Fluorescence Microscopy Research Articles

Carbon dot enhanced electrodeposited coatings for advanced early-stage corrosion sensing

This work explores a novel approach for metal corrosion by integrating carbon dots (CDs) into electrodeposited (ED) coatings. We detail the synthesis and properties of CDs, making them suitable for corrosion detection. Characterization techniques like Fourier Transform Infrared Spectroscopy (FTIR), Scanning electron microscope (SEM), Energy Dispersive X-ray Analysis (EDX), Fluorescent Microscopy and Photoluminescence Spectroscopy (PL).A Design of Experiment (DOE) optimized parameters like CD concentration, curing temperature, and voltage for improved coating performance. The optimized conditions (0.4 wt% M-CDots, 165 °C curing, and 36.2 V) resulted in enhanced adhesion and corrosion resistance.CD-infused coatings demonstrated superior adhesion and mechanical stability compared to controls. Electrochemical tests confirmed a 10 % improvement in corrosion resistance and PL study demonstrated an approximately 60 % enhancement in emission intensity. The adhesion was not affected by introducing carbon dots to the coating. Monitoring the onset of metal corrosion on samples in salt spray cabinets was achieved through the use of a UV source. The photoluminescence of M-CDs offered real-time, non-destructive monitoring of early-stage corrosion through quenching in the presence of exposed metal surfaces.This research presents a significant advancement by integrating CD-based sensing into corrosion-resistant coatings, holding promise for various industries and leading to improved safety, reduced maintenance costs, and extended equipment lifespans. Notably, the biocompatible and disposable nature of CD-based sensors further enhances their practical application.

Quantum dots as a fluorescent labeling tool for live-cell imaging of Leptospira.

Leptospirosis is a global public health problem caused by Gram-negative pathogenic bacteria belonging to the genus Leptospira. The disease is transmitted through the urine of infected animals, which contaminates water and soil, leading to the infection of other animals and humans. Currently, several approaches exist to detect these bacteria; however, a new sensitive method for the live-cell imaging of Leptospira is required. In this study, we report the green synthesis of cadmium telluride quantum dots (CdTe QDs) which are unique fluorescent nanocrystals with a high fluorescence quantum yield capable of modifying cell surfaces and are biocompatible with cells. The fabrication of QDs with concanavalin A (ConA), a carbohydrate-binding lectin and known biological probe for Gram-negative bacteria, produced ConA-QDs which can effectively bind on Leptospira and exhibit strong fluorescence under simple fluorescence microscopy, allowing the live-cell imaging of the bacteria. Overall, we performed the simple synthesis of ConA-QDs and demonstrated their potential use as versatile fluorescent probes for the live-cell imaging of Leptospira. This technique could be further applied to track leptospiral cells and study the infection mechanism, contributing to a more thorough understanding of leptospirosis and how to control it in the future.

Effect of oxidative stress on antigen productivity in B. Pertussis cultures

Whooping cough, also known as pertussis, is an infectious respiratory disease caused by Bordetella pertussis, a type of gram-negative, aerobic, pathogenic bacteria with a coccobacillus shape and an outer capsule. The acellular vaccine for this disease contains a combination of pertussis toxin, fimbriae, filamentous hemagglutinin, and pertactin. The main constraint in the manufacturing of the vaccine relates to pertactin productivity due to its low abundance in the fermentation broth. Being the most abundant nutrient in the media, variability in initial glutamate concentration is hypothesized to be a major cause of process variability. This study proposes the combined use of cytometry and chromatography based separation to study the impact of glutamate-induced oxidative stress on the growth and productivity of pertactin antigen. Surface expression of pertactin was observed using fluorescence microscopy and flow cytometry while extracellular concentration was quantified using affinity chromatography. Oxidative stress levels and secretion of NADPH, a crucial reactant in antioxidant reactions, were monitored in flask and bioreactor experiments. The results established clear correlations between oxidative stress with growth and productivity and differentiate between the reduction in productivity to growth and to synthesis rate of pertactin. These findings are industrially relevant for improving productivity and reducing process variability.

Clotrimazole inhibits growth of multiple myeloma cells in vitro via G0/G1 arrest and mitochondrial apoptosis

Patients with multiple myeloma (MM) experience relapse and drug resistance; therefore, novel treatments are essential. Clotrimazole (CTZ) is a wide-spectrum antifungal drug with antitumor activity. However, CTZ’s effects on MM are unclear. We investigated CTZ’s effect on MM cell proliferation and apoptosis induction mechanisms. CTZ’s effects on MM.1S, NCI- H929, KMS-11, and U266 cell growth were investigated using Cell Counting Kit-8 (CCK-8) assay. The apoptotic cell percentage was quantified with annexin V-fluorescein isothiocyanate/7-amino actinomycin D staining. Mitochondrial membrane potential (MMP) and cell cycle progression were evaluated. Reactive oxygen species (ROS) levels were measured via fluorescence microscopy. Expression of apoptosis-related and nuclear factor (NF)-κB signaling proteins was analyzed using western blotting. The CCK-8 assay indicated that CTZ inhibited cell proliferation based on both dose and exposure time. Flow cytometry revealed that CTZ decreased apoptosis and MMP and induced G0/G1 arrest. Immunofluorescence demonstrated that CTZ dose-dependently elevated in both total and mitochondrial ROS production. Western blotting showed that CTZ enhanced Bax and cleaved poly ADP-ribose polymerase and caspase-3 while decreasing Bcl-2, p-p65, and p-IκBα. Therefore, CTZ inhibits MM cell proliferation by promoting ROS-mediated mitochondrial apoptosis, inducing G0/G1 arrest, inhibiting the NF-κB pathway, and has the potential for treating MM.

AI-based digital pathology provides newer insights into lifestyle intervention-induced fibrosis regression in MASLD: An exploratory study.

Lifestyle intervention is the mainstay of therapy for metabolic dysfunction-associated steatohepatitis (MASH), and liver fibrosis is a key consequence of MASH that predicts adverse clinical outcomes. The placebo response plays a pivotal role in the outcome of MASH clinical trials. Second harmonic generation/two-photon excitation fluorescence (SHG/TPEF) microscopy with artificial intelligence analyses can provide an automated quantitative assessment of fibrosis features on a continuous scale called qFibrosis. In this exploratory study, we used this approach to gain insight into the effect of lifestyle intervention-induced fibrosis changes in MASH. We examined unstained sections from paired liver biopsies (baseline and end-of-intervention) from MASH individuals who had received either routine lifestyle intervention (RLI) (n = 35) or strengthened lifestyle intervention (SLI) (n = 17). We quantified liver fibrosis with qFibrosis in the portal tract, periportal, transitional, pericentral, and central vein regions. About 20% (7/35) and 65% (11/17) of patients had fibrosis regression in the RLI and SLI groups, respectively. Liver fibrosis tended towards no change or regression after each lifestyle intervention, and this phenomenon was more prominent in the SLI group. SLI-induced liver fibrosis regression was concentrated in the periportal region. Using digital pathology, we could detect a more pronounced fibrosis regression with SLI, mainly in the periportal region. With changes in fibrosis area in the periportal region, we could differentiate RLI and SLI patients in the placebo group in the MASH clinical trial. Digital pathology provides new insight into lifestyle-induced fibrosis regression and placebo responses, which is not captured by conventional histological staging.

The cytoplasmic tail of myelin protein zero induces morphological changes in lipid membranes

The major myelin protein expressed by the peripheral nervous system Schwann cells is protein zero (P0), which represents 50 % of the total protein content in myelin. This 30-kDa integral membrane protein consists of an immunoglobulin (Ig)-like domain, a transmembrane helix, and a 69-residue C-terminal cytoplasmic tail (P0ct). The basic residues in P0ct contribute to the tight packing of myelin lipid bilayers, and alterations in the tail affect how P0 functions as an adhesion molecule necessary for the stability of compact myelin. Several neurodegenerative neuropathies are related to P0, including the more common Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS) as well as rare cases of motor and sensory polyneuropathy. We found that high P0ct concentrations affected the membrane properties of bicelles and induced a lamellar-to-inverted hexagonal phase transition, which caused bicelles to fuse into long, protein-containing filament-like structures. These structures likely reflect the formation of semicrystalline lipid domains with potential relevance for myelination. Not only is P0ct important for stacking lipid membranes, but time-lapse fluorescence microscopy also shows that it might affect membrane properties during myelination. We further describe recombinant production and low-resolution structural characterisation of full-length human P0. Our findings shed light on P0ct effects on membrane properties, and with the successful purification of full-length P0, we have new tools to study the role of P0 in myelin formation and maintenance in vitro.

Quantifying superimposed protein flow dynamics in live cells using spatial filtering and spatiotemporal image correlation spectroscopy.

Flow or collective movement is a frequently observed phenomenon for many cellular components including the cytoskeletal proteins actin and myosin. To study protein flow in living cells, we and others have previously used spatiotemporal image correlation spectroscopy (STICS) analysis on fluorescence microscopy image time series. Yet, in cells, multiple protein flows often occur simultaneously on different scales resulting in superimposed fluorescence intensity fluctuations that are challenging to separate using STICS. Here, we exploited the characteristic that distinct protein flows often occur at different spatial scales present in the image series to disentangle superimposed protein flow dynamics. We employed a newly developed and an established spatial filtering algorithm to alternatively accentuate or attenuate local image intensity heterogeneity across different spatial scales. Subsequently, we analysed the spatially filtered time series with STICS, allowing the quantification of two distinct superimposed flows within the image time series. As a proof of principle of our analysis approach, we used simulated fluorescence intensity fluctuations as well as time series of nonmuscle myosin II in endothelial cells and actin-based podosomes in dendritic cells and revealed simultaneously occurring contiguous and noncontiguous flow dynamics in each of these systems. Altogether, this work extends the application of STICS for the quantification of multiple protein flow dynamics in complex biological systems including the actomyosin cytoskeleton.

Myosin-1C differentially displaces tropomyosin isoforms altering their inhibition of motility

Force generation and motility by actomyosin in non-muscle cells are spatially regulated by ∼40 tropomyosin (Tpm) isoforms. The means by which Tpms are targeted to specific cellular regions and the mechanisms that result in differential activity of myosin paralogs are unknown. We show that Tpm3.1 and Tpm1.7 inhibit Myosin-IC (Myo1C), with Tpm1.7 more effectively reducing the number of gliding filaments compared to Tpm3.1. Strikingly, cosedimentation and fluorescence microscopy assays revealed that Tpm3.1 is displaced from actin by Myo1C and not by myosin-II. In contrast, Tpm1.7 is only weakly displaced by Myo1C. Unlike other characterized myosins, Myo1C motility is inhibited by Tpm when the Tpm-actin filament is activated by myosin-II. These results point to a mechanism for exclusion of myosin-I paralogs from cellular Tpm-decorated actin filaments that are activated by other myosins. Additionally, our results suggest a potential mechanism for myosin-induced Tpm sorting in cells.

Controlling Drug Partitioning in Individual Protein Condensates through Laser-Induced Microscale Phase Transitions.

Gelation of protein condensates formed by liquid-liquid phase separation occurs in a wide range of biological contexts, from the assembly of biomaterials to the formation of fibrillar aggregates, and is therefore of interest for biomedical applications. Soluble-to-gel (sol-gel) transitions are controlled through macroscopic processes such as changes in temperature or buffer composition, resulting in bulk conversion of liquid droplets into microgels within minutes to hours. Using microscopy and mass spectrometry, we show that condensates of an engineered mini-spidroin (NT2repCTYF) undergo a spontaneous sol-gel transition resulting in the loss of exchange of proteins between the soluble and the condensed phase. This feature enables us to specifically trap a silk-domain-tagged target protein in the spidroin microgels. Surprisingly, laser pulses trigger near-instant gelation. By loading the condensates with fluorescent dyes or drugs, we can control the wavelength at which gelation is triggered. Fluorescence microscopy reveals that laser-induced gelation significantly further increases the partitioning of the fluorescent molecules into the condensates. In summary, our findings demonstrate direct control of phase transitions in individual condensates, opening new avenues for functional and structural characterization.

O-307 Decrease of human sperm hypercondensed chromatin volume after slow freezing revealed by three-dimensional (3D) nuclear morphometry

Abstract Study question Are the 3D morphometry parameters of the human sperm nucleus affected by slow freezing? Summary answer The slow freezing-thawing cycle induces significant alterations on hypercondensed nucleus zone by reducing its volume and splits it into several small zones. What is known already Sperm cryopreservation is an essential technique for infertility care, but it seems to highly affect the genetic and epigenetic integrity of sperm nuclei with poorly identified mechanisms. Moreover, it is known that sperm nuclear alterations may be involved in pregnancy failures. Therefore, sperm nuclear biomarkers are an essential issue to assess the outcome of ART procedures when frozen sperm is used. Thereby, 3D visualization of chromatin is a good way to understand modifications in the organization and composition of sperm chromatin after a slow freezing procedure. Study design, size, duration Ten semen of patients were analyzed (Germetheque biobank) after informed consent. Standard semen parameters, DNA fragmentation, chromatin decondensation, sperm telomere length (STL) and 3D nuclear morphometry were performed before and after slow freezing. Morphometric measurements were performed from a digital representation of the sperm nucleus in 3 dimensions that allow us to measure the volume, flatness and elongation of each sperm nucleus and characterize hypercondensed zones, with live/dead spermatozoa distinction. Participants/materials, setting, methods After addition of cryoprotective medium v/v (Cryosperm®), sperm was frozen with a programmable freezer (NanoDigitcool®). Standards sperm parameters were measured in accordance with WHO guidelines 2021, DNA fragmentation by flow cytometry TUNEL assay, chromatin decondensation by epifluorescent microscopy with Chromomycin A3 and STL using qPCR. 3D nuclear representation was performed by a z-axis image acquisition series of DAPI/PI stained slides with optigrid epifluorescent microscopy. Analyses were carried out using the NucleusJ2.0 software with NODeJ plugin. Main results and the role of chance Freezing induces an alteration of sperm vitality (p < 0.0001) and progressive and total motility (p < 0.001 and p < 0.0001 respectively) and an increase in the percentage of sperm with DNA fragmentation (18.30% fresh vs 31.6% frozen, p < 0.05). We did not observe a modification of the median volume of the sperm nucleus before and after freezing (37.15 [IC = 36.5;38.14] and 37.10 [33.27;37.78] µm3 respectively), as the same for flatness and elongation (2.48 vs. 2.50 a.u. and 2.04 vs. 2.04 a.u. respectively). Nevertheless, five samples showed significant alterations of nucleus volume (p < 0.0001), due to diminution of flatness and more or less elongated. A significant decrease of the total volume of the hypercondensed chromatin zone was observed after freezing (0.94 [0.84;0.99] before vs. 0.61 [0.53;0.69] µm3 after freezing-thawing, p < 0.0001), with an effect size of -1.48 [-2.10; -0.86]. Decrease of total volume of hypercondensed region was measured homogeny in all the samples tested, particularly on live (1.00 vs. 0.68 µm3 respectively, p < 0.0001) compared with dead spermatozoa (0.72 vs. 0.57 µm3, p < 0.05). We showed also a significant increase of the number of hypercondensed zones in each nucleus (p < 0.001), reflecting a split into several parts of the hypercondensed chromatin. Limitations, reasons for caution A 3D-FISH analysis would allow to characterize the hypercondensed chromatin portion, probably as heterochromatin and which we suspect to be the nucleus ring. Moreover, a chromosomes specific marking would allow for the identification of whether whole chromosomes or specific parts of chromosomes (centromeres or telomeres) are affected by this decompaction. Wider implications of the findings This study is the first to make exacts measurements of human sperm nucleus because of a non-decondensing method. We demonstrate that slow freezing affects hypercondensed chromatin probably through a decondensation and modification of the nucleus-specific organization. This new assessment will allow a better proficiency of men fertility preservation techniques. Trial registration number NCT04715828

P-792 Human endometrial organoids embedded in hybrid endometrial-derived hydrogels as a biocompatible treatment for endometrial pathologies in preclinical models

Abstract Study question Are human endometrial organoids (hEOs) labelled with Molday-ION Rhodamine B (MIRB) within hybrid endometrial-derived hydrogels a biocompatible approach to treat endometrial pathologies in immunocompetent mice? Summary answer The direct intrauterine injection of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels demonstrated xenogeneic biocompatibility in preclinical models over a 2-week period. What is known already The lack of effective treatments for endometrial pathologies emphasizes the urgent need for novel bioengineering approaches. In this context, hEOs are presented as promising candidates for tissue regeneration, since they offer a native and partially undifferentiated cellular source with high proliferative capacity, which is fostered after its culture within hybrid endometrial-derived hydrogels (Gómez-Álvarez et al. 2023). Their concurrent injection aims to enhance proliferation and differentiation through its rich biochemical composition. No prior study has successfully labelled and injected hEOs in immunocompetent models, which is crucial to discern whether the therapeutic effect operates at paracrine level or involves direct tissue regeneration. Study design, size, duration This study was approved by an ethics committee. Decellularized porcine endometrium was used to create endometrial-derived hydrogels as previously described and then mixed with PuraMatrix to obtain the hybrid endometrial-derived hydrogel. After 7 days of culture, hEOs (passages 6-7) were labelled with MIRB for final tracking. Finally, hEOs were mixed with the hybrid hydrogel and injected into the uterine horns in immunocompetent mice (n = 3). After two weeks, biocompatibility was evaluated through blood and macroscopical analyses. Participants/materials, setting, methods After 7 days, hEOs were collected and labelled with MIRB (50 µg/ml) for their final tracking in the murine endometrium. Endometrial-derived hydrogel (8 mg/ml) was mixed with PuraMatrix 1% (50:50 ratio) to generate the hybrid hydrogel. After its characterization and in vitro validation (Gómez-Álvarez et al. 2023), it was used as the bioactive carrier. Each animal received a single intrauterine injection (50 µl, ∼100.000 cells). Blood, spleen, and uterus were collected after 2-week period. Main results and the role of chance Hybrid endometrial-derived hydrogels demonstrated biocompatibility and in vivo stability in mice over a 2-week period (Gómez-Álvarez et al. 2023). In this study, we aim to use this hybrid hydrogel with hEOs as a biocompatible treatment in preclinical models. After 7 days of culture, hEOs were successfully labelled with MIRB (∼85% efficacy), which was confirmed under fluorescence microscopy. The single intrauterine injection (50 µl, ∼100.000 cells) did not cause apparent adverse immune reactions in mice (n = 3) during the 2-week period. All mice exhibited no signs of distress and their behavior remained within normal parameters. Furthermore, the macroscopic examination of uterine horns revealed normal appearance, characterized by the absence of anomalies and tumors. At day 14 post-injection, haematocrit analyses showed normal white blood cell count: 0.89 ± 0.53 · 109/L neutrophils; 7.82 ± 2.23 · 109/L lymphocytes; 0.21 ± 0.13 · 109/L monocytes; 0.04 ± 0.04 · 109/L eosinophils; 0.00 ± 0.00 · 109/L basophils. In addition, no significant changes in red blood cells (9.31 ± 0.54 · 1012/L) and platelets (1063.00 ± 318.58 · 109/L) were observed. Histological analyses will be perform to assess the presence of hEOs labelled with MIRB in the murine endometrium and dilucidate their mechanism of action. Limitations, reasons for caution This study has been carried out in healthy C57BL/6 mice. To confirm the scope of this technique, the next step is to explore the regenerative potential of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels in murine models with endometrial damage. Wider implications of the findings These findings demonstrate the biocompatibility of hEOs labelled with MIRB within hybrid endometrial-derived hydrogels in immunocompetent C57BL/6 mice. This bioengineering approach holds the potential to improve the treatment landscape for specific endometrial pathologies. Trial registration number Not applicable

P-054 Paternal age and cryopreservation impact sperm chromatin status as indicated by changes in protamine and histone levels

Abstract Study question Do paternal age and cryopreservation alter sperm chromatin status in normospermic men? Summary answer Paternal age and cryopreservation appear to impact sperm chromatin condensation by altering protamine and histone DNA levels. What is known already Semen parameters do not accurately reflect male fertility and thus are not adequate to assess the impact of patient profile and semen manipulation/conservation on sperm functional quality. DNA protamination leads to sperm chromatin condensation, which is associated with sperm maturity but also appears to function as a defence mechanism against oxidative attack. In the face of contrasting/insufficient previous data, the impact of advanced paternal age (APA) and cryopreservation on sperm chromatin condensation and histone/protamine balance remains unclear. Study design, size, duration Twenty-two normospermic men, 11 of which with advanced paternal age (APA; ≥42) and 11 pre-APA (≤35), provided semen samples by masturbation (1-5 days abstinence). Fresh and cryopreserved/thawed samples from all patients were assessed for chromatin histone staining, while samples from a subgroup of 10 patients (5 pre-APA and 5 APA) were assessed for protamine presence. The effects of age and cryopreservation on the percentage of stained cells were assessed with the Fisher’s exact test. Participants/materials, setting, methods Semen samples were provided by male partners of couples treated at our fertility centre. Sample aliquots were cryopreserved and analyzed after thawing (CryoSperm & Sperm Preparation Medium, Origio). Histone presence was assessed through aniline blue staining and protamine presence was indirectly assessed through staining with the protamine competitor Chromomycin A3 (CMA3); stained cells were visualized by bright field and fluorescence microscopy, respectively. Two hundred sperm cells were evaluated in each sample by the same embryologist. Main results and the role of chance Mean paternal age was 31.5±3.9 and 44.4±2.1 years for pre-APA and APA patient-groups, respectively. Major parameters observed in fresh semen samples were: 2.3±0.7 vs. 3.6±1.6mL (volume); 39±28 vs. 38±12 x10ˆ6/mL (concentration); 7±12 vs. 6±5% (rapid progressive motility); 41±12 vs. 38±13% (slow progressive motility); 12±5 vs. 11±4% (non-progressive motility); 40±17 vs. 45±16% (immotile) and 5±1 vs. 6±2% (normal morphology), for pre-APA and APA, respectively. The percentage of aniline blue (histone marker) positive cells was less than half in fresh samples from APA as compared to pre-APA patients (10.2% and 23.4%, respectively, p < 0.0001). Consistently, the percentage of CMA3 (protamine absence marker) positive cells was less than half in APA as compared to pre-APA patients (6.4% and 16.4%, respectively, p < 0.0001). In both age groups, the percentages of aniline blue and CMA3 positive cells were higher in cryopreserved/thawed samples as compared to their fresh counterparts (aniline blue positive: 35.2% vs. 23.4% and 22.0% vs. 10.2%; CMA3 positive: 27.2% vs. 16.4% and 18.8% vs. 6.4%, in cryopreserved vs. fresh in pre-APA and APA, respectively, p < 0.0001). Limitations, reasons for caution Our study is limited by the potential interference of confounding factors not equally distributed in age groups. The conclusions from this study must be confirmed in other patient populations with different race/genetics and habits. Wider implications of the findings Our findings suggest that APA increases, while cryopreservation decreases, sperm chromatin condensation, as indicated by changes in DNA histone and protamine abundance. Our study thus sheds light on the impact of paternal age and cryopreservation on sperm quality, providing new valuable parameters for reproductive medicine practice and research. Trial registration number na

P-544 Viability assessment using fluorescent markers, ultrastructure and implantation of human embryos following blastocyst biopsy using three different laser systems

Abstract Study question Are there any differences in viability, ultrastructure and implantation of human blastocysts biopsied using three different laser systems? Summary answer No statistically significant differences are observed in viability, ultrastructure and implantation of human blastocysts biopsied using three different laser systems What is known already Most studies in human embryos concentrate on assessing the success of biopsy/vitrification on survival rates and clinical outcomes following transfer. Our previous published studies examined in addition to the survival rates, the cytoskeleton, ultrastructure and viability of cleavage and blastocyst stage biopsied embryos, using a specific laser system, emphasizing differences between the two methods and alerting on effective strategies to minimise potential laser induced damage. This is the first study to investigate the safety of 3 different laser systems for blastocyst biopsy by comparing implantation rates and by analysing cell viability using Fluorescence Microscopy and ultrastructure by Transmission Electron Microscopy(TEM). Study design, size, duration 405 human blastocysts were biopsied on day 5 for PGT-A or PGT-SR or PGT-M using the Saturn laser (n = 145-58 cycles) or the Octax laser (n = 132-55 cycles) or the Lykos laser (n = 128-46 cycles). Following transfer of normal embryos, spare embryos, rejected for transfer due to being diagnosed as abnormal were processed following vitrification/warming for viability analysis (n = 30 Saturn laser, n = 30 Octax laser, n = 30 Lykos laser) or TEM (n = 20 Saturn laser, n = 20 Octax laser, n = 20 Lykos laser). Participants/materials, setting, methods The study was conducted in an academic hospital with an IVF/PGT laboratory, 2 private IVF Units and an IVF Unit of a Naval hospital. Blastocyst viability after biopsy/vitrification/warming was assessed using two fluorescent markers: carboxyfluorescein-diacetate succinimidylester (CFSE-viable cells) and propidium iodide (PI–damaged cells) in combination with DAPI to visualise DNA. TEM analysis was carried out following embryo fixation in gluteraldehyde, incubation in osmium, aqueous uranyl acetate, dehydration through ethanol series, and immersion in Epon. Main results and the role of chance The implantation rates (+ve hCG / ET) were not statistically different among the 3 laser systems (64.4% -Saturn, 63.4%- Octax, 64.7%- Lykos p > 0.05). Viability staining with CFSE/PI following vitrification/warming revealed that all biopsied blastocysts had damaged cells at the position of cutting (range 4-8cells) and additional PI stained cells in other parts (range 2-15), possibly due to damage sustained during vitrification/warming. The majority of embryos across the 3 groups had ≤7% PI stained damaged cells (73.3% Saturn, 73.3% Octax, 76.7% Lykos), indicating no statistically significant difference in blastocyst viability among the 3 laser systems (p > 0.05). No difference was also observed in the incidence of lysed biopsied TE cells or the PGT results. TEM analysis revealed similar ultrastracture among the 3 groups, as documented by the presence of normal organelles’ structures including smooth/rough endoplasmic reticulum, Golgi apparatus, mature mitochondria with cisternae, zona and microvilli. Multivesicular bodies, residual bodies, lipofuscins and autophagic vacuoles were also evident and indicative of elimination and rescue process. Excessive accumulation of glycogen granules and distension of mitochondria were more prominent in Grade B blastocysts compared to Grade A blastocysts, which may indicate a ‘block’ of glycogenolysis secondary to the mitochondrial alterations, suggesting changes in normal embryo metabolism. Limitations, reasons for caution The biopsied blastocysts used for viability and ultrastructural analysis in this study were all diagnosed with either chromosomal abnormalities or single gene defects following PGT-A or PGT-SR or PGT-M. Wider implications of the findings The similarities observed in implantation rates, the minimal levels of cell damage and the embryo quality dependent limited ultrastuctural anomalies identified confirm the safety of all three laser systems and emphasize the blastocysts’ ability to initiate elimination/rescue processes in their attempt to fully recover from ‘traumas’ sustained during biopsy/vitrification. Trial registration number not applicable

A project‐based learning mode for science students: Fluorescent protein expression in trypanosomes

AbstractAn effective, practical approach for incorporating molecular biology tools to biology and biochemistry students is described. For 6 weeks, students are exposed to bacterial transformation, plasmid DNA purification and analysis, parasite transfection, ectopic expression of fluorescent proteins, fluorescence microscopy, and flow cytometry analysis. This research‐oriented laboratory course delves into theoretical and practical concepts of basic techniques in molecular biology to emphasize the logical connections between the obtained results and the resolution of problems that arise daily in a research laboratory, training the students in planning, conducting, discussing, and presenting their own experiments and results.

P-453 Non-autologous human platelet lysate as a therapy for recurrent implantation failure: lessons from in vitro models

Abstract Study question Which biological pathways are modulated by primary human endometrial cells in response to in vitro treatment with non-autologous human platelet lysate (HPL)? Summary answer HPL treatment stimulates endometrial growth and trophoblast attachment by activating cell proliferation, and modulating cell-cell signaling and extracellular matrix organization. What is known already Inadequate endometrial receptivity and a thin endometrium (TE) are contributing factors to recurrent implantation failure (RIF), which occurs in 10% of in vitro fertilization cycles. Studies have reported that autologous platelet-rich plasma stimulates endometrial growth and enhances receptivity, thereby improving implantation rate in RIF patients. We previously showed that non-autologous human platelet lysate (HPL) promotes in vitro proliferation of primary endometrial epithelial (EECs) and stromal cells (ESCs). Our data suggest that HPL may standardize and improve future clinical treatments for a TE and endometrial origins of RIF. Study design, size, duration Endometrial tissue was collected from five proliferative phase control and eighteen RIF patients at the CReATe Fertility Centre, Toronto, Canada. The RIF patients were separated into three groups (N = 6 each): 1) proliferative phase RIF, 2) secretory phase RIF, and 3) secretory phase RIF+TE. Primary EECs and ESCs were enzymatically isolated, cultured separately, and treated with either serum-free media (SFM), or SFM + 1% HPL (EECs), or 10% HPL (ESCs). Participants/materials, setting, methods Cell proliferation was assessed using PrestoBlueTM reagent and immunocytochemistry for Ki67. Pooled RNA libraries were prepared and sequenced for 150 cycles paired-end. Differential expression was performed using DESeq2 and pathway analysis with Enrichr. Using an in vitro model of embryo attachment, attachment between HTR-8/SVneo trophoblast spheroids and EECs was assessed by fluorescent microscopy and ImageJ™ software. Main results and the role of chance For all groups, 48-hour treatment with non-autologous HPL stimulates a significant increase (1.24-1.49-fold, P<0.05) in EEC proliferation. Whereas ESCs experienced a significant 1.29-fold and 1.92-fold increase (P<0.05) in proliferation for proliferative phase RIF and secretory phase RIF+TE respectively. HPL-treated EECs also exhibited significantly increased attachment to HTR-8 spheroids from 57.52% to 86.5% (P<0.01) in RIF+TE cultures and 42.58% to 68.90% (P<0.01) in RIF only cultures. Comparing SFM vs. HPL treatment identified 45 significantly upregulated (log2FoldChange >2 and padj <0.05) genes, including MMP1 and MMP9, and 30 significantly downregulated genes including KL and ADRA2A. Whereas ESCs had 429 significantly upregulated genes, including BUB1 and PLK1, and 378 significantly downregulated genes, including PTGIS and ADAMTS16. Upregulated pathways included extracellular matrix organization and mitotic cell cycle. Downregulated pathways included NOTCH1 signaling and synthesis of prostaglandins. Limitations, reasons for caution Our data suggests in vitro HPL treatment significantly stimulates endometrial cell proliferation, trophoblast attachment, and modulates receptivity. However, the small sample size per group (N = 6) underscores the need for larger samples and randomized controlled trials to establish the clinical efficacy of HPL as a treatment for endometrial origins of RIF. Wider implications of the findings HPL treatment for RIF and TE is gaining global acceptance by fertility clinics. Our study establishes the molecular groundwork to understand how HPL impacts endometrial growth and receptivity, offering mechanistic insights into this therapeutic approach and informing future treatments aimed at enhancing the success of embryo transfers. Trial registration number not applicable

P-091 The Dopamine Transporter (DAT) and it’s role in male fertility- an in vitro investigation

Abstract Study question Does Ritalin (DAT antagonist) and dopamine (DAT agonist), influence human sperm parameters, and do these act via the DAT protein? Summary answer DAT is positively expressed on human sperm membranes with dopamine regulating sperm functions through mechanisms involving DAT whereas Ritalin does not affect sperm parameters. What is known already Spermatozoa exhibit a biphasic dose-response to dopamine: low concentrations enhance key sperm functions such as motility and viability, while high concentrations impair them. This biphasic response suggests the presence of a dopamine transporter (DAT), however its role in human sperm function remains under-investigated. Concerns regarding the unprescribed use of Ritalin, a dopamine reuptake inhibitor, are growing among men of reproductive age, given its potential to mimic dopamine’s negative effects on sperm function. Further research is crucial to elucidate the mechanisms and potential consequences of Ritalin exposure on male fertility. Study design, size, duration Twenty samples were collected and utilized to identify and localize DAT via indirect Immunofluorescence (IF) assays. Thirty samples were collected and sperm were treated at 37 °C to 100 nM, 10 μM, and 1 mM dopamine concentrations for 1, 3 and 6 hours; similarly sperm were exposed to 4 ng/mL, 30 ng/mL, and 100 ng/mL Ritalin concentrations for 1, 2 and 3 hours, whereafter the effects on basic and advanced sperm parameters were investigated. Participants/materials, setting, methods Participants were recruited from the Faculty of Medicine and Health Sciences at Stellenbosch University, Cape Town, South Africa. Male students aged 18-29 years donated a semen sample via masturbation. An indirect IF assay, adapted from Ramírez et al. (2009), was utilized to identify DAT. Computer-Aided Sperm Analysis (CASA) was employed to determine motility parameters. Viability was assessed via a dye-exclusion staining technique and the acrosome reaction was assessed via fluorescent microscopy. Main results and the role of chance Immunofluorescence confirmed the presence of the Dopamine Transporter (DAT) on human sperm, primarily localized in the mid-piece and tail regions. Interestingly, dopamine exhibited a biphasic effect on sperm function. Low doses offered protection, while high doses (1 mM) negatively impacted motility (total and progressive motility) and kinematic parameters (Curvilinear Velocity (VCL) and Average Path Velocity (VAP)) as early as 60 minutes, and significantly decreased viability after 3 hours. Additionally, the high dose prematurely triggered the acrosome reaction. Conversely, Ritalin, even at supraphysiological concentrations (100 ng/mL), showed no significant impact on basic or advanced sperm parameters. This suggests that, unlike high-dose dopamine, Ritalin exposure might not negatively affect sperm function within the tested parameters. This study highlights the complex role of dopamine in sperm function and emphasizes the need for further research to clarify potential consequences of high dopamine exposure or dopaminergic drugs such as Ritalin. Limitations, reasons for caution Ejaculated spermatozoa are transcriptionally and translationally silent, therefore the in vitro nature of this study is a limitation. The physiological relevance needs to be assessed in an in vivo system, additionally the treatments (dopamine plus Ritalin) should be investigated in combination to demonstrate their concurrent existence within a system. Wider implications of the findings These findings suggest a possible regulatory role for DAT inhibitory drugs on sperm parameters and cautions users thereof. Dopamine may be utilized as a physiological regulator of in vivo fertilization and should be further investigated with regards to Assisted Reproductive Techniques (ART’s). Trial registration number not applicable

O-069 The first mitotic spindle instability impacts nucleation, fragmentation, and development in human embryos

Abstract Study question How multinucleation and fragmentation occur in the first mitosis in human embryos? Summary answer The first mitotic spindles had heterogeneity, and the shapes and movements of the spindles were strongly associated with multinucleation and fragmentation. What is known already Studies have shown that many problems occur during the first mitosis, such as fragmentation and multinucleation. However, the cell biologic mechanisms whereby these errors occur during the first mitosis in human embryos remain unknown. To clarify this aspect, it is necessary to analysis the dynamics of DNA and cytoskeleton using live human embryos. Although microinjections of DNA or mRNA into mammalian embryos were widely used for live imaging during early mitosis, the method into human poses the risk of ethical issues. Therefore, we established a method used for real-time imaging in human embryos using chemical fluorescent labeling. Study design, size, duration Frozen human two-pronuclear stage embryos were donated for research by couples who had completed fertility treatment. We obtained written informed consent from patients who donated human embryos. The mean age of patients at embryo freezing was 32.00 ± 3.03 (SD) years. We imaged thirty-one living human embryos using chemical fluorescent labeling and confocal microscopy to visualize DNA and tubulin. Participants/materials, setting, methods Thirty-one human embryos were cultured in medium containing the live-cell stain probes SPY555-DNA and SPY650-Tubulin. The Embryos were imaged using an LSM980 laser scanning confocal microscope every 10–20 min for a period of eight hours. Three-dimensional (3D) visualizations of human embryos were performed using the Imaris software. The first spindle shapes were quantified by image analysis and calculated the aspect ratio of the spindles using ImageJ. Main results and the role of chance We discovered that over 81% of two-pronuclear zygotes became multinucleated 2-cell stage embryos. We demonstrated that there was variation in the first mitotic spindles. There was a significant difference between the aspect ratio (AR) of the spindles leading to the mononuclear and multinuclear types at the 2-cell stage (p < 0.005). In addition, the low-AR spindles were unstable and often had more defocused poles than the high-AR spindles, which lead to multinucleation at the 2-cell stage (55 % vs. 0%, p < 0.05). More embryo having high-aspect ratio spindles tend to develop blastocysts than those having low aspect-ratio spindles (p < 0.05). Moreover, the movement of the central spindles varied after chromosome segregation. The embryos with more fragments at two cell stage had a significantly longer period for the central spindles to transform into midbodies compared to those with fewer fragments. The central spindles of embryos with more fragments moved a longer distance from the beginning of furrow ingression to the completion of the first division (p < 0.05). These results suggest that the first mitotic spindle instability and heterogeneity cause many errors such as multinucleation, fragmentation, and embryonic development. Limitations, reasons for caution Imaged human embryos were donated from a fertility clinic, and detailed patient information, such as underlying disease and method of ovarian stimulation, was not available. It is possible that the human zygotes used in this study had a bias in patient characteristics. Wider implications of the findings Our study showed that there were instability and heterogeneity in the first mitotic spindle, which was strongly associated with multinucleation, fragmentation, and embryonic development. Revealing the mechanism underlying spindle formation in cleavage-stage embryos will contribute to improving the success rate of assisted reproduction technologies in humans. Trial registration number not applicable

FLUORESCEIN ISOTHIOCYANATE LABELLED PCL‐PEG‐PCL COPOLYMER AS DELIVERY SYSTEM OF CAPSAICIN

Amphiphilic block copolymers, made of biocompatible polycaprolactone (PCL) and polyethylene glycol (PEG), due to their ability to self‐assemble in water into nanoscopic micelles, have been largely exploited for drug delivery systems (DDS). This study introduces a novel approach by synthesizing a fluorescein isothiocyanate FITC‐labelled PCL‐PEG‐PCL triblock copolymer, with the aim to develop a drug delivery system for natural bioactive. As a proof of concept, the FITC‐labelled PCL‐PEG‐PCL copolymer is applied for the preparation of micelles encapsulating into the core capsaicin (CP), a pungent alkaloid found in chili peppers with diverse therapeutic applications. Challenges associated with CP's solubility, bioavailability, and stability are addressed using this DDS. Comprehensive characterization of FITC‐labelled copolymer is conducted using a range of analytical techniques, including nuclear magnetic resonance (NMR), dynamic light scattering (DLS), high‐performance liquid chromatography (HPLC), Fourier‐transform infrared spectroscopy (FTIR), fluorescence, and confocal laser scanning microscopy (CLSM). Key properties such as critical micelle concentration, CP loading, and release behavior are thoroughly investigated and compared with the characteristics of the unlabeled parent copolymer. This research pioneers the investigation of PCL‐PEG‐PCL triblock copolymers for CP delivery, along with the use of FITC‐labelled variants, opening new avenues for research in drug delivery and nanomedicine.

P-024 Revitalizing male fertility: Crocin’s dual role in antioxidant mastery and mitochondrial activation to enhance sperm motility

Abstract Study question The study aims to investigate the potential effect of crocin, a major constituent of Crocus sativus (saffron), on improving sperm motility in patients with asthenozoospermia. Summary answer In vitro crocin treatment enhances sperm motility, reduces ROS, improves mitochondrial membrane potential, and boosts mitochondrial activity, suggesting the potential role of enhancing sperm motility. What is known already Male infertility can result from various factors, with excessive production of reactive oxygen species (ROS) adversely affecting sperm motility. Crocin, known for its antioxidant properties in scavenging ROS, has led to increasing interest in natural compounds to enhance sperm quality. Study design, size, duration The current study involved the evaluation of 95 patients at the Service of Reproductive Biology at the Maternity and Neonatology Center of Monastir. Sperm samples were collected and incubated for 3 hours at 37 °C with increasing concentrations of Crocin (0, 0.2, 0.5, 1, 1.5 mM). Participants/materials, setting, methods This study included asthenozoospermic patients, categorized into two groups: G1 with progressive motility (PM) between 20% and 30%, and G2 with PM between 10% and 15%. Sperm samples underwent treatment with Crocin. Evaluation encompassed sperm motility, considering the distance covered and velocity via video-microscopy. Additionally, intracellular ROS levels were assessed using DCFH-DA fluorochrome, mitochondrial membrane potential was measured with rh123, and mitochondrial activity was determined through succinate dehydrogenase activity via the MTT assay. Main results and the role of chance The results demonstrate that sperm motility is significantly enhanced after in vitro treatment with 1mM Crocin for 3 hours, with 1.9-fold (p < 0.001) and 1.7-fold (p < 0.001) increases in sperm velocity and distance traveled, respectively. Additionally, Crocin reduces intracellular ROS levels, exhibiting a reduction of 1.5-fold (p < 0.001) in G1 and 2.4-fold (p < 0.001) in G2. Fluorescence microscopy of spermatozoa from a patient with severe asthenozoospermia showed intense ROS-induced fluorescence, which was attenuated after incubation with 1 mM Crocin for 3 hours. Furthermore, Crocin demonstrates a noteworthy improvement in mitochondrial membrane potential, with 1.7-fold (p < 0.001) for G1 and 1.9-fold (p < 0.001) for G2, suggesting its capability to prevent the decline of mitochondrial potential. Microscopic fluorescence revealed a marked increase in fluorescence, particularly in the midpiece after Crocin treatment, confirming the protective effect of Crocin on mitochondrial membrane potential. Moreover, there is a substantial increase in mitochondrial activity with Crocin, particularly in G1, showing a 2.14-fold (p < 0.001) increase compared to the G2 group’s 1.85-fold (p < 0.001) increase. These findings collectively highlight the multifaceted benefits of Crocin, encompassing enhanced sperm motility, reduced ROS levels, preserved mitochondrial membrane potential, and heightened mitochondrial activity, underscoring its potential as a therapeutic agent for male infertility, particularly in cases of asthenozoospermia. Limitations, reasons for caution While promising, this study’s limitations include the in vitro treatment, and not fully replicating complex in vivo conditions. Focused on asthenozoospermia, generalizing findings to other male infertility forms necessitates further investigation. Validation of Crocin’s therapeutic potential in male infertility requires additional clinical trials. Wider implications of the findings Our findings suggest that in vitro enhancement of sperm quality with crocin may serve as a promising solution to improve assisted reproductive techniques, mitigating exogenous oxidative stress during laboratory procedures. This insight holds potential for advancing reproductive interventions and warrants further exploration in clinical settings. Trial registration number not applicable

Decoupling of rotation and translation at the colloidal glass transition.

In dense particle systems, the coupling of rotation and translation motion becomes intricate. Here, we report the results of confocal fluorescence microscopy where simultaneous recording of translational and rotational particle trajectories from a bidisperse colloidal dispersion is achieved by spiking the samples with rotational probe particles. The latter consist of colloidal particles containing two fluorescently labeled cores suited for tracking the particle's orientation. A comparison of the experimental data with event driven Brownian simulations gives insights into the system's structure and dynamics close to the glass transition and sheds new light onto the translation-rotation coupling. The data show that with increasing volume fractions, translational dynamics slows down drastically, whereas rotational dynamics changes very little. We find convincing agreement between simulation and experiments, even though the simulations neglect far-field hydrodynamic interactions. An additional analysis of the glass transition following mode coupling theory works well for the structural dynamics but indicates a decoupling of the diffusion of the smaller particle species. Shear stress correlations do not decorrelate in the simulated glass states and are not affected by rotational motion.