Generation of porcine endometrial organoids and their use as a model for enhancing embryonic attachment and elongation.

In brief: Porcine endometrial organoids (EOs) were isolated and characterized, revealing distinctive features such as unique extracellular matrix formation, fusion into uterine bud-like structures, and facilitation of embryo elongation. The yield of EOs was significantly enhanced by cryopreservation medium supplemented with the rock inhibitor (Y-27632), resulting in reduced expression of apoptotic mRNAs and microRNAs. Abstract: Endometrial organoids (EOs) are acceptable models for understanding maternal-embryonic cross talk. This study was conducted to generate EOs and optimize their cryopreservation and provide coculture modeling with embryos. The endometrial tissues were used for culturing the organoids inside domes of Matrigel®. To improve the long-term storage of EOs, 10 µM ROCK inhibitor (RI) was added to the cryopreservation medium. Day 7 parthenogenetically activated embryos were cocultured with EOs or EO outgrowths, and embryonic cell numbers and embryo attachment were monitored. Spherical EOs 100-300 µm in size can be retrieved on day 7 of culture, and larger EOs, approximately 1.5 mm in diameter, can be maintained in the Matrigel® dome for 21 days. The nuclear expression of Ki67 indicates that more than 80% of EOs nuclei were proliferative. EOs exhibit unique novel characters such as formation of extracellular matrix and ability for fusion. RI increased the yield and quality of organoids after freezing or thawing. The cell number of cocultured embryos increased five-fold, and the proportion of trophoblast outgrowths increased seven-fold compared with those of control embryos. The embryos cultured with EO-conditioned medium showed a better attachment rate than the other models, and - for the first time - we report embryonic elongation. Immunofluorescence staining of the attached embryos showed CDX2 in the periphery of EOs outgrowths. The 3D assembly and cryopreservation of EOs was optimized, and EO coculture supported embryo attachment, trophoblast outgrowth, and elongation, which would provide a valuable tool for studying the intricate processes involved in porcine embryo implantation.

Candidate reference measurement procedure based on HPAEC-PAD for the development of certified reference materials for monosaccharides in serum.

To achieve the measurement reliability of monosaccharides used as diagnostic markers in clinical fields, it is essential to establish certified reference materials (CRMs). The purpose of this study is to develop a serum CRM by adopting high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as a new candidate reference measurement procedure for the measurement of glucose and galactose, common diagnostic markers of diabetes and galactosemia, respectively. Using various monosaccharides as internal standards, the accuracy of the HPAEC-PAD method was tested by measuring glucose CRM following treatment with three different deproteinization methods: ultrafiltration, protein precipitation by trichloroacetic acid (TCA), and protein precipitation by acetonitrile. Results showed that ultrafiltration and 5% TCA provided good accuracy with every tested monosaccharide as the internal standard. Accordingly, serum samples in this study were treated by ultrafiltration after adding 2-deoxy-D-glucose and arabinose, which were selected as internal standards for galactose and glucose, respectively. Both intra- and inter-day recovery tests showed good precision and accuracy within 2%. From the serum CRM batches prepared at two levels, 11 units were analyzed by exact-matched calibration methods, and the mass fractions of galactose and glucose were determined via HPAEC-PAD. The between-unit relative standard deviations were not more than 1.5%, showing homogeneity. The expanded uncertainties (%) of galactose and glucose for both levels were less than 3.6% and 2.3% at 95% confidence. The HPAEC-PAD method presented in this study can significantly improve the accuracy and precision of simultaneous monosaccharide analysis, allowing for the development of further serum CRMs for monosaccharides. Graphical abstract.

Dried blood spot sampling coupled with liquid chromatography-tandem mass for simultaneous quantitative analysis of multiple cardiovascular drugs.

Dried Blood Spots (DBS) revolutionize therapeutic drug monitoring using LC-MS for the precise quantification of cardiovascular drugs (CDs), enabling personalized treatment adapted to patient-specific pharmacokinetics with minimal invasiveness. This study aims to achieve simultaneous quantification of eight CDs in DBS, overcoming physicochemical challenges. A two-step protein precipitation method was used for simple and precise sample preparation. The drugs were analyzed using LC-MS/MS in ESI positive-ion mode, showing high sensitivity and linearity, with a correlation coefficient (r2) exceeding 0.999, after being separated on a reversed-phase chromatography by gradient elution of DW-acetonitrile containing 0.1 % formic acid + 2 mM ammonium formate. The validation results indicate good selectivity, with no observed matrix effect and carry-over. The intra- and inter-day accuracy and precision were within 6 % for most drugs, except for digoxin and deslanoside at low therapeutic levels where the variation was within 20 %. Stability tests confirmed suitable DBS handling and storage conditions, indicating drug stability for at least 30 days at room temperature. The analysis of whole spot has demonstrated remarkable precision and reliability in all target drugs. The analysis of 3 mm internal diameter discs, punched in and out of DBS, presumed to contain 3 µL of blood, showed acceptable accuracy for most drugs, with less polar drugs like digoxin and deslanoside showing lower accuracy, indicating a need for further correction due to non-uniform drug distribution. Consequently, the developed LC-MS/MS method enables the quantification of multiple CDs in a single DBS analysis, while suggesting the potential for accuracy-based analysis.

Simultaneous quantification of 11 antiepileptic drugs using limited isotope-labeled internal standards in LC-MS/MS: An accuracy assessment.

This study aims to establish an LC-MS/MS method to simultaneously analyze 11 antiepileptic drugs with a particular focus on maintaining accuracy while reducing the number of isotope-labeled internal standards employed for cost-effectiveness. By applying a water/acetonitrile gradient elution containing 0.1 % formic acid and 2 mM ammonium formate as the mobile phase, optimal sensitivity for the target drugs could be obtained in positive ESI mode in LC-MS/MS. After optimizing various extraction techniques, extraction with 70 % acetonitrile was selected as it provided good recoveries (>93 %) for all targets without matrix effects. Accuracies within 3 % were achieved from the combination of six internal standards, while accuracies of 5 % and 10 % were obtained by reducing the number of internal standards to four and two, respectively, for more economical analysis. The accuracy of the established method was maintained in hyperglycemia, hyperlipidemia, and hyperalbuminemia sera, suggesting that it can be successfully applied to individual serum samples with various properties.

The reprotoxic adverse side effects of neurogenic and neuroprotective drugs: current use of human organoid modeling as a potential alternative to preclinical models.

The management of neurological disorders heavily relies on neurotherapeutic drugs, but notable concerns exist regarding their possible negative effects on reproductive health. Traditional preclinical models often fail to accurately predict reprotoxicity, highlighting the need for more physiologically relevant systems. Organoid models represent a promising approach for concurrently studying neurotoxicity and reprotoxicity, providing insights into the complex interplay between neurotherapeutic drugs and reproductive systems. Herein, we have examined the molecular mechanisms underlying neurotherapeutic drug-induced reprotoxicity and discussed experimental findings from case studies. Additionally, we explore the utility of organoid models in elucidating the reproductive complications of neurodrug exposure. Have discussed the principles of organoid models, highlighting their ability to recapitulate neurodevelopmental processes and simulate drug-induced toxicity in a controlled environment. Challenges and future perspectives in the field have been addressed with a focus on advancing organoid technologies to improve reprotoxicity assessment and enhance drug safety screening. This review underscores the importance of organoid models in unraveling the complex relationship between neurotherapeutic drugs and reproductive health.

Melatonin and resveratrol alleviate molecular and metabolic toxicity induced by Bisphenol A in endometrial organoids.

Bisphenol A (BPA), a widespread environmental contaminant, poses concerns due to its disruptive effects on physiological functions of the uterine endometrium. In contrast, melatonin (MT) and Resveratrol (RSV) are under scrutiny for their potential protective roles against BPA-induced damage. For the efficacy and ethical concerns in the animal test, endometrial organoids, three-dimensional models mimicking endometrium, serve as crucial tools for unraveling the impact of environmental factors on reproductive health. This study aimed to comprehensively characterize the morphological, molecular and metabolic responses of porcine endometrial organoids to BPA and assess the potential protective effects of MT and RSV. Porcine uteri were prepared, digested with collagenase, mixed with Matrigel, and incubated at 38°C with 5 % CO2. Passaging involved dissociation through trypsin-EDTA treatment and subculturing. The culture medium was refreshed every 2-3 days. To investigate the environmental impact on reproductive health, endometrial organoids were treated with BPA (0.5 µM), MT (with/without BPA at 0.1 µM), and/or RSV (10 µM). Various molecular screening using gene expression, western blotting, immunofluorescence staining, and metabolites profiling were assessed the effects of BPA, MT, and RSV in terms of cell viability, morphology, reproductivity, and metabolism alteration in the endometrial organoids. As expected, BPA induced structural and molecular disruptions in organoids, affecting cytoskeletal proteins, Wnt/ß-catenin signaling, and epithelial/mesenchymal markers. It triggered oxidative stress and apoptotic pathways, altered miRNA expression, and disrupted the endocannabinoid system. The level of glucose, galactose, and essential amino acids were increased or decreased by approximately 1.5-3 times in BPA-treated groups compared to the control groups (p-value < 0.05), indicating metabolic changes. Moreover, MT and RSV treated groups exhibited protective effects, mitigating BPA-induced disruptions across multiple pathways. For the first time, our study models endometrial organoids, advancing understanding of environmental impacts on reproductive health.

Development and validation of an analytical method using liquid chromatography-tandem mass spectrometry for the therapeutic drug monitoring of seven cardiovascular drugs in clinical usage.

Therapeutic drug monitoring (TDM) of cardiovascular drugs is essential to improve treatment efficacy and minimize toxicity because of the usage of multiple drugs with a very limited therapeutic range and the high pharmacokinetic variation in patients. We developed and validated a reliable and economical liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of seven cardiovascular drugs-procainamide, lidocaine, quinidine, deslanoside, digoxin, atorvastatin, and digitoxin-for clinical usage. Serum samples were prepared by simple protein precipitation with an organic solvent consisting of acetonitrile and methanol (2:1 v/v) and analyzed under optimized LC-MS/MS conditions. The chromatographic separations were accomplished within 15 min on a reversed-phase C18 column with a gradient elution of aqueous solvent and acetonitrile while maintaining 0.1 (v/v) % formic acid and 2 mM ammonium formate. The optimized MS/MS conditions in ESI-positive mode offered sufficient sensitivity for the seven cardiovascular drugs (LOQs between 0.5 and 1 ng/mL). This method was fully validated including linearity, selectivity, accuracy, precision, carry-over, and matrix effects. Additionally, stability under several conditions was tested to determine how to handle the standard solutions and serum samples. The seven cardiovascular drugs, simultaneously, were precisely and accurately analyzed in intra- and inter-day assays (RSD < 6 % and recovery between 96.3 and 102.8 %) using only two isotope-labeled internal standards (lidocaine-(diethyl-d10) and digoxin-21, 21, 22-d3). The presented method also showed good accuracy in analyzing the seven drugs in hyperlipidemia, hyperalbuminemia, and hyperglycemia serum, allowing it to be recommended as a common and routine analysis method for cardiovascular drugs in clinical practice.

Berberine Nanoencapsulation Attenuates Hallmarks of Scoplomine Induced Alzheimer's-Like Disease in Rats.

BACKGROUND: Berberine (BBR), an isoquinoline alkaloid, acts as a multipotent active pharmaceutical ingredient to counteract several types of dementia based on its numerous pharmacological actions including antioxidant, anti-inflammatory, cholesterol-lowering effect, and inhibition of Aß production and AChE. However, BBR suffers from poor absorption, bioavailability and brain drug uptake. The present study is directed for the formulation and characterization of Chitosan BBR-Nanoparticles (BBR-NPs) as well as the estimation of its neuroprotective effects against scopolamine induced cognitive impairments. METHODS: BBR-NPs were formulated using the ionic gelation method, and tripolyphosphate was chosen as a crosslinker. Nanoparticles size, zeta potential, encapsulation efficiency and releasing profile were estimated. To investigate the neuroprotective effects, adult fifty-six Wistar male rats were randomly distributed into three control groups, received saline, polyethylene glycol or Chitosan- NPs, respectively; induced group, received scopolamine (2 mg/ kg, i.p.) and three treated groups were orally administrated BBR (50 mg/ kg), BBR- NP (7 mg/ kg) and donepezil (2.25 mg/ kg, as positive control) followed by scopolamine injection after 40 min, daily for 4 weeks. Morris water maze test, oxidative stress parameters, cholinergic and amyloid-ß processing intermediates, as well as neuroplasticity markers and histopathological examination were assessed. RESULTS: Our results showed that BBR- NPs were better than BBR and donepezil as BBR- NPs were powerful inhibitory ligands towards AChE and Aß42 formation and significantly down-regulated Tau, iNOS and BACE gene expression in rats' hippocampus. BBR-NPs administration, at 1/6 of BBR therapeutic recommended dose, significantly improved learning and memory function. This could be accredited to the diminution of oxidative stress and amyloid-ß toxicity in addition to the improvement of the neuroplasticity markers. CONCLUSION: The enhancing effect of BBR- NPs could be related to the enhancing of its bioavailability, absorption and brain drug uptake, which need more investigation in future work.

Neuroprotective effect of ipriflavone against scopolamine-induced memory impairment in rats.

BACKGROUND: Alzheimer's disease is an age-related neurodegenerative disorder characterized clinically by a progressive loss of memory and cognitive functions resulting in severe dementia. Ipriflavone (IPRI) is a non-hormonal, semi-synthetic isoflavone, clinically used in some countries for the treatment and prevention of postmenopausal osteoporosis. Moreover, ipriflavone is a non-peptidomimetic small molecule AChE inhibitor with an improved bioavailability after systemic administration, due to its efficient blood-brain barrier permeability in comparison with peptidomimetic inhibitors. OBJECTIVE: The present study aimed to evaluate the possible enhancing effects of IPRI on memory impairments caused by scopolamine administration. METHODS: Male rats were administered IPRI (50 mg/kg, oral) 2 h before scopolamine injection (2 mg/kg, intraperitoneally injected) daily for 4 weeks. Effects of IPRI on acetylcholinesterase activity, amyloid-ß precursor processing, and neuroplasticity in the rats' hippocampus were investigated. RESULTS: Daily administration of IPRI reverted memory impairment caused by scopolamine as measured by the reduction of the escape latency. IPRI significantly alleviated the oxidative stress and restored the mRNA expression of both cAMP-response element-binding protein and brain-derived neurotrophic factor in the hippocampus. Furthermore, it significantly increased the expression of ADAM10 and ADAM17 (two putative α-secretase enzymes) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) that associated with decreased expression of ß-secretase (BACE) in the hippocampus. Finally, both the amyloid-ß (Aß) and Tau pathologies were reduced. CONCLUSIONS: IPRI showed promising neuroprotective effects against scopolamine-induced memory dysfunction in rats. These findings contributed to the stimulation of α-secretase enzymes, the activation of MAPK/ERK1/2, and the alleviation of oxidative stress.