčová slova
• IVF/ICSI, infertility, sperm analysis, sperm separation, spermatozoa,
• MeSH
• analýza spermatu metody   MeSH
• dospělí MeSH
• fragmentace DNA MeSH
• intracytoplazmatické injekce spermie * metody   MeSH
• lidé MeSH
• membránový potenciál mitochondrií MeSH
• mikrofluidika * metody   MeSH
• motilita spermií * MeSH
• mužská infertilita terapie   MeSH
• separace buněk * metody   MeSH
• spermie * cytologie   metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH

BACKGROUND: Sperm metabolic pathways that generate energy for motility are compartmentalized within the flagellum. Dysfunctions in metabolic compartments, namely mitochondrial respiration and glycolysis, can compromise motility and male fertility. Studying these compartments is thus required for fertility treatment. However, it is very challenging to capture images of metabolic compartments in motile spermatozoa because the fast beating of the flagellum introduces motion blur. Therefore, most approaches immobilize spermatozoa prior to imaging. RESULTS: Our findings indicate that immobilizing sperm alters their metabolic profile, highlighting the necessity for measuring metabolism in spermatozoa during movement. We achieved this by encapsulating mouse epididymis in a hydrogel followed by two-photon fluorescence lifetime imaging microscopy for imaging motile sperm in situ. The autofluorescence of endogenous metabolites-FAD, NADH, and NADPH-enabled us to visualize sperm metabolic compartments without staining. We trained machine learning for automated image segmentation and generated metabolic fingerprints using object-based phasor analysis. We show that metabolic fingerprints of spermatozoa and the mitochondrial compartment (1) can distinguish individual males by genetic background, age, or fecundity status, (2) correlate with fertility, and (3) change with age likely due to increased oxidative metabolism. CONCLUSIONS: Our approach eliminates the need for sperm immobilization and labeling and captures the native state of sperm metabolism. This technique could be adapted for metabolism-based sperm selection for assisted reproduction.

• Klíčová slova
• Artificial intelligence, FLIM, Fertility, Metabolism, NADH, NADPH, Sperm,
• MeSH
• metabolom * MeSH
• mitochondrie metabolismus   MeSH
• motilita spermií * fyziologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• spermie * metabolismus   fyziologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Accurate analysis of sperm cell flagellar dynamics plays a crucial role in understanding sperm motility as flagella parameters determine cell behavior in the spatiotemporal domain. In this study, we introduce a novel approach by harnessing Genetic Algorithms (GA) to analyze sperm flagellar motion characteristics and compare the results with the traditional decomposition method based on Fourier analysis. Our analysis focuses on extracting key parameters of the equation approximating flagellar shape, including beating period time, bending amplitude, mean curvature, and wavelength. Additionally, we delve into the extraction of phase constants and initial swimming directions, vital for the comprehensive study of sperm cell pairs and bundling phenomena. One significant advantage of GA over Fourier analysis is its ability to integrate sperm cell motion data, enabling a more comprehensive analysis. In contrast, Fourier analysis neglects sperm cell motion by transitioning to a sperm-centered coordinate system (material system). In our comparative study, GA consistently outperform the Fourier analysis-based method, yielding a remarkable reduction in fitting error of up to 70% and on average by 45%. An in-depth exploration of the sperm cell motion becomes indispensable in a wide range of applications from complexities of reproductive biology and medicine, to developing soft flagellated microrobots.

• Klíčová slova
• Biological motion, Flagellum deformation, Genetic algorithm, Motion analysis, Sperm cell dynamics,
• Publikační typ
• časopisecké články MeSH

More suitable and efficient methods to protect gametes from external harmful effects during in vitro handling can be achieved by adding preovulatory porcine oviductal fluid (pOF) to in vitro culture media. The objective of this study was to assess the swim-up procedure's suitability as a sperm selection method using a medium supplemented with 1mg/mL BSA, 1% preovulatory pOF (v/v), 1% v/v pOF plus 1mg/mL BSA, and 5mg/mL BSA. After selection, various sperm parameters were studied, such as sperm recovery rate, sperm morphology, motility (by CASA), vitality, acrosome status and intracellular calcium (by flow cytometry) and ability to penetrate oocytes in vitro. Around 2% of sperm were recovered after swim-up, and the replacement of BSA by pOF showed a beneficial reduction of motility parameters calcium concentration, resulting in an increased penetration rate. The combination of albumin and oviductal fluid in the medium did not improve the sperm parameters results, whereas a high concentration of BSA increased sperm morphological abnormalities, motility, and acrosome damage, with a reduction of calcium concentration and penetration rate. In conclusion, the replacement of albumin by preovulatory oviductal fluid in the swim-up sperm preparation method modifies boar sperm parameters and improves the in vitro penetration of oocytes.

• Klíčová slova
• albumin, oviductal fluid, porcine, sperm selection, swim-up,
• Publikační typ
• časopisecké články MeSH

Out of millions of ejaculated sperm, a few reach the fertilization site in mammals. Flagellar Ca2+ signaling nanodomains, organized by multi-subunit CatSper calcium channel complexes, are pivotal for sperm migration in the female tract, implicating CatSper-dependent mechanisms in sperm selection. Here using biochemical and pharmacological studies, we demonstrate that CatSper1 is an O-linked glycosylated protein, undergoing capacitation-induced processing dependent on Ca2+ and phosphorylation cascades. CatSper1 processing correlates with protein tyrosine phosphorylation (pY) development in sperm cells capacitated in vitro and in vivo. Using 3D in situ molecular imaging and ANN-based automatic detection of sperm distributed along the cleared female tract, we demonstrate that spermatozoa past the utero-tubal junction possess the intact CatSper1 signals. Together, we reveal that fertilizing mouse spermatozoa in situ are characterized by intact CatSper channel, lack of pY, and reacted acrosomes. These findings provide molecular insight into sperm selection for successful fertilization in the female reproductive tract.

When mammals mate, males ejaculate millions of sperm cells into the females’ reproductive tract. But as the sperm travel up the tract, only a handful of the ‘fittest’ sperm will actually manage to reach the egg. This process of elimination prevents the egg from being fertilized by multiple sperm cells and stops the eggs from being fertilized outside of the womb. A lot of what is known about fertilization in mammals has come from studying how sperm and eggs cells interact in a Petri dish. However, this approach cannot explain how sperm are selected and removed as they journey towards the egg. Previous work suggests that a calcium channel, which sits in the membrane surrounding the sperm tail, may provide some answers. The core of this channel, known as CatSper, is made up of four proteins arranged into a unique pattern similar to racing stripes. Without this specific arrangement, sperm cells cannot move forward and fertilize the egg in time. To investigate the role of this protein in more depth, Ded et al. established a new way to image the minute structures of sperm cells, such as CatSper, in the reproductive tract of female mice. Experiments in a Petri dish revealed that sperm cells that have been primed to fertilize the egg are a diverse population: in some cells one of the proteins that make up the calcium channel, known as CatSper1, is cleaved, while in other cells this protein remains intact. Visualizing this protein in the female reproductive tract showed that sperm cells close to the site of fertilization contain non-cleaved CatSper1. Whereas sperm cells further away from the egg – and thus closer to the uterus – are more likely to contain broken down CatSper1. Taken together, these findings suggest that the state of the CatSper1 protein may be used to select sperm that are most likely to reach and fertilize the egg. Future studies should address what happens to the calcium channel once the CatSper1 protein is cleaved, and how this channel controls the movements and lifespan of sperm. This could help identify new targets for contraception and improve current strategies for assisted reproduction.

• Klíčová slova
• CatSper, capacitation, cell biology, fertilization, in situ imaging, molecular biophysics, molecular heterogeneity, mouse, sperm, structural biology,
• MeSH
• akrozomální reakce MeSH
• glykosylace MeSH
• molekulární zobrazování metody   MeSH
• myši MeSH
• regulace genové exprese MeSH
• spermie fyziologie   MeSH
• vápníková signalizace MeSH
• vápníkové kanály genetika   metabolismus   MeSH
• ženské pohlavní orgány diagnostické zobrazování   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• Catsper1 protein, mouse MeSH Prohlížeč
• vápníkové kanály MeSH

Fertilization is a multistep process during which two terminally differentiated haploid cells, an egg and a sperm, combine to produce a totipotent diploid zygote. In the early 1950s, it became possible to fertilize mammalian eggs in vitro and study the sequence of cellular and molecular events leading to embryo development. Despite all the achievements of assisted reproduction in the last four decades, remarkably little is known about the molecular aspects of human conception. Current fertility research in animal models is casting more light on the complexity of the process all our lives start with. This review article provides an update on the investigation of mammalian fertilization and highlights the practical implications of scientific discoveries in the context of human reproduction and reproductive medicine.

• Klíčová slova
• Fertilization, Gamete biology, Human reproduction, In vitro fertilization,
• MeSH
• asistovaná reprodukce trendy   MeSH
• diploidie MeSH
• embryonální vývoj genetika   MeSH
• fertilizace in vitro trendy   MeSH
• lidé MeSH
• modely u zvířat MeSH
• ovarium růst a vývoj   MeSH
• spermie růst a vývoj   MeSH
• zvířata MeSH
• zygota růst a vývoj   MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH