Metabolites In Medium Research Articles

Differences in the physiology of Pichia pastoris grown on glycerol/glucose using polyurethane foam slabs as solid support

AbstractBACKGROUNDGrowth physiology and metabolic flux of Pichia pastoris were analyzed using glass columns containing slabs of polyurethane foam as a solid support and culture medium with glycerol or glucose as a carbon source.RESULTSMedia with glycerol produced an average of 30.91% more biomass than media where glucose was used. However, the specific growth rate registered higher values for media with glucose than those with glycerol at low substrate concentrations: 0.46 versus 0.22 h−1. Media with glycerol produced a large amount of d‐arabitol, whereas ethanol and citrate were the predominant metabolites in media with glucose. Both types of culture systems generated biphasic curves in terms of the respiratory quotient value, whose inflection point coincided with the transition from aerobic metabolism to a mixed one.CONCLUSIONAdequate oxygen transfer occurred in both culture systems; therefore, the differences found in growth physiology can be explained by the transport mechanism that internalizes glycerol, the access point to primary metabolism, and the degree of glycerol oxidation. The arrangement of cells in the polyurethane matrix allowed an attenuation of the repression phenomenon due to excess substrate for both carbon sources. This is the first work to study metabolic flux comparisons using this system. © 2024 Society of Chemical Industry (SCI).

HPLC/ESI-QTOF-MS/MS based untargeted metabolomics authentication of Taxus × media six tissues.

Taxus media (Taxus × media Rehder) is renowned for its high paclitaxel content, serving as a major source for industrial paclitaxel production. In addition to paclitaxel, T. media contains a diverse range of metabolites, including flavonoids, alkaloids, and terpenoids, which have been shown to possess antioxidant, antibacterial, anti-inflammatory, and immunomodulatory effects. However, these compounds have not been thoroughly studied as key metabolites in T. media. The untargeted metabolomics analysis of six T. media tissues provides new insights into the development and utilization of T. media metabolites. The extracts from six tissues of T. media were analyzed and subjected to analysis using high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (HPLC-Q-TOF-MS/MS) and chemometric techniques. Using a reliable HPLC-Q-TOF-MS/MS method, we identified 312 compounds in six T. media tissues, including 214 previously unreported in T. media. To identify characteristic compounds across different tissues, 34 metabolites were further screened. KEGG metabolic pathway analysis revealed that these compounds primarily occur in the metabolic pathways of terpene glycosides, flavans, and O-methylated flavonoids. This study initially utilized an HPLC-QTOF-MS/MS-based metabolomics approach to assess the metabolites in different tissues of T. media, providing a basis for their utilization and management.

Impact of preovulatory follicle maturity on oocyte metabolism and embryo development.

Improved oocyte competence for embryo development and pregnancy was observed following ovulation of preovulatory follicles with greater physiological maturity, as indicated by estradiol production, prior to the gonadotropin-releasing hormone (GnRH)-induced luteinizing hormone (LH) surge. It was hypothesized that follicular fluid from preovulatory follicles of greater maturity better supports the maturing oocyte's metabolic requirements and improves embryo development. The objective was to determine if differences in preovulatory follicular fluid due to follicle maturity influence oocyte metabolism during in vitro maturation (IVM) and affect embryo development. Bovine preovulatory follicular fluid was collected 18 h after a GnRH-induced LH surge. Serum estradiol concentration at GnRH administration categorized follicles as greater or lesser maturity. Immature bovine oocytes were submitted to 24 h IVM in medium supplemented with 20% follicular fluid from preovulatory follicles of greater or lesser maturity. Embryo development was recorded. Oocyte maturation media and media conditioned by developing embryos were submitted for metabolomics. A randomized block design was utilized to determine differences in embryo development and media metabolites (P ≤ 0.05). Blastocysts from oocytes matured in greater vs. lesser maturity follicular fluid had a more moderate rate of development (P = 0.01). At the conclusion of 24 h IVM, abundance of 66 metabolites differed between greater and lesser follicle maturity treatments. Nine metabolites differed in media conditioned by developing embryos. Metabolome results suggest improved amino acid, purine, and glucose metabolism, followed by a more efficient rate of embryo development, in oocytes matured in greater vs lesser maturity follicular fluid.

Metabolomic changes in culture media with varying passage numbers of pig muscle stem cell culture for cultured meat production

Selecting the primary cells in an optimal state for cultured meat production is a crucial challenge in commercializing cultured meat. We investigated the metabolomic changes in culture media according to passage numbers for indirectly assessing the state of primary cells. Pig skeletal muscle stem cells (PSCs) harvested from the biceps femoris muscles of 7-d-old crossbred pigs (Landrace × Yorkshire × Duroc, LYD) were used for cell characterization. Fresh media (FM) and spent media (SM) of PSCs during passages 1 to 3 in vitro culture were prepared for metabolomics analysis. SM was collected on the third day of proliferation for each passage of PSCs. Cell characterization analysis revealed that the proliferation rate was highest at passage 2; however, a significant loss of expression of myogenic marker genes was observed at passage 3. Based on metabolomic profiles of culture media, FM and SM groups (SM1, SM2, and SM3) were clearly separated by partial least squares-discriminant analysis. A total of seven differentially abundant metabolites (DAMs) were identified from FM and SM for each passage, based on the following criteria: P < 0.05, fold change > 1.5 or < 0.66, and a variable importance in projection score > 1.5. All seven DAMs and their interconnected metabolites might be primarily used as substrates for energy production and most of them were relatively abundant in SM3. Among the seven DAMs, the three potential biomarkers (γ-glutamyl-L-leucine, cytosine, and ketoleucine), which showed significant changes exclusively in SM3, each had an area under the curve value of 1. Therefore, monitoring the levels of these key metabolites in culture media could serve as a quality control measure for cultured meat production by enabling the indirect detection of suboptimal PSCs based on their proliferation ability.

Metabolic stressful environment drives epigenetic modifications in oviduct epithelial cells

The oviduct provides a suitable microenvironment from the gametes’ final maturation until initial embryo development. Dynamic functional changes are observed in the oviduct cells, mainly controlled by steroid hormones and well-orchestrated during the estrous cycle. However, based on the roles played by the oviduct, additional layers of complexity might be present in its regulatory process. There is a cellular process that includes metabolic adaptation that can guide molecular modifications. This process is known as metaboloepigenetics. Therefore, we aimed to better understand how this crosstalk occurs in oviductal epithelial cells (OEC). Due to limited in situ access to the oviduct, we used the primary in vitro cell culture as a culture model and glucose as a metabolic disturbed factor. For that, cells derived from the oviductal epithelial layer were collected from cows at either follicular or luteal stages (n = 4 animals per group). They were cultured on a monolayer culture system under normoglycemic (2.7 mM glucose) or hyperglycemic conditions (27 mM glucose). On day five of culture, attached cells were submitted to analysis of mitochondrial metabolism (mitochondrial membrane potential - MMP) and epigenetics markers (5- methylcytosine - 5 mC and histone 3 lysine 9 acetylation - H3K9ac). Moreover, the culture media were submitted to the metabolites analysis profile by Raman spectrometry. Data were analyzed considering the effect of glucose level (normoglycemic vs. hyperglycemic), stages when OEC were harvested (follicular vs. luteal), and their interaction (glucose level * cycle stage) by two-way ANOVA. As a result, the high glucose level decreased the H3K9ac and MMP levels but did not affect the 5 mC. Regardless of the metabolic profile of the culture media, the glucose level was the only factor that changed the Raman shifts abundance. Although this present study evaluated oviductal epithelial cells after being submitted to an in vitro monolayer culture system, which is known to lead to cell dedifferentiation, yet, these results provide evidence of a relationship between epigenetic reprogramming and energy metabolism under these cell culture conditions. In conclusion, the levels of metabolites in culture media may be crucial for cellular function and differentiation, meaning that it should be considered in studies culturing oviductal cells.

Correlation between metabolomic profile and proliferation of Macrobrachium rosenbergii primary embryonic cell culture

Despite extensive research spanning several decades, a commercial cell line for prawns remains elusive, and we have yet to determine the ideal conditions required for successful in vitro cell cultures of crustaceans. This study marks the first application of mass spectrometry to analyze lipid content changes in prawn culture media, allowing us to investigate how specific metabolites in media correlate with cell cycle and proliferation status. To investigate these relationships, short-term cell culture experiments were conducted using three commercial media: Dulbecco's modified eagle medium (DMEM) high glucose, Opti-Minimal essential media (Opti-MEM), and DMEM:F12. We monitored metabolic activity, performed cell-cycle analysis, and used mass spectrometry to analyze lipid metabolic profiles of culture media. This comprehensive approach enables us to examine the interplay between media lipidomic content and cellular behavior. Our results revealed that all three media sustained cellular metabolic activity. DMEM high glucose and Opti-MEM showed peaks in metabolic activity at day 4, while DMEM:F12 exhibited a peak at day 7. Opti-MEM supported the shortest doubling time, making it ideal for short-term culture. Interestingly, DMEM high glucose retained more attached cells on day 1 and had the highest count on day 4, with a noticeable trend towards the G2/M cell-cycle phase. Moreover, our lipidomic analysis of Opti-MEM revealed significantly higher concentrations of 26 annotated lipids compared to the other media. Notably, lipids like phosphatidylcholine and phosphatidylserine were consistently abundant in all three media, and palmitate levels correlated with proliferation phase. These findings have significant implications for establishing sustainable crustacean cell culture lines. Understanding the lipidomic compositions and their associations with cell-cycle dynamics and proliferation allows for precise conditioning of culture media to promote sustainable growth and delay cellular entry into quiescence phases. This knowledge contributes to the long-term goal of maintaining robust prawn cell cultures for various research and biotechnological applications.

The Effect of Plasma-Treated Water on Microbial Growth and Biosynthesis of Gamma-Decalactones by Yarrowia lipolytica Yeast.

In recent years, the production of plasma-treated water (PTW) by low-temperature low-pressure glow plasma (LPGP) has been increasingly gaining in popularity. LPGP-treated water changes its physical and physiochemical properties compared to standard distilled water. In this study, a non-conventional lipolytic yeast species Yarrowia lipolytica was cultivated in culture media based on Nantes plasma water with heightened singlet oxygen content (Nantes PW) or in water treated with low-temperature, low-pressure glow plasma while in contact with air (PWTA) or nitrogen (PWTN). The research aimed to assess the influence of culture conditions on castor oil biotransformation to gamma-decalactone (GDL) and other secondary metabolites in media based on nanowater. The Nantes plasma water-based medium attained the highest concentration of gamma-decalactone (4.81 ± 0.51 g/L at 144 h of culture), maximum biomass concentration and biomass yield from the substrate. The amplified activity of lipases in the nanowater-based medium, in comparison to the control medium, is encouraging from the perspective of GDL biosynthesis, relying on the biotransformation of ricinoleic acid, which is the primary component of castor oil. Although lipid hydrolysis was enhanced, this step seemed not crucial for GDL concentration. Interestingly, the study validates the significance of oxygen in β-oxidation enzymes and its role in the bioconversion of ricinoleic acid to GDL and other lactones. Specifically, media with higher oxygen content (WPTA) and Nantes plasma water resulted in remarkably high concentrations of four lactones: gamma-decalactone, 3-hydroxy-gamma-decalactone, dec-2-en-4-olide and dec-3-en-4-olide.

Development of a Robust Consensus Modeling Approach for Identifying Cellular and Media Metabolites Predictive of Mesenchymal Stromal Cell Potency.

Mesenchymal stromal cells (MSCs) have shown promise in regenerative medicine applications due in part to their ability to modulate immune cells. However, MSCs demonstrate significant functional heterogeneity in terms of their immunomodulatory function because of differences in MSC donor/tissue source, as well as non-standardized manufacturing approaches. As MSC metabolism plays a critical role in their ability to expand to therapeutic numbers ex vivo, we comprehensively profiled intracellular and extracellular metabolites throughout the expansion process to identify predictors of immunomodulatory function (T-cell modulation and indoleamine-2,3-dehydrogenase (IDO) activity). Here, we profiled media metabolites in a non-destructive manner through daily sampling and nuclear magnetic resonance (NMR), as well as MSC intracellular metabolites at the end of expansion using mass spectrometry (MS). Using a robust consensus machine learning approach, we were able to identify panels of metabolites predictive of MSC immunomodulatory function for 10 independent MSC lines. This approach consisted of identifying metabolites in 2 or more machine learning models and then building consensus models based on these consensus metabolite panels. Consensus intracellular metabolites with high predictive value included multiple lipid classes (such as phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins) while consensus media metabolites included proline, phenylalanine, and pyruvate. Pathway enrichment identified metabolic pathways significantly associated with MSC function such as sphingolipid signaling and metabolism, arginine and proline metabolism, and autophagy. Overall, this work establishes a generalizable framework for identifying consensus predictive metabolites that predict MSC function, as well as guiding future MSC manufacturing efforts through identification of high-potency MSC lines and metabolic engineering.

Abstract 3710: Serum tryptophan metabolites mediate constitutive AHR activity in head and neck squamous cell carcinoma cells

Abstract The aryl hydrocarbon receptor (AHR) is transcription factor, that is activated upon binding to various exogenous and endogenous ligands. AHR activation leads to induction of target gene expression, including the members of cytochrome P450 enzyme family that metabolize many xenobiotics. Numerous studies have shown that the AHR is involved in multiple regulatory pathways important during carcinogenesis. High level of AHR protein and activation was observed in head and neck squamous cell carcinoma (HNSCC) and has been correlated with aggressive tumor phenotypes and migratory potential. Endogenous compounds derived from host tryptophan metabolism such as kynurenine, as well as the derivatives of indole derived from bacterial tryptophan metabolism such as indole-3-propionic acid are known to activate AHR. A fundamental question not previously addressed is whether these circulating tryptophan (Tryp) metabolites can mediate persistent AHR activation within tumor tissues and its effect on tumorigenesis. The focus of our research is to determine whether the continuous exposure of Tryp metabolites at relevant concentrations leads to sustained AHR activation in HNSCC cells and its subsequent effect on tumor cell phenotypes. Here, we present data demonstrating sustained AHR activation by Tryp metabolites in two HNSCC cell lines. A total of 6 tryptophan (Tryp) metabolites present at significant levels in serum with known AHR agonist activity were identified and quantified from 40 healthy individuals on a controlled diet by LC/MS/MS. HN30 and OSC19 cells were treated with a representative pool of 6 Tryp metabolites in a cell culture system to achieve continuous exposure to the Tryp metabolites, thus mimicking blood circulation in vivo. Treated cells were collected at six different time points and AHR activation within cells was determined by measuring mRNA and protein expression of AHR target genes. The effect of Tryp pool on HN30 and OSC19 cell viability was determined. The presence of Tryp metabolites within the cells and media was quantitated by LC/MS/MS at six time points over the course of 24 h. Continuous exposure of Tryp metabolites resulted in sustained AHR activation in HN30 and OSC19 cells, confirmed by induced mRNA expression of target genes and subsequent protein levels. The LC/MS data revealed the presence of all six Tryp metabolites in media collected at all time points, suggesting no spontaneous degradation of metabolites. Tryp metabolites were detected inside the cells starting from 2 h post-treatment to the last time point 24 h. Viability assays indicated no cytotoxic effects of Tryp metabolites on cells. Overall, our work has demonstrated a pattern of sustained AHR activation in HNSCC cells resulting from continuous exposure of Tryp metabolites, supporting the concept that endogenous Tryp metabolites in HNSCC patients would impact tumor progression, metastasis, and treatment outcomes. Citation Format: Dhwani Patel, Ethan W. Morgan, Fangcong Dong, Iain A. Murray, Gary H. Perdew. Serum tryptophan metabolites mediate constitutive AHR activity in head and neck squamous cell carcinoma cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3710.

Metabolomic approaches for the detection of Listeria monocytogenes and Staphylococcus aureus in culture media

This study aimed to determine the changes in metabolites in the culture media of foodborne pathogens using nuclear magnetic resonance (NMR) spectroscopy. Extracellular metabolites in tryptic soy broth inoculated with none, Listeria monocytogenes ATCC 19111, and Staphylococcus aureus ATCC 29213 and 27213 at 37 °C were collected and analyzed by 1D 1H NMR and 2D 1H–13C heteronuclear single quantum coherence (HSQC) NMR. Partial least squares discriminant analysis revealed an excellent variable influence on projection (VIP) score and predictive ability among the different culture media metabolites. Based on presence/absence, five extracellular metabolites (cadaverine, histidine, nicotinate, putrescine, and succinate) were only detected in culture media containing pathogens. According to the pathogen species, L. monocytogenes and S. aureus ATCC 29213 could be distinguished based on changes in 11 metabolites (tyrosine, betaine, glycine, pyroglutamate, fumarate, alanine, putrescine, arginine, inosine, threonine, and sucrose). Moreover, different strains of S. aureus (ATCC 29213 and 27213) were distinguished using hypoxanthine and putrescine. Overall, extracellular metabolites in culture media could be used to detect the presence/absence, species, and even different strains of foodborne pathogens.

Metabolomic analysis of cooperative adaptation between Co-cultured Lacticaseibacillus casei Zhang and Lactiplantibacillus plantarum P8

Microbial secondary metabolites have long been considered as potential sources of natural active molecules due to their rich chemical diversity and extensive biological activities. Increasing evidence shows that coculture from probiotic strains had synergistic effect promoting the production of bioactive molecules, and secondary metabolites that had higher stability and/or functional performance. The purpose of this study was to explore the interaction between Lacticaseibacillus casei Zhang (LcZ) and Lactiplantibacillus plantarum P8 (P8) during co-culture using non-targeted metabonomics approach. Results showed stronger growth in the LcZ and P8 co-culture (LcZ + P8) compared with either bacterium alone. Multivariate analysis revealed that significant differences in metabolic profiles between strains, and by and within the same strain at different growth stages. In addition, the alteration of cell and media metabolites during cell growth was strongly associated with energy production. Moreover, some new bioactive substances and substances with higher expression levels that might have beneficial effect on human health were identified in LcZ + P8.This is the first study to systematically identify a symbiotic interaction of metabolites in an ecosystem established by P8 and LcZ. The results provide novel insight into the system-level cooperative adaptation between strains and have implications for the further development of microbial natural products.

Sunitinib versus Pazopanib Dilemma in Renal Cell Carcinoma: New Insights into the In Vitro Metabolic Impact, Efficacy, and Safety.

Sunitinib and pazopanib are tyrosine kinase inhibitors (TKIs) used as first-line therapy for metastatic renal cell carcinoma (RCC). Although these TKIs are associated with similar survival outcomes, some differences have been reported in their safety profiles. In this work, traditional toxicological endpoints (cell viability and growth, oxidative stress, and nuclear morphology) and 1H NMR spectroscopy-based metabolomics analysis were used to provide new insights into the cytotoxicity and metabolic mechanisms underlying sunitinib and pazopanib treatments. Tumoral (Caki-1) and non-tumoral (HK-2) human renal cells were exposed to clinically relevant concentrations of sunitinib (2 µM) or pazopanib (50 µM). Sunitinib showed selectivity for cancer cells, inhibiting proliferation, and inducing apoptotic death of Caki-1 cells, whereas pazopanib had a similar cytotoxic effect in both tumoral and non-tumoral cells. 1H-NMR metabolomics unveiled a higher impact of sunitinib on the levels of intracellular metabolites of Caki-1 cells (seven dysregulated metabolites), suggesting dysregulations on amino acid, glutathione and glycerophospholipid metabolisms. In contrast, pazopanib had a higher impact on the levels of extracellular metabolites of Caki-1 cells (seven dysregulated metabolites in culture medium), unveiling alterations on amino acid and energetic metabolisms. In HK-2 cells, sunitinib caused only a minor increase in intracellular isoleucine levels, whereas pazopanib induced several alterations on the intracellular (three dysregulated metabolites) and extracellular (three dysregulated metabolites) compartments suggesting changes on amino acid, glycerophospholipid, and energy metabolisms. Our results demonstrate that these TKIs elicit distinct cellular and metabolic responses, with sunitinib showing better in vitro efficacy against target RCC cells and lesser nephrotoxic potential than pazopanib.

In silico genome-scale metabolic modeling and in vitro static time-kill studies of exogenous metabolites alone and with polymyxin B against Klebsiella pneumoniae.

Multidrug-resistant (MDR) Klebsiella pneumoniae is a top-prioritized Gram-negative pathogen with a high incidence in hospital-acquired infections. Polymyxins have resurged as a last-line therapy to combat Gram-negative “superbugs”, including MDR K. pneumoniae. However, the emergence of polymyxin resistance has increasingly been reported over the past decades when used as monotherapy, and thus combination therapy with non-antibiotics (e.g., metabolites) becomes a promising approach owing to the lower risk of resistance development. Genome-scale metabolic models (GSMMs) were constructed to delineate the altered metabolism of New Delhi metallo-β-lactamase- or extended spectrum β-lactamase-producing K. pneumoniae strains upon addition of exogenous metabolites in media. The metabolites that caused significant metabolic perturbations were then selected to examine their adjuvant effects using in vitro static time–kill studies. Metabolic network simulation shows that feeding of 3-phosphoglycerate and ribose 5-phosphate would lead to enhanced central carbon metabolism, ATP demand, and energy consumption, which is converged with metabolic disruptions by polymyxin treatment. Further static time–kill studies demonstrated enhanced antimicrobial killing of 10 mM 3-phosphoglycerate (1.26 and 1.82 log10 CFU/ml) and 10 mM ribose 5-phosphate (0.53 and 0.91 log10 CFU/ml) combination with 2 mg/L polymyxin B against K. pneumoniae strains. Overall, exogenous metabolite feeding could possibly improve polymyxin B activity via metabolic modulation and hence offers an attractive approach to enhance polymyxin B efficacy. With the application of GSMM in bridging the metabolic analysis and time–kill assay, biological insights into metabolite feeding can be inferred from comparative analyses of both results. Taken together, a systematic framework has been developed to facilitate the clinical translation of antibiotic-resistant infection management.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter metabolomics signature

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media metabolites in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. Metabolomic signatures between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. Metabolite levels showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the metabolite until 120h. No significant differences in the levels of metabolites were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of metabolites observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter metabolomics signature.

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media metabolites in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. Metabolomic signatures between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. Metabolite levels showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the metabolite until 120h. No significant differences in the levels of metabolites were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of metabolites observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

Duration of dry and humidified incubation of single-step embryo culture medium and oxygen tension during sham culture do not alter metabolomics signature.

Background: The extended embryo culture using single-step medium gained popularity in clinical in vitro fertilisation (IVF). However, there are concerns about the degradation of unstable medium components and their negative effects on the developing embryos. Further, dry-incubation can increase osmolality, which can in-turn enhance the concentration of constituents of the media and their stability. Hence, this study was conducted to understand the immediate changes in the culture media metabolites in relation to clinically comparable situations such as single-step extended embryo culture and use of dry and humidified-incubation in two-different gaseous conditions. Methods: Commercially available single-step medium was sham-cultured in droplets under oil in two different conditions viz. dry (37°C; 6%CO 2; 5%O 2) and humidified (37°C; 6% CO 2; atmospheric O 2) for 0h, 72h, and 120h intervals. Droplets were subjected to the sensitivity-enhanced nuclear magnetic resonance (NMR)-based profiling using 800 MHz NMR equipped with a cryogenically cooled micro-coil (1.7mm) probe. Metabolomic signatures between the two groups were comprehensively assessed. Results: A total of ten amino acids and four energy substrates were identified from the culture medium. Metabolite levels showed a non-significant increase in the dry-incubation group at 72h and then declined at 120h. Humidified incubation had no effects on the level of the metabolite until 120h. No significant differences in the levels of metabolites were observed between the dry and humidified-groups at various time-points tested. Conclusions: A non-significant variation in the levels of metabolites observed in the dry-incubation of single-step medium most unlikely to influence a clinical outcome. However, the impact of these subtle changes on the (epi)genetic integrity of the embryos in a clinical set-up to be addressed.

AMP deamination is sufficient to replicate an atrophy-like metabolic phenotype in skeletal muscle

BackgroundSkeletal muscle atrophy, whether caused by chronic disease, acute critical illness, disuse or aging, is characterized by tissue-specific decrease in oxidative capacity and broad alterations in metabolism that contribute to functional decline. However, the underlying mechanisms responsible for these metabolic changes are largely unknown. One of the most highly upregulated genes in atrophic muscle is AMP deaminase 3 (AMPD3: AMP → IMP + NH3), which controls the content of intracellular adenine nucleotides (AdN; ATP + ADP + AMP). Given the central role of AdN in signaling mitochondrial gene expression and directly regulating metabolism, we hypothesized that overexpressing AMPD3 in muscle cells would be sufficient to alter their metabolic phenotype similar to that of atrophic muscle. MethodsAMPD3 and GFP (control) were overexpressed in mouse tibialis anterior (TA) muscles via plasmid electroporation and in C2C12 myotubes using adenovirus vectors. TA muscles were excised one week later, and AdN were quantified by UPLC. In myotubes, targeted measures of AdN, AMPK/PGC-1α/mitochondrial protein synthesis rates, unbiased metabolomics, and transcriptomics by RNA sequencing were measured after 24 h of AMPD3 overexpression. Media metabolites were measured as an indicator of net metabolic flux. At 48 h, the AMPK/PGC-1α/mitochondrial protein synthesis rates, and myotube respiratory function/capacity were measured. ResultsTA muscles overexpressing AMPD3 had significantly less ATP than contralateral controls (−25%). In myotubes, increasing AMPD3 expression for 24 h was sufficient to significantly decrease ATP concentrations (−16%), increase IMP, and increase efflux of IMP catabolites into the culture media, without decreasing the ATP/ADP or ATP/AMP ratios. When myotubes were treated with dinitrophenol (mitochondrial uncoupler), AMPD3 overexpression blunted decreases in ATP/ADP and ATP/AMP ratios but exacerbated AdN degradation. As such, pAMPK/AMPK, pACC/ACC, and phosphorylation of AMPK substrates, were unchanged by AMPD3 at this timepoint. AMPD3 significantly altered 191 out of 639 detected intracellular metabolites, but only 30 transcripts, none of which encoded metabolic enzymes. The most altered metabolites were those within purine nucleotide, BCAA, glycolysis, and ceramide metabolic pathways. After 48 h, AMPD3 overexpression significantly reduced pAMPK/AMPK (−24%), phosphorylation of AMPK substrates (−14%), and PGC-1α protein (−22%). Moreover, AMPD3 significantly reduced myotube mitochondrial protein synthesis rates (−55%), basal ATP synthase-dependent (−13%), and maximal uncoupled oxygen consumption (−15%). ConclusionsIncreased expression of AMPD3 significantly decreased mitochondrial protein synthesis rates and broadly altered cellular metabolites in a manner similar to that of atrophic muscle. Importantly, the changes in metabolites occurred prior to reductions in AMPK signaling, gene expression, and mitochondrial protein synthesis, suggesting metabolism is not dependent on reductions in oxidative capacity, but may be consequence of increased AMP deamination. Therefore, AMP deamination in skeletal muscle may be a mechanism that alters the metabolic phenotype of skeletal muscle during atrophy and could be a target to improve muscle function during muscle wasting.

P–272 The aneuploid embryo secretome

Abstract Study question Does the metabolomic analysis of the embryonic culture medium predict the embryo aneuploidy? Summary answer The presence and quantity of some metabolites in the culture medium can select euploid embryos for transfer. What is known already Advances in analytical techniques for metabolomics have brought the possibility of better tools for the characterization of molecules. Embryonic metabolism can be used as a good indicator of viability, regardless of the morphology of the blastocysts, since differences were observed in the metabolic activities between the days of embryo development and in the rates of live births. Study design, size, duration 17 patients had their embryos biopsied between January to July 2019 in a human reproduction laboratory. All cases had PGT-A indication and after the biopsy, the embryos were frozen. The culture medium samples were individually prepared for metabolites extraction according to the Bligh and Dyer protocol. Controlled ovarian stimulation and dose adjustments according to the response of each patient. The metabolomics analysis was performed by mass spectrometry. Participants/materials, setting, methods Ovum pick up will be performed 35 hours after r-hCG administration. The embryos were kept in individual 50ul drops until the blastocyst stage. The biopsy was performed in 26 blastocysts. The samples were sent to the 337 metabolites analysis by mass spectrometry. 15 molecules with the highest score on the PLS-Da was submitted the ROC curves to illustrate the power of the metabolic ploidy analysis. Besides, we performed the functional enrichment analysis for each group. Main results and the role of chance After the genetic analysis by PGT-a, 10 aneuploid embryos and 16 euploid embryos were found. Comparing the quantitative target metabolomic analysis of the 337 metabolites in the embryo culture medium, we observed the L-Alanine, Cytosine, Guanosine monophosphate, Homocysteine, Hypoxanthine, and Xanthine hiperrepresented in the aneuploid embryos, and the Citrulline, L-Glutamic acid, Kynurenine, L-Leucine, Methionine, Ornithine, L-Phenylalanine, L-Tyrosine, L-Valine were hiperrepresented in the euploid embryos. Through the ROC curve, we can verify AUC = 0.987. This result suggests that the analysis of euploid embryos through the metabolomic analysis of the culture medium is valid to be used as a noninvasive aneuploid diagnostic. The functional enrichment analysis shows the urea cycle and the glycine and serine metabolism as the principal function alter by aneuploid. Limitations, reasons for caution Small number of samples and not validate sample group. Wider implications of the findings: Further studies are needed to validate these findings for the diagnostic of embryo euploidy. Trial registration number N/A

P–170 The secretomy of embryo sex

Abstract Study question Does the analysis of the metabolites of the embryonic culture medium can predict the sex of the embryo? Summary answer The presence and quantity of some metabolites in the culture medium can predict the sex of the human embryos. What is known already Advances in analytical techniques for metabolomics have brought the possibility of better tools for the characterization of molecules. Embryonic metabolism can be used as a good indicator of viability, regardless of the morphology of the blastocysts, since differences were observed in the metabolic activities between the days of embryo development and in the rates of live births. Study design, size, duration 16 patients had their embryos biopsied between the months of January to July 2019 in a human reproduction laboratory. All cases had PGT-A indication and after the biopsy, the embryos were frozen. The culture medium samples were individually prepared for metabolites extraction according to the Bligh and Dyer protocol. Controlled ovarian stimulation and dose adjustments according to the response of each patient. The metabolomics analysis was performed by mass spectrometry. Participants/materials, setting, methods Follicular puncture were performed 35 hours after r-hCG. The eggs were kept in individual culture until the blastocyst stage. The blastocysts biopsy was performed (20). After the culture medium was sent to the 337 metabolites analysis by mass spectrometry. 14 molecules with the highest score on the PLS-Da was submitted to the ROC curves showing the power of metabolic analysis to predict the sex of euploid embryos. Besides, we performed the functional enrichment analysis. Main results and the role of chance After the genetic analysis by PGT-a, we obtain 20 euploid embryos, being 12 female embryos and 08 male embryos. Comparing the quantitative target metabolomic analysis of the 337 metabolites in the embryo culture medium, we observed the Asymmetric dimethylarginine, FAD, Malic Acid, Serotonin, increased in female embryos and Adenosine monophosphate, L-Alanine, L-Arginine, Cysteamine, DL-Dopa, Flavin Mononucleotide, Methionine sulfone, Nicotinic acid, L-Tyrosine, Uracil in male embryos. Through the ROC curve, we can verify AUC = 0.937. This result suggests that the metabolomic analysis of the culture medium is valid to be used as a complement of PGT-A to know embryo sex diagnostic. The functional enrichment analysis shows the Asymmetric dimethylarginine and Malic Sulfone metabolism as the principal function alter by female embryos. Limitations, reasons for caution Small number of samples Wider implications of the findings: Further studies are needed to validate these findings for the diagnostic of sex embryos Trial registration number N/A

Tricarboxylic Acid Cycle Metabolites as Mediators of DNA Methylation Reprogramming in Bovine Preimplantation Embryos.

In many cell types, epigenetic changes are partially regulated by the availability of metabolites involved in the activity of chromatin-modifying enzymes. Even so, the association between metabolism and the typical epigenetic reprogramming that occurs during preimplantation embryo development remains poorly understood. In this work, we explore the link between energy metabolism, more specifically the tricarboxylic acid cycle (TCA), and epigenetic regulation in bovine preimplantation embryos. Using a morphokinetics model of embryonic development (fast- and slow-developing embryos), we show that DNA methylation (5mC) and hydroxymethylation (5hmC) are dynamically regulated and altered by the speed of the first cleavages. More specifically, slow-developing embryos fail to perform the typical reprogramming that is necessary to ensure the generation of blastocysts with higher ability to establish specific cell lineages. Transcriptome analysis revealed that such differences were mainly associated with enzymes involved in the TCA cycle rather than specific writers/erasers of DNA methylation marks. This relationship was later confirmed by disturbing the embryonic metabolism through changes in α-ketoglutarate or succinate availability in culture media. This was sufficient to interfere with the DNA methylation dynamics despite the fact that blastocyst rates and total cell number were not quite affected. These results provide the first evidence of a relationship between epigenetic reprogramming and energy metabolism in bovine embryos. Likewise, levels of metabolites in culture media may be crucial for precise epigenetic reprogramming, with possible further consequences in the molecular control and differentiation of cells.