Search Results (3,196)

This study aimed to establish a model for investigating X chromosome inactivation using transgenic mouse strains expressing green fluorescent protein (GFP). The D4/XGFP-Tg (XGFP) strain carries the GFP transgene on the X chromosome; therefore, due to random X chromosome inactivation, female offspring from crosses between XGFP males and CD-1 females exhibit mosaic GFP expression. In contrast, the B5/EGFP-Tg (EGFP) strain harbours autosomal integration of the same reporter construct, resulting in uniform GFP expression in progenies. Analysis of CD-1 × XGFP attached blastocysts revealed strong GFP expression in giant trophoblast cells and primordial germ cells (PGCs) at E6.5, demonstrating paternal X-chromosome reactivation. In 14.5-day-old CD-1 × XGFP female embryos and CD-1 × EGFP embryos, intense CAG promoter-driven GFP signals were detected in the brain, heart, gonads, somites, and limbs. In line with random X-chromosome inactivation, only 56% of embryonic fibroblast cells, derived from CD-1 × XGFP female embryos, exhibited GFP expression. These findings validate that CD-1 × XGFP mice represent a valuable in vivo model for studying X chromosome inactivation during early embryonic development and PGC specification. Furthermore, CD-1 × XGFP embryonic fibroblasts represent a valuable in vitro model for investigating the molecular mechanisms governing X-chromosome activation and inactivation. Full article

The formation and expansion capacity of blastocysts plays a very important role in successful implantation. During mammalian embryo development derived from in vitro production (IVP), early embryos are highly susceptible to various cellular stresses, including endoplasmic reticulum (ER) stress, which has been identified in IVP embryos, suggesting that ER stress modulation is crucial for IVP embryo development. In this study, we aimed to evaluate the influences of ER stress on preimplantation embryos during blastocyst formation and expansion potential in pigs. Tunicamycin (TM), an ER stress inducer, was employed in porcine embryos, significantly increasing the mRNA levels of ER stress-related markers ATF6, CHOP, and GRP78. When one-cell embryos were cultured in the presence of TM, the blastocyst formation and diameter (reflecting the blastocyst expansion capacity) were significantly inhibited in a dose-dependent manner. When morula-stage porcine embryos were cultured in TM, the blastocyst formation rate, blastocyst diameter, total cells and EdU-positive cell numbers were significantly lower than the TM-free control group. TM reduced the potential of blastocoel recovery (ex-expansion) in blastocysts collapsed by cytochalasin D and impeded blastocyst expansion. In addition, TM reduced the mRNA levels of CDH1 and TJP1 and affected the normal expression pattern of E-cadherin, Oct4, Sox2 and Cdx2 in porcine blastocysts. Taken together, these findings suggest that TM treatment during embryo development in vitro interferes with the formation and expansion capacity of the blastocoel in pigs. Full article

Oxidative stress (OS) and sperm DNA fragmentation (SDF) are complementary contributors to male infertility. OS characterizes a compromised seminal redox status, whereas SDF quantifies downstream genomic damage. Human sperm are highly susceptible to redox damage due to lipid-rich membranes and disrupted post-meiotic DNA-repair capacity. Excess reactive oxygen species (ROS) can cause lipid peroxidation, oxidative base lesions, and DNA strand breaks that impair fertilization, embryo development, and pregnancy outcomes. This review explains how OS promotes genomic instability and summarizes the main laboratory assays that assess redox status and SDF in semen. These include direct ROS chemiluminescence assay, oxidation–reduction potential, total antioxidant capacity/ferric reducing antioxidant power, and lipid peroxidation biomarkers, alongside SDF platforms (Sperm Chromatin Structure Assay, terminal deoxynucleotidyl transferase dUTP nick-end labeling, alkaline/neutral Comet, and sperm chromatin dispersion). Additionally, guideline-aligned indications are highlighted to clarify the conditions for testing OS and SDF. OS testing is most relevant in men with leukocytospermia or suspected genital tract infection or inflammation, including dysbiosis; in cases of major modifiable exposures such as smoking or heat; and for early monitoring after treatment. SDF testing is particularly informative in couples with recurrent pregnancy loss and in unexplained infertility with normal semen parameters. Combined OS and SDF testing is recommended in clinical varicocele, repeated in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) failure, poor embryo development, and follow-up after targeted therapy. Management centers on treating infection and inflammation, improving lifestyle and environmental factors, considering varicocelectomy when indicated, using targeted antioxidant therapy in men with documented OS, and selectively applying sperm selection technologies or testicular sperm for ICSI when SDF remains high. Priorities include assay standardization, etiologic attribution of DNA damage, and trials testing OS/SDF-guided pathways with live birth as the primary endpoint. When used selectively and in the appropriate context, OS and SDF testing can help refine diagnosis, improve counseling, and help personalize care of infertile couples. Full article

Assessing human embryo quality is a critical step in in vitro fertilization (IVF), yet traditional manual grading remains subjective and physically limited by the shallow depth-of-field in conventional microscopy. This study develops a novel “soft optical sensor” architecture that transforms standard optical microscopy into an automated, high-precision instrument for embryo quality assessment. The proposed system integrates two key computational innovations: (1) a multi-focal image fusion module that reconstructs lost morphological details from Z-stack focal planes, effectively creating a 3D-aware representation from 2D inputs; and (2) a retrieval-augmented generation (RAG) framework coupled with a Swin Transformer to provide both high-accuracy classification and explainable clinical rationales. Validated on a large-scale clinical dataset of 102,308 images (prior to augmentation), the system achieves a diagnostic accuracy of 94.11%. This performance surpasses standard single-plane analysis methods by 9.43%, demonstrating the critical importance of fusing multi-focal data. Furthermore, the RAG module successfully grounds model predictions in standard ESHRE consensus guidelines, generating natural language explanations. The results demonstrate that this soft sensor approach significantly reduces inter-observer variability and offers a robust tool for standardized morphological assessment, though prospective validation against live birth outcomes remains essential for clinical adoption. Full article

Background/Objectives: The attention of world science has been focused on human umbilical cord blood cell (hUCB) products for the treatment of various human diseases. The prospects for using hUCB stem from the availability of the material, non-invasive collection procedure, low immunogenicity, multipotency and non-tumorigenicity. But information about the acute toxicity, reproductive and developmental toxicity of hUCB mononuclear cells (MNCs) remains insufficient. Thus, the aim of this study is to assess the reproductive and developmental toxicity of human umbilical cord blood mononuclear cells on Wistar rats. Methods: In the fertility and early embryonic development study, human umbilical cord mononuclear blood cells (hUCB-MNCs) were administered at dose levels of 4.28 × 10⁸ cells/kg and 8.57 × 10⁸ cells/kg to male and female rats during the pre-mating, mating and gestation period. In the embryo–fetal development study, the pregnant female rats also received hUC-MNCs at doses of 4.28 × 10⁸ cells/kg and 8.57 × 10⁸ cells/kg. Results: In gestational data, including fertility rate, pregnancy rate, corpora lutea and implantation sites counts, dead and absorption fetuses’ number, body weight and craniocaudal size of fetuses, anomalies in fetal development showed no statistically significant changes in 4.28 × 10⁸ cells/kg (low dose) and 8.57 × 10⁸ cells/kg (high dose) dose groups of hUCB-MNCs to negative control group. External, visceral and skeletal examination of the fetuses in all experimental groups also showed no changes. Embryo–fetal development study in low and high groups of hUCB-MNCs application also showed no changes in the negative control group. Conclusions: This reproductive and developmental toxicity study demonstrates that human umbilical cord blood mononuclear cells (hUCB-MNCs) administered intravenously at doses up to 8.57 × 10⁸ cells/kg do not cause adverse effects on fertility, embryo–fetal development, or postnatal offspring viability in Wistar rats. The absence of reproductive toxicity is mechanistically attributable to three intrinsic properties of hUCB-MNCs: their low immunogenicity, which prevents maternal immune activation; the protective function of the intact placental barrier; and their transient, paracrine-dominant mode of action, which limits exposure duration. Full article

Background: Endometriosis is associated with infertility and impaired assisted reproductive technology (ART) outcomes, potentially due to an altered follicular microenvironment characterized by chronic inflammation. This study investigates the systemic and local inflammatory profiles in women with endometriosis and assesses their impact on oocyte and embryo quality using both static and dynamic embryo evaluation. Methods: A prospective, monocentric observational study enrolled 47 women undergoing controlled ovarian stimulation for ART, including 29 with laparoscopically confirmed endometriosis and 18 controls with tubal or male-factor infertility. Serum and follicular fluid cytokines (TGF-β1, NF-κB, IL-10, HIF-1α) were quantified. A sub-study analyzed embryo quality and development in 36 patients subdivided into static morphological assessment and dynamic time-lapse monitoring cohorts. Results: Endometriosis patients exhibited significantly elevated pro-inflammatory cytokines (TGF-β1, NF-κB) and reduced anti-inflammatory IL-10 in serum, alongside decreased NF-κB in follicular fluid. These alterations correlated with diminished ovarian reserve, reduced oocyte yield, and lower fertilization rates. Embryos from endometriosis patients showed increased multinucleation and persistent fragmentation, features more sensitively detected via dynamic time-lapse imaging. Clinical pregnancy rates were significantly lower in the endometriosis group. Conclusions: Endometriosis induces a dysregulated inflammatory follicular milieu that adversely affects oocyte competence and embryo morphodynamics. Dynamic embryo assessment provides enhanced detection of subtle developmental abnormalities. Integration of immunomodulatory strategies and advanced embryo monitoring may improve ART success in this population. Full article

Congenital valve defects account for a substantial proportion of cardiovascular malformations, yet the molecular mechanisms orchestrating cardiac valve development remain incompletely elucidated. While Bone morphogenetic protein (BMP) signaling is essential for valvulogenesis, the specific contributions of individual BMP ligands, particularly the teleost-specific bmp16, have not been characterized. Using the CRISPR/Cas9 system, we generated a bmp16 null knockout and delineated critical roles of this ligand in valvular morphogenesis. bmp16 knockout embryos display a significant reduction in Sox9-positive valvular cells and exhibit severely dysplastic arterial valves, characterized by increased interleaflet distance, thickened leaflets, and shortened leaflet lengths. These morphological abnormalities correlate with impaired valve function, culminating in progressive blood regurgitation, ventricular dilation, and pericardial edema. Mechanistically, loss of bmp16 or pharmacological inhibition of BMP signaling significantly downregulates notch1b expression in developing valves, while pharmacological activation of Notch signaling rescues the regurgitation phenotype in bmp16 mutants. Collectively, our findings establish bmp16 as a novel regulator of valve development and uncover a functional BMP-Notch signaling axis required for vertebrate valvulogenesis, providing new insights into the molecular mechanisms that govern cardiac valve formation and pathogenesis. Full article

Oxygen (O₂) tension is a critical factor influencing in vitro development of pre-implantation embryos. The in vivo environment has lower O₂ tension (2–10%) than atmospheric air (~20%), along the female reproductive tract, from the oviducts (8–10%) to the uterus (2–5%), supporting development of early-stage embryos. As the female reproductive tract is inherently hypoxic, replicating low-O₂ conditions in vitro may enhance embryo development. In contrast, culturing embryos under non-physiological O₂ tension may impair stress adaptation and reduce developmental competence. Optimal O₂ tension likely varies with species and embryo stage, suggesting a single uniform O₂ tension throughout in vitro culture may not be ideal; conditions beneficial at one stage may be detrimental at another. Although atmospheric O₂ harms embryo development and redox balance, specific advantages of low (5%) or ultra-low (≤2%) O₂ remain uncertain, despite many studies documenting improved development under hypoxia. This review examines the current literature on effects of atmospheric, low, and ultra-low O₂ tension during in vitro embryo culture, emphasizing impacts on in vitro fertilization (IVF) outcomes, and the regulation of transcription and epigenomics during pre-implantation embryo development. Full article

Receptive endometrium (RE) is essential for mammalian embryo implantation. The establishment of RE is a complex and precise dynamic process regulated by various cytokines, including non-coding RNAs (miRNAs, lncRNAs, and circRNAs). We identified candidate miR-26b-3p and circRNA3890 from our previous endometrial non-coding RNA sequencing data. CircRNA3890 adsorbs miR-26b-3p and inhibits its activity. Mouse double minute 4 (MDM4) is a target gene of miR-26b-3p, and circRNA3890 up-regulates the expression level of MDM4 by inhibiting the activity of miR-26b-3p in dairy goat endometrial epithelial cells (gEECs) in vitro. circRNA3890/miR-26b-3p/MDM4 could promote the proliferation of gEECs through the p53 signaling pathway. MiR-26b-3p could regulate the expression levels of vascular endothelial growth factor A (VEGFA) and leukemia inhibitory factor (LIF) through MDM4 in gEECs, which contributes to the development of endometrial receptivity. Furthermore, the results showed that miR-26b-3p significantly promoted the development of RE and embryo implantation. These findings demonstrate that circRNA3890 targets and adsorbs miR-26b-3p to relieve MDM4 inhibition and promotes EEC proliferation through the p53 signaling pathway. They reveal the regulatory effect of miR-26b-3p on receptive endometrial development and embryo implantation in vitro and in vivo. Full article

Background/Objectives: The physiological disorder known as “orange spot” in Juglans regia L. cv. Chandler is an emerging kernel quality issue of increasing concern for the Chilean walnut industry. Characterized by a localized orange discoloration of the embryo, its etiology remains unknown. This study aimed to determine whether orange spot is associated with bacterial infection and to elucidate the metabolic alterations underlying its development, testing the hypothesis that it results from oxidative imbalance and lipid disorganization. Methods: Untargeted metabolomic profiling and fatty acid methyl ester (FAME) analysis were applied to compare affected (orange) and unaffected (white) walnut kernels collected from a commercial orchard in Melipilla, Chile, during the 2018 and 2022 seasons. Bacterial DNA was screened by PCR amplification of the 16S rRNA gene (799F/1193R). Results: Orange spot incidence reached 31–34% across seasons. No bacterial-sized amplicons were detected in either kernel type, indicating the absence of detectable bacterial DNA under the analyzed conditions. Metabolomic analyses revealed significant alterations in glyoxylate and dicarboxylate metabolism, glutamate-related pathways, and glycerophospholipid metabolism. Affected kernels exhibited reduced levels of L-glutamic acid and antioxidant-associated metabolites, alongside increased gluconic, citric and quinic acid, consistent with the redox imbalance and membrane oxidative degradation. FAME analysis showed higher total fatty acid content in affected kernels, suggesting intensified lipid turnover associated under oxidative stress. Conclusions: Orange spot is a physiological, non-infectious disorder linked to premature oxidative stress and lipid disorganization, negatively affecting walnut kernel quality. Full article

Genetic engineering tools have the potential to rapidly and precisely improve the genome of slow-to-breed cacao. We previously developed an efficient protocol for transforming cacao using cotyledonary explants derived from secondary somatic embryos via Agrobacterium tumefaciens. In this study, we demonstrate that our transformation protocol is successful in elite cultivars, INIAPG-038 and Matina 1-6, producing fertile seeds with stable visual marker inheritance regardless of whether the transgenic plants were used as the pollen or ovule donor. Three vectors were used in the transformations, each containing genes for enhanced yellow fluorescent protein (eyfp) and neomycin phosphotransferase II (nptII). Three transgenic INIAPG-038 events and one transgenic Matina 1-6 event were used to evaluate seed fertility and the stability of transgene inheritance in cacao seeds and plants. The T₁ progeny of these four transgenic events were analyzed for YFP expression and transgene presence. YFP expression segregated at a 1:1 ratio in all events when the transgenic plants were crossed with non-transgenic plants, while a 3:1 segregation was observed when transgenic events were crossed with each other. The transgenic plants exhibited a normal phenotype compared to non-transgenic control plants, producing seeds with a 97% germination rate. Full article

Fermentation utilizing a combination of lactic acid bacteria (LAB) is known to enhance the nutritional value of wheat germ extract via the production of functional bioactive compounds. In this work, fermentation via compound LAB significantly enhanced the antioxidant activity of wheat germ extract. Compared to the unfermented group (CON), the in vitro antioxidant indices of Lactobacillus fermented wheat embryo extract were increased significantly: DPPH^·+/ABTS^·+ clearance (67.87 ± 3.48%/71.44 ± 5.90%), FRAP value (1.33 ± 0.02 μmol Trolox/10 mg), and active substance content including GSH (78.04 ± 1.43 μmol/g), total phenols (0.53 ± 0.01 mg GAE/10 mg), and total flavonoids (0.032 ± 0.01 mg/10 mg). Moreover, the antioxidant activity and substances of lactic acid bacteria-fermented wheat embryo extract were improved after gastrointestinal digestion compared with CON. In the erastin-induced Drosophila oxidative stress model, LFWGC intervention significantly improved behavioral performance (12.6 ± 3.78 s of tube climbing and 101.2 ± 7.98 jumps) and increased in vivo antioxidant levels: DPPH^·+·+ clearance by 31.75 ± 0.62%, ABTS^·+ clearance by 50.11 ± 0.82%, FRAP to 0.89 ± 0.03 μmol Trolox/10 mg, and GSH (116.30 ± 1.95 μmol/g), total phenols (0.117 ± 0.01 mg GAE/mg), and total flavonoids (0.027 ± 0.002 mg/g). Mechanistically, LFWGC enriched the intestinal flora with Levilactobacillus and Pseudomonas by restoring Tsf1 protein function, upregulating the expression of the TSF1 and GPX4 genes, and activating the pentose phosphate and a-lanine–aspartate-glutamate metabolic pathways, thereby synergistically enhancing the antioxidant defense system. LAB fermentation effectively enhanced the antioxidant capacity of wheat germ extract, providing a theoretical foundation for the development of functional foods. Full article

The proper regulation of signaling pathways, including WNT signaling, during early embryonic development is critical for whole-organism development. In particular, maternally enriched WNTs play critical roles in cell cleavage and axis formation through non-canonical and canonical pathways during early embryogenesis. However, early developmental processes related to maternal WNTs and their underlying mechanisms have remained unstudied in avian species. In this study, we investigated WNT signaling-mediated early development in the chicken embryo. We found that WNT4 and WNT6, different ligands from other species, exhibited expression patterns consistent with maternal enrichment in chicken. The chemical inhibition of maternal WNT signaling in intrauterine embryos led to aberrant zygotic expression of WNT8C, which is important for primitive streak formation. Combined with in vitro functional studies, we demonstrated that WNT4 increased WNT8C expression through the non-canonical JNK pathway and that WNT8C subsequently promoted the canonical β-catenin pathway. Our results indicate that maternal WNT4 activates zygotic WNT8C and potentially regulates embryonic polarity in chicken. Full article

Assisted reproductive technologies (ART) use has increased over the past decades. However, reports concerning ART’s low efficiency continue to emerge, citing causes related to lower embryo quality and pregnancy rates compared to their in vivo counterparts. One of the setbacks of ART is oxidative stress, which can impair embryo developmental rates. Mitochondrial redox and energetic homeostasis determine both cell survival and death, so mitochondria are a key target for therapeutic intervention strategies. In the present work, our objective was to improve the quality of viable embryos by adding new mitochondria-targeted antioxidants in the embryo culture media to reduce oxidative stress. Two naturally derived antioxidants synthesized by our team, AntiOxBEN₂ and AntiOxCIN₄, based on hydroxybenzoic and hydroxycinnamic scaffolds, respectively, were studied in two different experimental protocols (here called experiments). The first experiment investigated the effects of the antioxidants on embryo development to determine their optimal concentrations. The first assay of the first experiment focused on the effects of AntiOxCIN₄ at concentrations of 1, 2.5, and 10 μM, while the second assay focused on the effects of AntiOxBEN₂ at the same concentrations. A control group without supplementation was run simultaneously. The second experiment aimed to compare the best concentrations of these antioxidant molecules in the embryo culture media and their effect on embryos’ resistance to vitrification/warming. In each experiment, the embryos were morphologically evaluated, and the total and viable cell numbers were examined. Reactive oxygen species (ROS) and mitochondrial polarization were also evaluated using specific fluorescent dyes. In experiment 1, an increased embryo quality was identified by using 2.5 μM AntiOxCIN₄ (p = 0.03) and 2.5 μM AntiOxBEN₂ (p = 0.001). Moreover, blastocysts supplemented with 2.5 μM AntiOxCIN₄ had higher viability (p = 0.008), while those supplemented with 2.5 μM AntiOxBEN₂ presented a greater total cell number (p = 0.01). An improvement in embryo cryosurvival following the supplementation during the culture process with either antioxidant was identified in experiment 2, with superior expansion scores after vitrification/warming and culture (2.5 μM AntiOxCIN₄, p = 0.056 and 2.5 μM AntiOxBEN₂, p = 0.059). In conclusion, both AntiOxCIN₄ and AntiOxBEN₂ had a beneficial effect on embryo development and cryosurvival, suggesting a potential intervention to reduce oxidative stress in assisted reproductive technologies. Full article

Myostatin (MSTN) is a key negative regulator of skeletal muscle development, and its deficiency can markedly enhance muscle growth. However, the application of CRISPR/Cas12Mix in large livestock remains limited. In this study, 11 Xinjiang Brown calves were generated via embryo-stage CRISPR/Cas12Mix editing, among which five were confirmed as MSTN-knockout. These five edited calves were compared with five unedited controls to evaluate early growth traits and serum untargeted metabolomics at one month of age. MSTN-knockout calves exhibited significantly higher body weight, hip width, chest girth, and abdominal girth than controls (p < 0.05). Untargeted metabolomics identified 225 and 129 differential metabolites in positive and negative ion modes, mainly enriched in lipid, organic acid, and amino acid metabolism. KEGG pathway analysis revealed significant alterations in arginine and proline, tryptophan, and carbohydrate metabolism. These results demonstrate that CRISPR/Cas12Mix efficiently mediates MSTN knockout in cattle, conferring early growth advantages, accompanied by systemic metabolic reprogramming. Full article