Application of a single-cell-RNA-based biological-inspired graph neural network in diagnosis of primary liver tumors.

Single-cell technology depicts integrated tumor profiles including both tumor cells and tumor microenvironments, which theoretically enables more robust diagnosis than traditional diagnostic standards based on only pathology. However, the inherent challenges of single-cell RNA sequencing (scRNA-seq) data, such as high dimensionality, low signal-to-noise ratio (SNR), sparse and non-Euclidean nature, pose significant obstacles for traditional diagnostic approaches. The diagnostic value of single-cell technology has been largely unexplored despite the potential advantages. Here, we present a graph neural network-based framework tailored for molecular diagnosis of primary liver tumors using scRNA-seq data. Our approach capitalizes on the biological plausibility inherent in the intercellular communication networks within tumor samples. By integrating pathway activation features within cell clusters and modeling unidirectional inter-cellular communication, we achieve robust discrimination between malignant tumors (including hepatocellular carcinoma, HCC, and intrahepatic cholangiocarcinoma, iCCA) and benign tumors (focal nodular hyperplasia, FNH) by scRNA data of all tissue cells and immunocytes only. The efficacy to distinguish iCCA from HCC was further validated on public datasets. Through extending the application of high-throughput scRNA-seq data into diagnosis approaches focusing on integrated tumor microenvironment profiles rather than a few tumor markers, this framework also sheds light on minimal-invasive diagnostic methods based on migrating/circulating immunocytes.

Liquid-liquid phase separation-related gene in gliomas: FABP5 is a potential prognostic marker.

BACKGROUND: The glioma is the most malignant human brain tumor. Early glioma detection and treatment are still difficult. New biomarkers are desperately required to aid in the evaluation of diagnosis and prognosis. METHODS: The single cell sequencing dataset scRNA-6148 for glioblastoma was obtained from the Chinese Glioma Genome Atlas database. Data were gathered for the transcriptome sequencing project. Genes involved in liquid-liquid phase separation (LLPS) were taken out of the DrLLPS database. To find the modules connected to LLPS, the weighted co-expression network was analyzed. Differential expression analysis was used to identify the differentially expressed genes (DEGs) in gliomas. Pseudo-time series analysis, gene set enrichment analysis (GSEA) and immune cell infiltration analysis were used to investigate the role of important genes in the immunological microenvironment. We examined the function of key glioma genes using polymerase chain reaction (PCR) testing, CCK-8 assays, clone generation assays, transwell assays and wound healing assays. RESULTS: FABP5 was identified as a key gene in glioblastoma by multiomics research. Pseudo-time series analysis showed that FABP5 was highly linked with the differentiation of many different types of cells. GSEA revealed that FABP5 was strongly linked to several hallmark pathways in glioblastoma. We looked at immune cell infiltration and discovered a significant link between FABP5, macrophages and T cell follicular helpers. The PCR experiment results demonstrated that FABP5 expression was elevated in glioma samples. Cell experiments showed that FABP5 knockdown dramatically decreased the viability, proliferation, invasion and migration of the LN229 and U87 glioma cell lines. CONCLUSIONS: Our study provides a new biomarker, FABP5, for glioma diagnosis and treatment.

FOSL1 promotes proneural-to-mesenchymal transition of glioblastoma stem cells via UBC9/CYLD/NF-κB axis.

Proneural (PN) to mesenchymal (MES) transition (PMT) is a crucial phenotypic shift in glioblastoma stem cells (GSCs). However, the mechanisms driving this process remain poorly understood. Here, we report that Fos-like antigen 1 (FOSL1), a component of AP1 transcription factor complexes, is a key player in regulating PMT. FOSL1 is predominantly expressed in the MES subtype, but not PN subtype, of GSCs. Knocking down FOSL1 expression in MES GSCs leads to the loss of MES features and tumor-initiating ability, whereas ectopic expression of FOSL1 in PN GSCs is able to induce PMT and maintain MES features. Moreover, FOSL1 facilitates ionizing radiation (IR)-induced PMT and radioresistance of PN GSCs. Inhibition of FOSL1 enhances the anti-tumor effects of IR by preventing IR-induced PMT. Mechanistically, we find that FOSL1 promotes UBC9-dependent CYLD SUMOylation, thereby inducing K63-linked polyubiquitination of major nuclear factor κB (NF-κB) intermediaries and subsequent NF-κB activation, which results in PMT induction in GSCs. Our study underscores the importance of FOSL1 in the regulation of PMT and suggests that therapeutic targeting of FOSL1 holds promise to attenuate molecular subtype switching in patients with glioblastomas.

Current applications and future perspective of CRISPR/Cas9 gene editing in cancer.

Clustered regularly interspaced short palindromic repeats (CRISPR) system provides adaptive immunity against plasmids and phages in prokaryotes. This system inspires the development of a powerful genome engineering tool, the CRISPR/CRISPR-associated nuclease 9 (CRISPR/Cas9) genome editing system. Due to its high efficiency and precision, the CRISPR/Cas9 technique has been employed to explore the functions of cancer-related genes, establish tumor-bearing animal models and probe drug targets, vastly increasing our understanding of cancer genomics. Here, we review current status of CRISPR/Cas9 gene editing technology in oncological research. We first explain the basic principles of CRISPR/Cas9 gene editing and introduce several new CRISPR-based gene editing modes. We next detail the rapid progress of CRISPR screening in revealing tumorigenesis, metastasis, and drug resistance mechanisms. In addition, we introduce CRISPR/Cas9 system delivery vectors and finally demonstrate the potential of CRISPR/Cas9 engineering to enhance the effect of adoptive T cell therapy (ACT) and reduce adverse reactions.

Circular RNA circMET drives immunosuppression and anti-PD1 therapy resistance in hepatocellular carcinoma via the miR-30-5p/snail/DPP4 axis.

BACKGROUND: Amplification of chromosome 7q21-7q31 is associated with tumor recurrence and multidrug resistance, and several genes in this region are powerful drivers of hepatocellular carcinoma (HCC). We aimed to investigate the key circular RNAs (circRNAs) in this region that regulate the initiation and development of HCC. METHODS: We used qRT-PCR to assess the expression of 43 putative circRNAs in this chromosomal region in human HCC and matched nontumor tissues. In addition, we used cultured HCC cells to modify circRNA expression and assessed the effects in several cell-based assays as well as gene expression analyses via RNA-seq. Modified cells were implanted into immunocompetent mice to assess the effects on tumor development. We performed additional experiments to determine the mechanism of action of these effects. RESULTS: circMET (hsa_circ_0082002) was overexpressed in HCC tumors, and circMET expression was associated with survival and recurrence in HCC patients. By modifying the expression of circMET in HCC cells in vitro, we found that circMET overexpression promoted HCC development by inducing an epithelial to mesenchymal transition and enhancing the immunosuppressive tumor microenvironment. Mechanistically, circMET induced this microenvironment through the miR-30-5p/Snail/ dipeptidyl peptidase 4(DPP4)/CXCL10 axis. In addition, the combination of the DPP4 inhibitor sitagliptin and anti-PD1 antibody improved antitumor immunity in immunocompetent mice. Clinically, HCC tissues from diabetic patients receiving sitagliptin showed higher CD8+ T cell infiltration than those from HCC patients with diabetes without sitagliptin treatment. CONCLUSIONS: circMET is an onco-circRNA that induces HCC development and immune tolerance via the Snail/DPP4/CXCL10 axis. Furthermore, sitagliptin may enhance the efficacy of anti-PD1 therapy in a subgroup of patients with HCC.

Cancer cell-derived exosomal circUHRF1 induces natural killer cell exhaustion and may cause resistance to anti-PD1 therapy in hepatocellular carcinoma.

OBJECTIVE: Natural killer (NK) cells play a critical role in the innate antitumor immune response. Recently, NK cell dysfunction has been verified in various malignant tumors, including hepatocellular carcinoma (HCC). However, the molecular biological mechanisms of NK cell dysfunction in human HCC are still obscure. METHODS: The expression of circular ubiquitin-like with PHD and ring finger domain 1 RNA (circUHRF1) in HCC tissues, exosomes, and cell lines was detected by qRT-PCR. Exosomes were isolated from the culture medium of HCC cells and plasma of HCC patients using an ultracentrifugation method and the ExoQuick Exosome Precipitation Solution kit and then characterized by transmission electronic microscopy, NanoSight and western blotting. The role of circUHRF1 in NK cell dysfunction was assessed by ELISA. In vivo circRNA precipitation, RNA immunoprecipitation, and luciferase reporter assays were performed to explore the molecular mechanisms of circUHRF1 in NK cells. In a retrospective study, the clinical characteristics and prognostic significance of circUHRF1 were determined in HCC tissues. RESULTS: Here, we report that the expression of circUHRF1 is higher in human HCC tissues than in matched adjacent nontumor tissues. Increased levels of circUHRF1 indicate poor clinical prognosis and NK cell dysfunction in patients with HCC. In HCC patient plasma, circUHRF1 is predominantly secreted by HCC cells in an exosomal manner, and circUHRF1 inhibits NK cell-derived IFN-γ and TNF-α secretion. A high level of plasma exosomal circUHRF1 is associated with a decreased NK cell proportion and decreased NK cell tumor infiltration. Moreover, circUHRF1 inhibits NK cell function by upregulating the expression of TIM-3 via degradation of miR-449c-5p. Finally, we show that circUHRF1 may drive resistance to anti-PD1 immunotherapy in HCC patients. CONCLUSIONS: Exosomal circUHRF1 is predominantly secreted by HCC cells and contributes to immunosuppression by inducing NK cell dysfunction in HCC. CircUHRF1 may drive resistance to anti-PD1 immunotherapy, providing a potential therapeutic strategy for patients with HCC.

The long noncoding RNA NORAD enhances the TGF-ß pathway to promote hepatocellular carcinoma progression by targeting miR-202-5p.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers with common features of invasion and metastasis. Recent evidence indicate that the long noncoding RNA NORAD is a potential oncogene and is significantly upregulated in several cancers. However, the general biological role and clinical value of NORAD in HCC remains unknown. Here, NORAD expression was measured in 29 paired tumor and paratumor tissues via quantitative real-time polymerase chain reaction (qPCR). The effects of NORAD on HCC cell malignant potential were investigated via NORAD overexpression and knockdown both in vitro and in vivo. The mechanism of competitive endogenous RNAs (ceRNAs) was acquired and identified by bioinformatics analyses and luciferase assays. Moreover, the impact of NORAD level on the transforming growth factor ß (TGF-ß) pathway was further determined by qPCR. We found that HCC tissues had a high level of NORAD compared with the paratumor tissues, and NORAD upregulation was associated with the shorter overall survival of patients with HCC. Furthermore, NORAD overexpression was demonstrated to promote HCC cell migration and invasion. Mechanically, NORAD might function as a ceRNA to regulate miR-202-5p, which served as a tumor-suppressing microRNA via the TGF-ß pathway. We address that NORAD has a tumor-promoting effect in HCC and describes a novel mechanism whereby NORAD regulates the TGF-ß pathway as a ceRNA of Homo sapiens (hsa)-miR-202-5p.

Circular RNA circTRIM33-12 acts as the sponge of MicroRNA-191 to suppress hepatocellular carcinoma progression.

BACKGROUND: Recently, the dysregulation of circular RNA (circRNA) have been shown to have important regulatory roles in cancer development and progression, including hepatocellular carcinoma (HCC). However, the roles of most circRNAs in HCC are still unknown. METHODS: The expression of circular tripartite motif containing 33-12 (circTRIM33-12) in HCC tissues and cell lines was detected by qRT-PCR. The role of circTRIM33-12 in HCC progression was assessed by western blotting, CCK-8, flow cytometry, transwell and a subcutaneous tumor mouse assays both in vitro and in vivo. In vivo circRNA precipitation, RNA immunoprecipitation, luciferase reporter assays were performed to evaluate the interaction between circTRIM33-12 and miR-191. RESULTS: Here, we found that circTRIM33-12, is downregulated in HCC tissues and cell lines. The downregulation of circTRIM33-12 in HCC was significantly correlated with malignant characteristics and served as an independent risk factor for the overall survival (OS) and recurrence-free survival (RFS) of patients with HCC after surgery. The reduced expression of circTRIM33-12 in HCC cells increases tumor proliferation, migration, invasion and immune evasion. Mechanistically, we demonstrated that circTRIM33-12 upregulated TET1 expression by sponging miR-191, resulting in significantly reduced 5-hydroxymethylcytosine (5hmC) levels in HCC cells. CONCLUSIONS: These results reveal the important role of circTRIM33-12 in the proliferation, migration, invasion and immune evasion abilities of HCC cells and provide a new perspective on circRNAs in HCC progression.

Lymphoid-specific helicase promotes the growth and invasion of hepatocellular carcinoma by transcriptional regulation of centromere protein F expression.

Lymphoid-specific helicase (LSH) is overexpressed in tumor tissues and its overexpression is associated with poor prognosis in several cancers. However, the role and molecular mechanism of LSH in hepatocellular carcinoma (HCC) remains largely unknown. Herein, we report that LSH was overexpressed in tumor tissues of HCC, and overexpression of LSH was associated with poor prognosis from a public HCC database, and validated by clinical samples from our department. Ectopic LSH expression promoted the growth of HCC cells in vivo and in vitro. Mechanistically, LSH overexpression promoted tumor growth by activating transcription of centromere protein F (CENPF). Clinically, overexpression of LSH and/or CENPF correlated with shorter overall survival and higher cumulative recurrence rates of HCC. In conclusion, LSH promotes tumor growth of HCC through transcriptional regulation of CENPF expression. Therefore, LSH may be a novel predictor for prognosis and a potential therapeutic target for HCC.

Identification of 3 subpopulations of tumor-infiltrating immune cells for malignant transformation of low-grade glioma.

BACKGROUND: Tumor-infiltrating immune cells (TIICs) are highly relevant to clinical outcome of glioma. However, previous studies cannot account for the diverse functions that make up the immune response in malignant transformation (MT) from low-grade glioma (LGG) to high-grade glioma (HGG). METHODS: Transcriptome level, genomic profiles and its relationship with clinical practice were obtained from TCGA and CGGA database. The "Cell type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT)" algorithm was used to estimate the fraction of 22 immune cell types. We divided the TCGA and CGGA set into an experiment set (n = 174) and a validation set (n = 74) by random number table method. Univariate and multivariate analyses were performed to evaluate the 22 TIICs' value for MT in LGG. ROC curve was plotted to calculate area under curve (AUC) and cut-off value. RESULTS: Heterogeneity between TIICs exists in both intra- and inter-groups. Several TIICs are notably associated with tumor grade, molecular subtypes and survival. T follicular helper (TFH) cells, activated NK Cells and M0 macrophages were screened out to be independent predictors for MT in LGG and formed an immune risk score (IRS) (AUC = 0.732, p < 0.001, 95% CI 0.657-0.808 cut-off value = 0.191). In addition, the IRS model was validated by validation group, Immunohistochemistry (IHC) and functional enrichment analyses. CONCLUSIONS: The proposed IRS model provides promising novel signatures for predicting MT from LGG to HGG and may bring a better design of glioma immunotherapy studies in years to come.

Cuproptosis: Advances in Stimulus-Responsive Nanomaterials for Cancer Therapy.

Cuproptosis, a recently identified non-apoptotic programmed cell death modality, attracts considerable attention in the realm of cancer therapeutics owing to its unique cellular demise mechanisms. Since its initial report in 2022, strategies inducing or amplifying cuproptosis for cancer treatment emerge. The engineering of nano-systems to elicit cuproptosis effectively circumvents constraints associated with conventional small-molecule pharmaceutical interventions, presenting novel prospects for oncological therapy. Stimulus-responsive nanomaterials, leveraging their distinctive spatiotemporal control attributes, are investigated for their role in modulating the induction or augmentation of cuproptosis. In this comprehensive review, the physiological characteristics of cuproptosis, encompassing facets such as copper overload and depletion, coupled with regulatory factors intrinsic to cuproptosis, are expounded upon. Subsequently, design methodologies for stimulus-responsive induction or enhancement of cuproptosis, employing stimuli such as light, ultrasound, X-ray, and the tumor microenvironment, are systematically delineated. This review encompasses intricacies in nanomaterial design, insights into the therapeutic processes, and the associated advantages. Finally, challenges inherent in stimulus-responsive induction/enhancement of cuproptosis are deliberated upon and prospective insights into the future trajectory of copper-mediated cancer therapy are provided.

Recreational gymnastics exercise of moderate intensity enhances executive function in Chinese preschoolers: A randomized controlled trial.

The current study aimed to investigate the impact of recreational gymnastics on executive function in Chinese preschoolers, with a focus on gymnastics potential to enhance core components of executive function. A total of 63 preschool children who received full-time education were randomly assigned to either an intervention group (N = 31, mean age = 66.27 months, SD = 3.12 months) or a control group (N = 32, mean age = 66.79 months, SD = 3.34 months). The intervention group engaged in recreational gymnastics for 60 min, three times a week for 12 weeks. Meanwhile, the control group continued with their typical outdoor activities at kindergarten and did not participate in any organized sports. The intervention program was primarily conducted through group play and was facilitated by teachers who underwent standardized training. Various simple and complex tasks were utilized to evaluate delay gratification (Snack delay and Wrapped gift), inhibitory control (Stop signal task and Circle drawing task), working memory (Letter memory task and Keep track task), and cognitive flexibility (Go/No-Go task and Dots task). The analysis of covariance revealed that the children who participated in the intervention outperformed the control group on most simple and complex executive function tasks. Specifically, these children demonstrated an enhanced ability to regulate persistent responses, process and update information, and manage high cognitive conflict. The findings of this investigation lend support to the hypothesis that moderate-intensity recreational gymnastics is an efficacious means of enhancing executive function in early childhood. Future research should employ a larger sample size, incorporate a long-term follow-up design, and utilize a multi-method approach to further substantiate the impact of moderate-intensity gymnastics on the executive function of young children, as well as to investigate its underlying mechanism and generalizability.

A Low Mismatch Current Charge Pump Applied to Phase-Locked Loops.

This paper presents a charge pump circuit with a wide output range and low current mismatch applied to phase-locked loops. In this designed structure, T-shaped analog switches are adopted to suppress the non-ideal effects of clock feedthrough, switching time mismatch, and charge injection. A source follower and current splitting circuits are proposed to improve the matching accuracy of the charging and discharging currents and reduce the current mismatch rate. A rail-to-rail high-gain amplifier with a negative feedback connection is introduced to suppress the charge-sharing effect of the charge pump. A cascode current mirror with a high output impedance is used to provide the charge and discharge currents for the charge pump, which not only improves the current accuracy of the charge pump but also increases the output voltage range. The proposed charge pump is designed and simulated based on a 65 nm CMOS process. The results show that when the power supply voltage is 1.2 V, the output current of the charge pump is 100 µA, the output voltage is in the range of 0.2~1 V, and the maximum current mismatch rate and current variation rate are only 0.21% and 1.4%, respectively.

Engineering the nanozyme hydrogel beads by polyvinyl alcohol/chitosan encapsulation with recyclable and sustainable catalytic activity for visual analysis of hydrogen peroxide.

BACKGROUND: Hydrogen peroxide (H2O2) plays a vital role in human health and have been regarded as a crucial analyte in metabolic processes, redox transformations, foods research and medical fields. Especially, the long-time and excessive digestion of H2O2 may even cause severe diseases. Although conventional instrumental methods and nanozymes-based colorimetric methods have been developed to accomplish the quantitative analysis of H2O2, the drawbacks of instrument dependence, cost-effectiveness, short lifespan, non-portable and unsustainable detection efficacies will limit their applications in different detection scenarios. RESULTS: Herein, to address these challenges, we have proposed a novel strategy for nanozyme (RuO2) hydrogel preparation by the solid support from cross-linked polyvinyl alcohol (PVA) and chitosan (CS) to both inherit the dominant peroxidase-like (POD) activity and protect the RuO2 from losing efficacies. Taking advantages from the hydrogel, the encapsulated RuO2 were further prepared as the regularly spherical beads (PCRO) to exhibit the sustainable, recyclable, and robust catalysis. Moreover, the intrinsic color interferences which originated from RuO2 can be avoided by the encapsulation strategy to promote the detection accuracy. Meanwhile, the high mechanical strength of PCRO shows the high stability, reproducibility, and cyclic catalysis to achieve the recyclable detection performance and long lifetime storage (40 days), which enables the sensitively detection of H2O2 with the detection limit as lower to 15 µM and the wide detection linear range from 0.025 to 1.0 mM. SIGNIFICANCE: On the basis of the unique properties, PCRO has been further adopted to construct a smartphone detection platform to realize the instrument-free and visual analysis of H2O2 in multi-types of milk and real water samples through capturing, processing, and analyzing the RGB values from the colorimetric photographs. Therefore, PCRO with the advanced detection efficacies holds the great potential in achieving the portable and on-site analysis of targets-of-interest.

Deubiquitinase PSMD14 promotes tumorigenicity of glioblastoma by deubiquitinating and stabilizing ß-catenin.

The deubiquitinating enzyme 26S proteasome non-ATPase regulatory subunit 14 (PSMD14), a member of the JAB1/MPN/Mov34 metalloenzyme (JAMM) family, has been shown to function as an oncogene in various human cancers. However, the function of PSMD14 in glioma and the underlying mechanism remain unclear. In this study, our findings reveal a dramatic upregulation of PSMD14 in GBMs, which is associated with poor survival outcomes. Knocking down PSMD14 is associated with decreased proliferation and invasion of GBM cells in vitro and inhibited tumor growth in a xenograft mouse model. Mechanistically, PSMD14 directly interacts with ß-catenin, leading to a decrease in the K48-linked ubiquitination of ß-catenin and subsequent ß-catenin stabilization. Increased ß-catenin expression significantly reverses the inhibitory effects of PSMD14 knockdown on the migration, invasion, and tumor growth of GBM cells. Moreover, we observed a significant correlation between PSMD14 and ß-catenin expression in human GBM samples. In summary, our results reveal that PSMD14 is a crucial deubiquitinase that is responsible for stabilizing the ß-catenin protein, highlighting its potential for use as a therapeutic target for GBM.

Topochemical-like bandgap regulation engineering: A bismuth thiooxide nanocatalyst for breast cancer phototherapy.

The physical property tuning of nanomaterials is of great importance in energy, medicine, environment, catalysis, and other fields. Topochemical synthesis of nanomaterials can achieve precise control of material properties. Here, we synthesized a kind of element-doped bismuth-based nanomaterial (BOS) by topochemical-like synthesis and used it for the phototherapy of tumors. In this study, we employed bismuth fluoride nanoflowers as a template and fabricated element-doped bismuth oxide nanoflowers by reduction conditions. The product is consistent with the precursor in crystal structure and nanomorphology, realizing topochemical-like synthesis under mild conditions. BOS can generate reactive oxygen species, consume glutathione, and perform photothermal conversion under 730 nm light irradiation. In vitro and in vivo studies demonstrate that BOS could suppress tumor growth by inducing apoptosis and ferroptosis through phototherapy. Therefore, this study offers a general regulation method for tuning the physical properties of nanomaterials by using a topochemical-like synthesis strategy.

High-Z elements dominated bismuth-based heterojunction nano-semiconductor for radiotherapy-enhanced sonodynamic breast cancer therapy.

Ultrasound and X-rays possess remarkable tissue penetration capabilities, making them promising candidates for cancer therapy. Sonodynamic therapy, which utilizes ultrasound excitation, offers a safer alternative to radiotherapy and can be combined with X-rays to mitigate the adverse effects on normal tissues. In this study, we developed a bismuth-based heterostructure semiconductor (BFIP) to enhance the efficacy of radiotherapy and sonodynamic therapy in treating breast cancer. The semiconductor is fabricated through a two-step process involving the synthesis of porous spherical bismuth fluoride and partially reduced to bismuth oxyiodide. Then, followed by surface modification with amphiphilic polyethylene glycol, BFIP is fabricated. Incorporating heavy atoms in the BFIP enhances radiosensitivity. The BFIP exhibits superior carrier separation efficiency compared to bismuth fluoride, generating a substantial quantity of reactive oxygen species upon ultrasound stimulation. Moreover, the BFIP effectively depletes glutathione through coordination and hole-mediated oxidation pathways, disrupting the tumor microenvironment and inducing oxidative stress. Encouraging results are acquired in both in vitro cell and in vivo tumor models. Our study provides a de-risking strategy by utilizing ultrasound as a partial substitute for X-rays in treating deep-seated tumors, offering a viable research direction for constructing a unified nanoplatform.

A novel approach to detecting microduplication in split hand/foot malformation type 3 at the single-cell level: SHFM as a case study.

BACKGROUND: Split hand/foot malformation (SHFM) is a congenital limb deficiency characterized by missing or shortened central digits. Several gene loci have been associated with SHFM. Identifying microduplications at the single-cell level is challenging in clinical practice, and traditional detection methods may lead to misdiagnoses in embryos and pregnant women. RESULTS: In this research, we utilized a low cell count and whole-genome amplification products to employ single nucleotide polymorphism arrays, next-generation sequencing, and third-generation sequencing methods to detect copy number variants of microduplications in a SHFM3 case with limited DNA. Additionally, Karyomapping and combined linkage analysis were conducted to validate the results. CONCLUSIONS: This study establishes a new strategy for identifying microduplications or microdeletions at the single-cell level in clinical preimplantation genetic testing, enhancing the efficiency and accuracy of diagnosing microduplication or microdeletion diseases during IVF-PGT and prenatal diagnosis.