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At least 19 recordsLinked to original sources
PubMed2025

Transcript-guided targeted cell enrichment for scalable single-nucleus RNA sequencing.

Large-scale single-cell atlases have revealed many aging- and disease-associated cell types, yet these populations are often underrepresented in heterogeneous tissues, limiting detailed molecular analyses. To address this, we developed EnrichSci-a scalable, microfluidics-free platform that combines hybridization chain reaction RNA fluorescence in situ hybridization (FISH) with combinatorial indexing to profile single-nucleus transcriptomes of target cell types with full gene-body coverage. Applied to oligodendrocytes in the aging mouse brain, EnrichSci uncovered aging-associated molecular dynamics across distinct oligodendrocyte subtypes, revealing both shared and subtype-specific gene expression changes. Additionally, we identified aging-associated exon-level signatures missed by conventional gene-level analyses, highlighting post-transcriptional regulation as a critical dimension of cell-state dynamics in aging. By coupling transcript-guided enrichment with a scalable sequencing workflow, EnrichSci provides a versatile approach to decode dynamic regulatory landscapes in diverse cell types from complex tissues.

Animals↗
PubMed2026

Spatially confined electrochemical strategy with DNA-assembled nanogaps for SNP detection.

Accurate detection of low-abundance single nucleotide polymorphisms (SNPs) against a large excess of homologous wild-type sequences requires both selective molecular recognition and effective transduction of small sequence differences into measurable signals. Here, we report a spatially confined electrochemical strategy that couples sequence-selective recognition with size-dependent mass-transport gating. DNA-hybridization-driven self-assembly of gold nanoparticles (AuNPs) forms a three-dimensional self-assembled electrode (3D-SAE) with a DNA-defined interparticle architecture. Competitive probes (SP/WP) convert single-base recognition into distinct molecular-size states: the SNP-associated pathway preferentially triggers a hybridization chain reaction (HCR), generating bulky AuNP-anchored HCR/methylene blue complexes (Au@HCR/MB) with reduced electrochemical accessibility through the porous 3D-SAE, whereas the wild-type pathway does not trigger HCR and maintains a high-current response from more readily accessible MB-containing species. Thus, sequence recognition is translated into a molecular-size difference and subsequently into an electrochemical signal through differential mass transport. Under buffer conditions, the platform achieved a statistically estimated detection limit of ∼0.47 fM and a quantitative range of 1 fM-100 pM. It discriminated a 0.1% mutant abundance in a fragmented genomic-DNA background. The downstream signal-transduction chemistry is enzyme-free and isothermal. This work establishes a mechanistical recognition-size-conversion-mass-transport-gating architecture for electrochemical nucleic acid analysis.

Polymorphism, Single Nucleotide↗
PubMed2025

Probing orthobunyavirus reassortment using Bunyamwera and Batai viruses as models.

Reassortment is a critical evolutionary mechanism for segmented viruses, enabling the exchange of intact genome segments during co-infection and driving orthobunyavirus evolution; however, the molecular mechanisms underpinning this process remain unclear. With over 100 orthobunyavirus species, many of which are significant human and veterinary pathogens, understanding how reassortment influences transmissibility and virulence is essential for preempting the emergence of novel pathogens. Here, we use Bunyamwera virus (BUNV) and Batai virus (BATV) as models to explore orthobunyavirus reassortment through reverse genetics. We established the first reverse genetics system for BATV, generated reassortants, and employed minigenome assays to assess replication machinery compatibility. Additionally, we developed a novel hybridization chain reaction assay for high-resolution visualization of viral RNA segments. Our findings revealed that all six reassortants between BUNV and BATV are viable, exhibiting notable phenotypic differences in interferon-deficient (IFNAR-/-) mice. This work introduces essential tools and new insights into orthobunyavirus reassortment and pathogenesis, laying the groundwork for understanding this critical evolutionary process.

Animals↗
PubMed1979

Mutual homology of mouse immunoglobulin gamma-chain gene sequences.

We have assessed the relative homology of mouse immunoglobulin heavy-chain gene sequence using complementary DNAs (cDNAs) synthesized against gamma-chain mRNAs (gamma 1, gamma 2a, gamma 2b, and gamma 3) purified from mouse myelomas. cDNAs complementary to the gamma-chain mRNAs did not cross-hybridize with the mu- and alpha-chain mRNAs, whereas they cross-hybridized to significant extents (22--66%) with the gamma-chain mRNAs of other subclasses. The heterologous hybrids formed, however, melt at 5--13 degrees C lower temperatures as compared to the homologous hybrids, indicating that significant portions of the heterologous hybrids are mismatched. The rates of the cross-hybridization reactions are 2- to 17-fold slower than those of the homologous hybridization reactions. Therefore, the gamma-chain gene sequences of four subclasses share a part of homology with each other, but they are different enough to be measured separately. Cross-hybridization analysis indicate that the gamma 2a and gamma 2b genes are the most closely related, while the gamma 1 and gamma 3 genes are the least related among the gamma subclass genes.

Animals↗
PubMed2025

Innovations in Transgene Integration Analysis: A Comprehensive Review of Enrichment and Sequencing Strategies in Biotechnology.

Understanding the integration of transgene DNA (T-DNA) in transgenic crops, animals, and clinical applications is paramount for ensuring the stability and expression of inserted genes, which directly influence desired traits and therapeutic outcomes. Analyzing T-DNA integration patterns is essential for identifying potential unintended effects and evaluating the safety and environmental implications of genetically modified organisms (GMOs). This knowledge is crucial for regulatory compliance and fostering public trust in biotechnology by demonstrating transparency in genetic modifications. This review highlights recent advancements in T-DNA integration analysis, specifically focusing on targeted DNA enrichment and sequencing strategies. We examine key technologies, such as polymerase chain reaction (PCR)-based methods, hybridization capture, RNA/DNA-guided endonuclease-mediated enrichment, and high-throughput resequencing, emphasizing their contributions to enhancing precision and efficiency in transgene integration analysis. We discuss the principles, applications, and recent developments in these techniques, underscoring their critical role in advancing biotechnological products. Additionally, we address the existing challenges and future directions in the field, offering a comprehensive overview of how innovative DNA-targeted enrichment and sequencing strategies are reshaping biotechnology and genomics.

Transgenes↗
PubMed2026

Scrape Cytology of DEK::AFF2 Fusion-Associated Papillary Squamous Cell Carcinoma of the Sinonasal Tract Masquerading as Schneiderian Papilloma: A Case Report.

INTRODUCTION: DEK::AFF2 fusion-associated papillary squamous cell carcinoma is a recently characterized sinonasal neoplasm that closely mimics Schneiderian papilloma. Although one report has described fine-needle aspiration cytology of a metastatic lymph node, scrape cytological features from the primary site remain undocumented. CASE PRESENTATION: We report the scrape cytology of this tumor in a 26-year-old woman. While certain features (perivascular arrangement and intracytoplasmic nuclear debris-like structures) overlapped with Schneiderian papilloma, several findings diverged: a predominantly discohesive pattern, nuclear enlargement with anisonucleosis, prominent nucleoli, stippled chromatin distinct from the neuroendocrine pattern, and a crackled cytoplasmic appearance. Immunohistochemistry for synaptophysin and chromogranin A was negative, excluding neuroendocrine differentiation. CD163 immunohistochemistry confirmed that the debris-containing cells were not histiocytes. E-cadherin showed heterogeneous downregulation in the initial biopsy, suggesting a candidate molecular basis for the discohesive pattern. The diagnosis was confirmed by AFF2 immunohistochemistry, DEK break-apart fluorescence in situ hybridization, and reverse transcription polymerase chain reaction with Sanger sequencing. Retrospective AFF2 immunohistochemistry of the initial biopsy, originally diagnosed as Schneiderian papilloma, was positive. The patient remained free of disease progression 34 months after completion of chemoradiotherapy for the antecedent lacrimal sac carcinoma. CONCLUSION: These findings provide the first comprehensive cytological documentation of this entity from a primary sinonasal site and delineate features diverging from Schneiderian papilloma that may prompt ancillary investigations.

Case report↗
PubMed2025

Novel photoreceptor-specific promoters for gene therapy in mid- to late-stage retinal degeneration.

Inherited retinal degenerations (IRDs) cause progressive photoreceptor loss, leading to vision impairment. Gene therapy using adeno-associated viral (AAV) vectors holds immense promise for treating these conditions. However, achieving optimal gene expression at mid to late stages of retinal degeneration remains challenging due to scarcity of efficient photoreceptor-specific promoters expressed at these disease stages. This study aimed to identify and validate novel promoters capable of robust and specific transgene expression when ≥50% of photoreceptors are lost. Analysis of transcriptomic data from two naturally occurring canine IRD models, laser capture microdissection of retinal cryosections followed by qPCR, and RNA in situ hybridization identified six promising genes with sustained or upregulated expression in photoreceptors in late-stage disease. Upstream cis-regulatory elements of both canine and human orthologs were identified and characterized using in silico analyses and dual-luciferase assays. Short promoters (≤840 base pairs) derived from GNGT2, IMPG2, and PDE6H genes exhibited robust reporter gene expression in photoreceptors when delivered via AAV to the subretinal space of two non-allelic canine IRD models at mid and late disease stages. These findings provide a strategy to enhance AAV-mediated gene therapy by enabling sustained transgene expression in degenerating retinas, improving treatment outcomes for patients with progressive vision loss.

Retinal Degeneration↗
PubMed2025

OsIDD6, an INDETERMINATE DOMAIN containing transcription factor in rice, plays an essential role in reproductive development.

INDETERMINATE DOMAIN containing proteins (IDD) are plant-specific transcriptional factors with a diverse range of roles in plants. Among the 15 IDD genes in rice, a staple food crop for the world, only about half have been functionally characterized. To elucidate the function of the remaining members, we created loss-of-function mutants using the CRISPR genome editing technique. Although no mutant exhibited obvious growth phenotypes, the Osidd6 mutant was completely sterile. By genetic crossing, we showed that both the male and female gametophytes were defective in the mutant. Histochemical staining and thin sectioning revealed that microspore development was compromised, likely due to a delay in tapetum degeneration. We also showed that meiosis was impaired in the mutant, resulting in defective megaspore development. Through a series of experiments, including transcriptome analysis, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), in situ hybridization, β-glucuronidase (GUS) staining with promoter-GUS transgenic plants, yeast one-hybrid method, a dual-visible reporter assay, and transcriptional activity assay, we demonstrated that OsIDD6 is expressed in all cell types in the male and female reproductive organs and that the OsIDD6 protein directly regulates genes potentially having a role in meiosis and tapetum development. Since reproductive development is directly related to crop yield, OsIDD6 could be an important target for genetic manipulation in rice breeding.

Oryza↗
PubMed2025

An Immunosenescent CD8+ T Cell Subset in Patients with Axial Spondyloarthritis and Psoriatic Arthritis Links Spontaneous Motility to Telomere Shortening and Dysfunction.

OBJECTIVE: A pathogenetic role of CD8+ T lymphocytes in radiographic axial spondyloarthritis (r-axSpA) and other spondyloarthritis (SpA) is sustained by genome-wide association studies and by the expansion of public T cell clonotypes in the target tissues. This study investigates the migration of CD8+ T cells along with their phenotype and functions in patients with r-axSpA and psoriatic arthritis (PsA). METHODS: Peripheral blood CD8+ and CD4+ T cells were isolated from patients with r-axSpA (n = 128), PsA (n = 60), and rheumatoid arthritis (RA) (n = 74) and healthy donors (HDs) (n = 79). Transwell migration assay was performed in the presence of different chemokines. CD8+ T cell immunoprofiling and effector functions were assessed by multiparametric flow cytometry. Transcriptome signature was evaluated by RNA sequencing analysis, whereas telomere length and dysfunction were measured by reverse transcriptase-polymerase chain reaction and immunofluorescence-fluorescence in situ hybridization, respectively. RESULTS: A significantly higher number of CD8+ T cells migrating in the absence of chemokine stimuli was found in patients with SpA compared with HDs and patients with RA. This subset, producing cytotoxic (granzyme B, perforin, granulysin) and proinflammatory molecules (tumor necrosis factor), was significantly enriched in terminally differentiated (CCR7-CD45RA+) and senescent (CD28-CD57+) cells having a gene expression profile characterized by cytolytic signature and natural killer markers. Remarkably, these spontaneously migrating CD8+ T cells showed DNA damage response activation, telomere shortening, and dysfunction. CONCLUSION: These data describe a terminally differentiated CD8+ T cell subset with a senescent and cytotoxic/proinflammatory profile and an intrinsic invasive potential enriched in patients with SpA that represents a possible player in disease pathogenesis.

Humans↗
PubMed2026

A Novel Long Noncoding RNA-LNC000133 Associated With Steroid-Induced Osteonecrosis of the Femoral Head Promotes Osteoblast Differentiation Through Bone Marrow Mesenchymal Stem Cells-Derived Exosomes Pathway: A Bioinformatics Validation and Detailed Mechanistic Study.

Steroid-induced osteonecrosis of the femoral head (SONFH) is a debilitating disease caused by glucocorticoid abuse, characterized by complex pathogenesis and unclear molecular mechanisms. Dysfunction of bone marrow mesenchymal stem cells (BMSCs) and their exosome-mediated signalling is a key contributor to SONFH, although the precise mechanisms remain to be elucidated. In this study, the differential expression profiles of long noncoding RNAs (lncRNAs), microRNAs (miRNAs) and messenger RNAs (mRNAs) in exosomes derived from human BMSCs (hBMSCs) obtained from patients with SONFH compared to controls with femoral neck fractures were identified. Through next-generation sequencing, a novel lncRNA, LNC000133, associated with SONFH was discovered. Using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and competing endogenous RNA (ceRNA) network construction, the LNC000133/miR-362-5p/TGF-β3/SMAD3/BMP2 signalling axis was established. The definitive expression, localization and full-length sequence of LNC000133 in BMSCs were subsequently validated by Northern blot, quantitative real-time polymerase chain reaction (qRT-PCR), fluorescence in situ hybridization (FISH) and rapid amplification of cDNA ends (RACE). Most notably, mechanistic studies demonstrated that LNC000133-modified BMSCs-derived exosomes were efficiently taken up by osteoblasts, which promoted proliferation and osteogenic differentiation by targeting the miR-362-5p/TGF-β3/SMAD3/BMP2 signalling pathway.

Humans↗
PubMed2026

In Situ Hybridization and RT-PCR Detection of Nervous Necrosis Virus in Fourfinger Threadfin, Eleutheronema tetradactylum, in Taiwan.

Between April and July 2020, suspected outbreaks of nervous necrosis virus (NNV) infection were observed in fourfinger threadfin (Eleutheronema tetradactylum) fingerling hatcheries in Pingtung County, southern Taiwan. Affected fish exhibited spiral swimming behaviour and abdominal distension, resulting in mortality rates between 50% and 100%. Histopathological examination showed severe vacuolation in the brain and ocular tissues, with large oval and/or irregular basophilic cytoplasmic inclusion bodies in the brain. Phylogenetic analysis of the viral replicase (RNA1) and capsid protein (RNA2) genes revealed high nucleotide sequence identities among the isolates in this study, with sequence similarity rates of 96.9%-99% for RNA1 and 98.2%-99.0% for RNA2 compared to RGNNV reference strains available in the NCBI GenBank database. This is the first detection of betanodavirus in fourfinger threadfin in Taiwan, using RT-PCR and ISH. A positive correlation between elevated water temperatures and disease severity indicates the need for year-round surveillance and genomic analysis to clarify the epidemiology of FTNNV. The data suggest that infected eggs may facilitate the vertical transmission of Betanodavirus. Crucially, utilising virus-free broodstock, alongside routine health screening and environmental control, is essential for mitigating NNV risks in fourfinger threadfin aquaculture.

Animals↗
PubMed1976

No detectable reiteration of genes coding for mouse MOPC 41 immunoglobulin light-chain mRNA.

RNA fractions rich in immunoglobulin light (L)-chain mRNA were isolated from mouse myeloma MOPC 41 by procedures previously described, and chemically labeled with 125I. These RNA fractions were hybridized with MOPC 41 DNA under conditions of DNA excess. Hybridization conditions were chosen under which the entire sequence of the L-chain mRNA probe, thus including the variable region, remains available for hybridization throughout the reaction. The hybridization (C0t) curve showed double transition kinetics, with one component corresponding to about 250 gene copies and the other to about two to four copies. In contrast, when MOPC 41 L-chain mRNA was further purified as a single band by gel elecptrophoresis in 99% formamide, the hybridization curve showed only a single transition, corresponding to about two to four genes, with the disappearance of the "reiterated" component. That component resulted therefore from contaminating RNA species. The data indicate that no reiteration can be detected by RNase or by hydroxylapatite for the genes corresponding to the entire sequence of MOPC 41 L-chain mRNA, including the untranslated segments, within the limits of detectability of short reiterated segments. It thus appears that there is only one or very few genes corresponding to the 41 L-chain variable region "subgroup" in MOPC 41 DNA. The possibility that the variable genes of plasmocytes might result frm a combination of several nonreiterated germline genes is discussed.

Alleles↗
PubMed2026

A Novel piRNA-Mediated Epigenetic Axis: piR-36241 Exacerbates Pulpitis by Silencing the Protective Receptor ADGRG2 in Human Dental Pulp Stem Cells.

BACKGROUND: Pulpitis, a prevalent inflammatory dental disease, primarily results from bacterial infection, yet its epigenetic regulatory mechanisms remain poorly understood. PIWI-interacting RNAs (piRNAs), a class of small non-coding RNAs known to maintain genomic stability, have not been investigated in the context of pulpitis. OBJECTIVE: This study aimed to profile piRNA expression patterns in pulpitis and to elucidate the functional role and underlying mechanism of a specific piRNA, piR-36241 and its target gene ADGRG2 in disease progression. METHODS: piRNA and mRNA sequencing were performed on pulp tissues from patients with irreversible pulpitis and healthy controls. Potential piRNA targets were predicted bioinformatically. The interaction between piR-36241 and the 3'UTR of ADGRG2, an orphan adhesion G protein-coupled receptor (GPCR), was validated using dual-luciferase reporter assay, quantitative Real-Time PCR (qRT-PCR), fluorescence in situ hybridization (FISH), Western blot (WB), hematoxylin-eosin staining (HE), immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA). Functional analyses were conducted in LPS-stimulated human dental pulp cells (HDPCs) through gain-of-function and rescue experiments. RESULTS: We identified a distinct piRNA expression profile in pulpitis, with 21 piRNAs differentially expressed. piR-36241 was notably upregulated. It directly targeted the 3'UTR of ADGRG2 and suppressed its expression. ADGRG2, hypothesised to exert protective and homeostatic functions, was downregulated in pulpitis tissues and LPS-induced HDPCs. Overexpression of piR-36241 inhibited ADGRG2 expression and promoted the secretion of pro-inflammatory cytokines (IL-6, IL-8). Conversely, knockdown of ADGRG2 similarly enhanced inflammatory responses. Rescue experiments demonstrated that piR-36241 primarily regulates inflammation through ADGRG2 silencing. CONCLUSION: Our study reveals a novel pathogenic axis in pulpitis whereby upregulation of piR-36241 exacerbates inflammatory progression by repressing the protective receptor ADGRG2. These findings provide the first evidence of piRNA-mediated epigenetic regulation in pulpitis and highlight the piR-36241/ADGRG2 pathway as a potential therapeutic target for preserving pulp vitality.

Humans↗
PubMed2012

Limitations of encapsidation of recombinant self-complementary adeno-associated viral genomes in different serotype capsids and their quantitation.

We previously reported that self-complementary adeno-associated virus (scAAV) type 2 genomes of up to 3.3 kb can be successfully encapsidated into AAV2 serotype capsids. Here we report that such oversized AAV2 genomes fail to undergo packaging in other AAV serotype capsids, such as AAV1, AAV3, AAV6, and AAV8, as determined by Southern blot analyses of the vector genomes, although hybridization signals on quantitative DNA slot-blots could still be obtained. Recently, it has been reported that quantitative real-time PCR assays may result in substantial differences in determining titers of scAAV vectors depending on the distance between the primer sets and the terminal hairpin structure in the scAAV genomes. We also observed that the vector titers determined by the standard DNA slot-blot assays were highly dependent on the specific probe being used, with probes hybridizing to the ends of viral genomes being significantly overrepresented compared with the probes hybridizing close to the middle of the viral genomes. These differences among various probes were not observed using Southern blot assays. This overestimation of titer is a systemic error during scAAV genome quantification, regardless of viral genome sequences and capsid serotypes. Furthermore, different serotypes capsid and modification of capsid sequence may affect the ability of packaging intact, full-length AAV genomes. Although the discrepancy is modest with wild-type serotype capsid and short viral genomes, the measured titer could be as much as fivefold different with capsid mutant vectors and large genomes. Thus, based on our data, we suggest that Southern blot analyses should be performed routinely to more accurately determine the titers of recombinant AAV vectors. At the very least, the use of probes/primers hybridizing close to the mutant inverted terminal repeat in scAAV genomes is recommended to avoid possible overestimation of vector titers.

Blotting, Southern↗
PubMed2026

Cooperation of transposable elements to endow global networks of initiators of hybrid assembly pathways of endogenous multiprotein complexes.

Mechanisms governing initiation steps of the assembly of endogenous multi-protein complexes (EMC) remain incompletely understood. Here, multiple lines of observations are reported describing the function-aligned initiation sequence of hybrid assembly pathways (HAP) of EMC. The first step of HAP-guided chain reactions of protein-protein interactions (PPI) of EMC assemblies constitutes the creation of cell type-specific pools of hetero and homo dimers. The molecular anatomy of HAP was elucidated by defining qualitative and quantitative characteristics of protein binding to a compendium of 200,393 distinct genomic regulatory elements (GRE), including 49,667 sequences representing control sets of genomic loci as well as 150,726 GRE of different evolutionary origins. The consensus sequence of HAP actions consists of: a) Initiation on genomic DNA of the formation of metastable hetero- and homodimers of EMCs' protein constituents; b) Release of dimers from DNA templates for delivery to the EMC assembly compartments; c) Assembly of defined EMC by sequential on demand addition of proteins to preformed dimers serving as attractors of EMC-specific ensembles of monomers. Chromosome-naïve DNA scaffolds facilitating creation of intracellular dimer pools engage networks of ~700 transcription factors (TFs), 534 of which manifest region-specific patterns of significantly enriched expression in 1358 brain regions. HAP initiators appear to operate within nucleosome-depleted islands of transposable elements (TE) - derived sequences within heterochromatin. PPI assembly lines of EMCs operate in 2 concurrent modes: TF-TF PPI cascade and PPI HUB protein cascade. Regardless of the number of DNA-bound initiator TFs (ranging from one to 716 TFs), both modes of operations reached the equilibrium at the PPI constituents saturation levels of ~245 proteins for TF-TF PPI modes and of ~351 proteins for PPI HUB protein modes. Distinct panels of DNA-bound initiator TFs and proteins of PPI cascade ensembles are enriched in either defined sets of neuroanatomical structures (TF-TF mode) or among structural-functional constituents of synapses (HUB proteins mode). Thus, these bifurcated cascades appear biologically congruent: TF-TF constituents map to transcriptional signatures of hundreds of brain regions, whereas HUB constituents map to synaptogenesis and synaptic structures, suggesting the unified logic of genomic functions coordinating region identity and connectivity. Evidence-supported examples of default operations of PPI-guided assemblies of hetero- and homodimers of Yamanaka factors, neurogenesis constituents, and protein components of postsynaptic density of excitatory and inhibitory synaptogenesis are reported with detailed analytical focus on human Claustrum. The foundational set of observations reported in this contribution should facilitate experimental and theoretical explorations of TE-seeded genomic codes for initiators of PPI chain reactions of protein dimerization creating pools of attractors to guide and accelerate the EMC assemblies.

Humans↗
PubMed1975

Analysis of cooperativity in hemoglobin. Valency hybrids, oxidation, and methemoglobin replacement reactions.

An allosteric model proposed previously for structure-function relations in hemoglobin is applied to the analysis of low- and high-spin valency hybrids. By assuming that the low-spin oxidized chains have the tertiary structure of oxygenated chains while the high-spin oxidized chains have a tertiary structure intermediate between that of deoxygenated and oxygenated chains, the model parameters associated with the different valency hybrids can be obtained, and their equilibrium properties can be estimated. The hybrid results are used also to provide an interpretation of methemoglobin and its ligand replacement reactions and of the oxidation-reduction equilibrium of normal hemoglobin. For the various systems studied it is found that the effects of pH and 2,3-diphosphoglycerate are in agreement with the model.

Allosteric Regulation↗
PubMed2026

Non-invasive embryo assessment: Cell-free DNA-based genetic testing and amino acid metabolomics in relation to morphology: A case-control study.

BACKGROUND: Cell-free DNA (cfDNA) in spent culture medium (SCM) offers a non-invasive option for preimplantation genetic testing, but its low concentration and fragmentation reduce clinical reliability. Combining genetic assessment with metabolomic profiling may provide complementary information about embryo competence. OBJECTIVE: This study assessed pre-analytical cfDNA processing workflows and examined whether SCM amino acid metabolic patterns could act as practical markers of embryo quality. MATERIALS AND METHODS: In this case-control study (2021-2023), 90 embryos were evaluated using fluorescence in situ hybridization or array comparative genomic hybridization. SCM samples underwent rapid boiling, silica-based purification, or whole-genome amplification (WGA). Sex determination was performed using quantitative polymerase chain reaction (qPCR). For cfDNA quality control and aneuploidy screening, the multiplex IRFiling kit and quantitative fluorescent polymerase chain reaction (QF-PCR) were used. Amino acid profiles across embryonic developmental stages and quality grades were quantified via liquid chromatography-tandem mass spectrometry. RESULTS: Rapid boiling resulted in complete failure of DNA amplification. Conversely, silica-based purification yielded 70.0% concordance for qPCR-based sexing and 56.7% for QF-PCR. WGA achieved the highest efficacy (73.3% qPCR and 56.7% QF-PCR concordance), although quality control checks flagged occasional misclassifications. LC-MS/MS profiling revealed significantly elevated alanine and arginine levels in tripronuclear embryos. Furthermore, high-quality blastocysts exhibited elevated glutamic acid levels alongside a pronounced overall depletion of extracellular amino acids compared to low-quality counterparts and controls. CONCLUSION: WGA improves cfDNA detectability and qPCR accuracy compared with boiling or purification, but remains inadequate as a standalone screening approach. SCM amino acid profiling provides informative, complementary metabolic signatures of developmental competence, supporting a multimodal strategy for non-invasive embryo assessment.

Amino acid metabolism↗