Cost-Effective Whole Transcriptome Sequencing Landscape and Diagnostic Potential Biomarkers in Active Tuberculosis.

Tuberculosis (TB) is a prevalent and severe infectious disease that poses a significant threat to human health. However, it is frequently disregarded as there are not enough quick and accurate ways to diagnose tuberculosis. Here, we develop a strategy for tuberculosis detection to address the challenges, including an experimental strategy, namely, Double Adapter Directional Capture sequencing (DADCSeq), an easily operated and low-cost whole transcriptome sequencing method, and a computational method to identify hub differentially expressed genes as well as the diagnosis of TB based on whole transcriptome data using DADCSeq on peripheral blood mononuclear cells (PBMCs) from active TB and latent TB or healthy control. Applying our approach to create a robust and stable TB multi-mRNA risk probability model (TBMMRP) that can accurately distinguish active and latent TB patients, including active TB and healthy controls in clinical cohorts, this diagnostic biomarker was successfully validated by several independent cross-platform cohorts with favorable performance in differentiating active TB from latent TB or active TB from healthy controls and further demonstrated superior or similar diagnostic accuracy compared to previous diagnostic markers. Overall, we develop a low-cost and effective strategy for tuberculosis diagnosis; as the clinical cohort increases, we can expand to different disease kinds and learn new features through our disease diagnosis strategy.

Identification of extracellular matrix-related biomarkers in colon adenocarcinoma by bioinformatics and experimental validation.

Backgrounds: Extracellular matrix (ECM) is an important component of tumor microenvironment, and its abnormal expression promotes tumor formation, progression and metastasis. Methods: Weighted gene co-expression network analysis (WGCNA) was used to identify ECM-related hub genes based on The Cancer Genome Atlas (TCGA) colon adenocarcinoma (COAD) data. COAD clinical samples were used to verify the expression of potential biomarkers in tumor tissues, and siRNA was used to explore the role of potential biomarkers in cell proliferation and epithelial-mesenchymal transition (EMT). Results: Three potential biomarkers (LEP, NGF and PCOLCE2) related to prognosis of COAD patients were identified and used to construct ERGPI. Immunohistochemical analysis of clinical samples showed that the three potential biomarkers were highly expressed in tumor tissues of COAD patients. Knockdown of LEP, NGF or PCOLCE2 inhibited COAD cell proliferation and EMT. Dictamnine inhibited tumor cell growth by binding to these three potential biomarkers based on molecular docking and transplanted tumor model. Conclusion: The three biomarkers can provide new ideas for the diagnosis and targeted therapy of COAD patients.

A Biomimetic Fibrous Composite Scaffold with Nanotopography-Regulated Mineralization for Bone Defect Repair.

The effective regeneration of large bone defects via bone tissue engineering is challenging due to the difficulty in creating an osteogenic microenvironment. Inspired by the fibrillar architecture of the natural extracellular matrix, we developed a nanoscale bioengineering strategy to produce bone fibril-like composite scaffolds with enhanced osteogenic capability. To activate the surface for biofunctionalization, self-adaptive ridge-like nanolamellae were constructed on poly(ε-caprolactone) (PCL) electrospinning scaffolds via surface-directed epitaxial crystallization. This unique nanotopography with a markedly increased specific surface area offered abundant nucleation sites for Ca2+ recruitment, leading to a 5-fold greater deposition weight of hydroxyapatite than that of the pristine PCL scaffold under stimulated physiological conditions. Bone marrow mesenchymal stem cells (BMSCs) cultured on bone fibril-like scaffolds exhibited enhanced adhesion, proliferation, and osteogenic differentiation in vitro. In a rat calvarial defect model, the bone fibril-like scaffold significantly accelerated bone regeneration, as evidenced by micro-CT, histological histological and immunofluorescence staining. This work provides the way for recapitulating the osteogenic microenvironment in tissue-engineered scaffolds for bone repair.

Heterologous synthesis of ginsenoside F1 and its precursors in Nicotiana benthamiana.

Ginsenoside F1 has high medicinal values, which is a kind of rare triterpene saponin isolated from Panax plants. The extremely low content of ginsenoside F1 in herbs has limited its research and application in medical field. In this work, we constructed a pathway in tobacco for the biosynthesis of ginsenoside F1 by metabolic engineering. Four enzyme genes (PnDDS, CYP716A47, CYP716S1 and UGT71A56) isolated from Panax notoginseng were introduced into tobacco. Thus, a biosynthetic pathway for ginsenoside F1 synthesis was artificially constructed in tobacco cells; moreover, the four exogenous genes could be expressed in the roots, stems and leaves of transgenic plants. Consequently, ginsenoside F1 and its precursors were successfully synthesized in the transgenic tobacco, compared with Panax plants, the content of ginsenoside F1 in transgenic tobacco was doubled. In addition, accumulation of ginsenoside F1 and its precursors in transgenic tobacco shows organ specificity. Based on these results, a new approach was established to produce rare ginsenoside F1; meanwhile, such strategy could also be employed in plant hosts for the heterologous synthesis of other important or rare natural products.

Engineering of a compact, high-fidelity EbCas12a variant that can be packaged with its crRNA into an all-in-one AAV vector delivery system.

The CRISPR-associated endonuclease Cas12a has become a powerful genome-editing tool in biomedical research due to its ease of use and low off-targeting. However, the size of Cas12a severely limits clinical applications such as adeno-associated virus (AAV)-based gene therapy. Here, we characterized a novel compact Cas12a ortholog, termed EbCas12a, from the metagenome-assembled genome of a currently unclassified Erysipelotrichia. It has the PAM sequence of 5'-TTTV-3' (V = A, G, C) and the smallest size of approximately 3.47 kb among the Cas12a orthologs reported so far. In addition, enhanced EbCas12a (enEbCas12a) was also designed to have comparable editing efficiency with higher specificity to AsCas12a and LbCas12a in mammalian cells at multiple target sites. Based on the compact enEbCas12a, an all-in-one AAV delivery system with crRNA for Cas12a was developed for both in vitro and in vivo applications. Overall, the novel smallest high-fidelity enEbCas12a, this first case of the all-in-one AAV delivery for Cas12a could greatly boost future gene therapy and scientific research.

Regulatory emotional self-efficacy and self-compassion mediate anxiety, depression, body image distress and subjective well-being in women with polycystic ovary syndrome: A cross-sectional study.

AIMS: This study aims to investigate the mediating role of regulatory emotional self-efficacy and self-compassion in the relationship among anxiety, depression, body image distress and subjective well-being among women with polycystic ovary syndrome. DESIGN: A cross-sectional study. METHODS: The study recruited 510 women with polycystic ovary syndrome from a tertiary hospital affiliated with a university in Hunan Province, China. The study employed several tools to collect data, including the Generalized Anxiety Scale, the Patient Health Questionnaire-9, the Body Image States Scale, the Self-Compassion Scale, the Regulatory Emotional Self-Efficacy Scale and the Index of Well-being questionnaire. Data analysis was carried out using descriptive analysis, spearman correlation analysis, ordinary least squares regression and bootstrapping. RESULTS: The study's findings indicate that regulatory emotional self-efficacy and self-compassion both act as mediators in the connection between anxiety, depression, body image distress and subjective well-being among women with polycystic ovary syndrome. CONCLUSION: The study emphasizes the significance of regulatory emotional self-efficacy and self-compassion in promoting well-being among women with polycystic ovary syndrome. It also implies that interventions targeted at enhancing these factors could potentially enhance the subjective well-being of women affected by PCOS. IMPACT: Our study's primary contribution is to underscore the crucial mediating roles of regulatory emotional self-efficacy and self-compassion in the relationship among anxiety, depression, body image distress and subjective well-being. Our study indicates that clinical practitioners should prioritize improving the regulatory emotional self-efficacy and self-compassion of women with polycystic ovary syndrome, reducing their anxiety, depression and body image distress and improving their subjective well-being. REPORTING METHOD: This study was reported according to the Standards for Reporting Qualitative Research (SRQR). PATIENT OR PUBLIC CONTRIBUTION: No patient or public contribution outside of participation in the actual study for purposes of data collection.

Assessment of Left Ventricular Longitudinal Systolic Function Using Global Tissue Motion Annular Displacement in Patients with Chronic Severe Aortic Regurgitation.

Background: In patients with chronic aortic regurgitation (AR), the left ventricle (LV) develops compensatory mechanisms to sustain its function. LV global longitudinal strain (GLS) is a key means to detect subclinical LV dysfunction, even when LV ejection fraction (LVEF) remains within the normal range. Compared to GLS, Tissue motion annular displacement (TMAD) is a simpler strain-based method to assess LV systolic function. This study investigated the correlation among TMAD parameters, LVEF, and GLS, and determined the diagnostic value and threshold of TMAD parameters for left ventricular systolic dysfunction. Methods: A prospective study was conducted at a single center. The case and control groups consisted of patients with chronic severe AR and healthy volunteers, respectively. Speckle-tracking echocardiography (STE) was used to assess the GLS and TMAD parameters in the apical 4-chamber and apical 2-chamber. Subsets of participants were analyzed for inter- and intra-observer variability and analysis time. A correlation analysis was performed among the TMAD parameters, LVEF, and GLS. Receiver operating characteristic curves and the area under the curves (AUCs) were used to evaluate the predictive value of the TMAD parameters for LVEF <50% and GLS > -18%. Results: This study involved 96 patients with severe chronic AR and 45 healthy volunteers. Compared to GLS, TMAD demonstrated superior intra- and inter-observer consistency and shorter average analysis time. Biplane global Midpt% showed the highest correlation with GLS and LVEF among all the TMAD parameters, with r values of 0.81 and 0.74, respectively. Furthermore, global Midpt% had AUCs of 0.89 and 0.92 for predicting LVEF< 50% and GLS > -18%, respectively. Conclusion: The TMAD global Midpt% has the potential to replace GLS in clinical practice and find wide applications.

Engineering of Cas12a nuclease variants with enhanced genome-editing specificity.

The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.

Stigma and its influencing factors for seeking professional psychological help among pregnant women: A cross-sectional study.

BACKGROUND: Although the stigma associated with seeking professional psychological help has received significant attention, few studies have focused on the status and factors influencing this stigma among pregnant women, especially those with a Chinese cultural background. This study aimed to assess the status of stigma associated with seeking professional psychological help and its influencing factors. METHODS: A total of 1325 pregnant women were recruited from Hunan Province, China. The Stigma for Seeking Professional Psychological Help questionnaire, Patient Health Questionnaire-9, Perceived Social Support Scale, Simplified Coping Style Questionnaire, and questionnaires on sociodemographic characteristics and pregnancy-related information were used. Descriptive, univariate, correlation, and multivariate linear regression analyses were used to investigate the status of stigma and identify its influencing factors. FINDINGS: Pregnant Chinese women experienced a mild to moderate level of stigma associated with seeking professional psychological help, and self-stigma was more common than social stigma associated with seeking psychological help. The results of the multivariate analysis suggested that perceived social support, positive coping styles, negative coping styles, and depressive symptoms were the factors influencing self-stigma and social stigma associated with seeking professional psychological help. Advanced age and a low educational level were the only predictors associated with self-stigma, while a history of multiple pregnancies had an effect only on social stigma associated with seeking professional psychological help. CONCLUSION: Chinese pregnant women experienced a mild to moderate level of stigma associated with seeking professional psychological help. Destigmatizing interventions focusing on self-stigma and social stigma should be designed according to maternal characteristics, social support, coping style, and depressive symptoms.

Rheological properties of warm mixed high viscosity asphalt at high and low temperatures.

The rheological properties of asphalt can well reflect its road performance, but the rheological properties of warm mix high viscosity asphalt (HVA) are unclear. In order to study the effect of warm mixing agent on rheological properties of HVA, two kinds of warm mixing agent EC120 (EC) and Evotherm M1 (M1) were selected to prepare warm mix HVA. The rheological properties of warm mix HVA at high temperature (135~195°C), medium temperature (0~80°C) and low temperature (-6~18°C) were studied by Brinell rotary viscosity test, dynamic shear rheological test (including temperature scanning, frequency scanning, linear amplitude scanning) and bending beam rheological test. The test results show that both EC and M1 have good viscosity reduction effect on HVA at high temperature, and can effectively reduce the construction temperature. At medium temperature, M1 can effectively improve the fatigue resistance of HVA, and the fatigue life can be increased by about 30% when the dosage is 0.6%. EC can increase the rutting factor of HVA and improve its resistance to deformation, but it will reduce its fatigue performance. When the dosage is 4%, the fatigue life will be reduced by about 9%. At low temperature, M1 can reduce the creep stiffness S, increase the creep rate m, and improve the low temperature performance of HVA, while EC has the opposite effect, weakening the low temperature performance of HVA. The results are helpful to understand the rheological properties of warm mix HVA and promote its application.

Fetal growth restriction exhibits various mTOR signaling in different regions of mouse placentas with altered lipid metabolism.

Fetal growth restriction (FGR) is a common complication of pregnancy and can have significant impact on obstetric and neonatal outcomes. Increasing evidence has shown that the inhibited mechanistic target of rapamycin (mTOR) signaling in placenta is associated with FGR. However, interpretation of existing research is limited due to inconsistent methodologies and varying understanding of the mechanism by which mTOR activity contributes to FGR. Hereby, we have demonstrated that different anatomic regions of human and mouse placentas exhibited different levels of mTOR activity in normal compared to FGR pregnancies. When using the rapamycin-induced FGR mouse model, we found that placentas of FGR pregnancies exhibited abnormal morphological changes and reduced mTOR activity in the decidual-junctional layer. Using transcriptomics and lipidomics, we revealed that lipid and energy metabolism was significantly disrupted in the placentas of FGR mice. Finally, we demonstrated that maternal physical exercise during gestation in our FGR mouse model was associated with increased fetal and placental weight as well as increased placental mTOR activity and lipid metabolism. Collectively, our data indicate that the inhibited placental mTOR signaling contributes to FGR with altered lipid metabolism in mouse placentas, and maternal exercise could be an effective method to reduce the occurrence of FGR or alleviate the adverse outcomes associated with FGR.

Targeting myeloperoxidase to stabilize unruptured aneurysm: an imaging-guided approach.

Inflammation plays a key role in pathogenesis and rupture of aneurysms. Non-invasively and dynamically monitoring aneurysm inflammation is critical. This study evaluated myeloperoxidase (MPO) as an imaging biomarker and therapeutic target for aneurysm inflammation using an elastase-induced rabbit model treated with or without 4-aminobenzoic acid hydrazide (ABAH), an irreversible inhibitor of MPO. Myeloperoxidase-sensitive magnetic resonance imaging (MRI) using Mn-TyrEDTA, a peroxidase activity-dependent contrast agent, revealed weak contrast enhancement in contralateral arteries and decreased contrast enhancement in aneurysm walls with ABAH treatment, indicating MPO activity decreased and inflammation mitigated. This was supported by reduced immune cell infiltration, matrix metalloproteinases (MMP-2 and - 9) activity, ROS production and arterial wall destruction on histology. Finally, the aneurysm expansion rate remained < 50% throughout the study in the ABAH(+) group, but increased gradually in the ABAH(-) group. Our results suggest that inhibition of MPO attenuated inflammation and expansion of experimental aneurysm and MPO-sensitive MRI showed promise as a noninvasive tool for monitoring aneurysm inflammation.

Research on surface treatment technology for quickly improving the skid resistance of tunnel concrete pavement.

In order to solve the problem that the skid resistance of concrete pavement in tunnel deteriorates rapidly, which is easy to cause traffic accidents, the anti-skid rapid elevation technology of surface treatment is proposed. Wear tests were used to investigate the effects of concrete surface roughness, properties of modified emulsified asphalt binder and anti-skid fine aggregate type on long-term skid resistance of treated surfaces. The results show that the four coarsening methods of fine milling, milling, grooving and brooming can improve the skid resistance of concrete, and the skid resistance durability of fine milling and milling is better. The adhesive property of modified emulsified asphalt is the best when the content of water-based epoxy resin is 20%. In different aggregates, the anti-skid effect is better when silicon carbide is used as anti-skid aggregate and the particle size is 0.6mm:0.3mm = 2:3. The method of fine milling of concrete surface + spraying epoxy emulsified asphalt + spreading silicon carbide can effectively improve the anti-skid performance of the original concrete pavement, and the feasibility of the scheme is verified by the test road. The research results have a good reference value for improving the skid resistance of tunnel concrete pavement.

Transcriptome-Wide Identification and Integrated Analysis of a UGT Gene Involved in Ginsenoside Ro Biosynthesis in Panax ginseng.

Panax ginseng as a traditional medicinal plant with a long history of medicinal use. Ginsenoside Ro is the only oleanane-type ginsenoside in ginseng, and has various pharmacological activities, including anti-inflammatory, detoxification, and antithrombotic activities. UDP-dependent glycosyltransferase (UGT) plays a key role in the synthesis of ginsenoside, and the excavation of UGT genes involved in the biosynthesis of ginsenoside Ro has great significance in enriching ginsenoside genetic resources and further revealing the synthesis mechanism of ginsenoside. In this work, ginsenoside-Ro-synthesis-related genes were mined using the P. ginseng reference-free transcriptome database. Fourteen hub transcripts were identified by differential expression analysis and weighted gene co-expression network analysis. Phylogenetic and synteny block analyses of PgUGAT252645, a UGT transcript among the hub transcripts, showed that PgUGAT252645 belonged to the UGT73 subfamily and was relatively conserved in ginseng plants. Functional analysis showed that PgUGAT252645 encodes a glucuronosyltransferase that catalyzes the glucuronide modification of the C3 position of oleanolic acid using uridine diphosphate glucuronide as the substrate. Furthermore, the mutation at 622 bp of its open reading frame resulted in amino acid substitutions that may significantly affect the catalytic activity of the enzyme, and, as a consequence, affect the biosynthesis of ginsenoside Ro. Results of the in vitro enzyme activity assay of the heterologous expression product in E. coli of PgUGAT252645 verified the above analyses. The function of PgUGAT252645 was further verified by the result that its overexpression in ginseng adventitious roots significantly increased the content of ginsenoside Ro. The present work identified a new UGT gene involved in the biosynthesis of ginsenoside Ro, which not only enriches the functional genes in the ginsenoside synthesis pathway, but also provides the technical basis and theoretical basis for the in-depth excavation of ginsenoside-synthesis-related genes.

Healthcare professionals' perspectives on the challenges with managing polycystic ovary syndrome: A systematic review and meta-synthesis.

OBJECTIVE: To provide an overview of healthcare professionals' experience of PCOS management and identify the relevant facilitators and barriers. METHODS: A systematic search was conducted in MEDLINE, EMBASE, CINAHL, Web of Science, and Cochrane CENTRAL database from the earliest available date to April 2023. Qualitative and mixed methods studies that described healthcare professionals' experiences of PCOS management were included. RESULTS: A total of 74 findings were extracted from the 8 included studies, which were categorized into facilitators and barriers. The barriers were meta-aggregated into four themes: the weakness of clinical evidence; women's low adherence to PCOS management; various obstacles that healthcare professionals face, and the influence of social environment and culture. The facilitators were meta-aggregated into three themes: chronic disease healthcare plan, communication techniques and healthcare professionals' ability and awareness. CONCLUSION: The findings of this study have the potential to improve the care provided to women with PCOS. However, it is important for national health professionals and policy markers to consider the cultural context of their own country when implementing these findings. PRACTICAL IMPLICATIONS: This study illustrated several challenges in managing the heterogeneous condition of PCOS and provide insights for the development of medical policies and future research directions.

Transition readiness of adolescents with cancer: A cross-sectional study based on self-determination theory.

PURPOSE: This study aimed to assess the transition readiness of adolescents with cancer in central China and to explore the paths associated with transition readiness based on self-determination theory (SDT). METHODS: Self-management and transition to adulthood with Rx = treatment questionnaire, patient activation measure, perceived social support scale and general self-efficacy scale were used to measure transition readiness as well as constructs pertaining to SDT (competence, relatedness and autonomy). The factors influencing transition readiness were evaluated using multiple linear regression. Models 4 and 6 in PROCESS Macro 3.3 were used to test the mediating effects and chain mediating effects, respectively. RESULTS: A total of 217 adolescents with cancer were included; their mean transition readiness score was 59.95 (11.34). Age (t = 6.086, p < 0.000), duration of diagnosis (t = 2.218, p = 0.028), completion of treatment (t = -2.036, p = 0.043), insurance, and competence (t = 11.149, p < 0.000) were significantly associated with transition readiness. The direct effects of self-efficacy and perceived social support on transition readiness were not significant. However, two chain mediating paths were observed: perceived social support - self-efficacy - patient activation - transition readiness and self-efficacy - perceived social support - patient activation - transition readiness; the effect values of these paths were 0.0678 and 0.0703, respectively. CONCLUSIONS: The findings of this study add to the evidence supporting the use of SDT-related constructs to promote transition readiness among adolescents with cancer, highlight the importance of encouraging patient activation, and clarify the ancillary roles of social support and self-efficacy in patient activation development during transitional period.

ECM-inspired calcium/zinc laden cellulose scaffold for enhanced bone regeneration.

Cellulose-based polymer scaffolds are highly diverse for designing and fabricating artificial bone substitutes. However, realizing the multi-biological functions of cellulose-based scaffolds has long been challenging. In this work, inspired by the structure and function of the extracellular matrix (ECM) of bone, we developed a novel yet feasible strategy to prepare ECM-like scaffolds with hybrid calcium/zinc mineralization. The 3D porous structure was formed via selective oxidation and freeze drying of bacterial cellulose. Following the principle of electrostatic interaction, calcium/zinc hybrid hydroxyapatite nucleated, crystallized, and precipitated on the 3D scaffold in simulated physiological conditions, which was well confirmed by morphology and composition analysis. Compared with alternative scaffold cohorts, this hybrid ion-loaded cellulose scaffold exhibited a pronounced elevation in alkaline phosphatase (ALP) activity, osteogenic gene expression, and cranial defect regeneration. Notably, the hybrid ion-loaded cellulose scaffold effectively fostered an M2 macrophage milieu and had a strong immune effect in vivo. In summary, this study developed a hybrid multifunctional cellulose-based scaffold that appropriately simulates the ECM to regulate immunomodulatory and osteogenic differentiation, setting a measure for artificial bone substitutes.

Factors associated with stress among pregnant women with a second child in Hunan province under China's two-child policy: a mixed-method study.

OBJECTIVE: The purpose of the study was to investigate the status of pregnancy stress and to explore factors associated with pregnancy stress among women by China's two-child policy. METHODS: A mixed-method study involving both quantitative and qualitative methods was conducted using questionnaires and semi-structured interviews. The questionnaires encompassed socio-demographic and obstetric characteristics, as well as the Pregnancy Stress Rating Scale (PSRS) and the Social Support Rating Scale (SSRS). Initially, the participants were required to complete the questionnaires, enabling us to assess their respective pregnancy stress statuses. Subsequently, we selectively interviewed pregnant women with a second child and exhibited at least mild pregnancy stress. The qualitative study sought to uncover the factors contributing to their stress during pregnancy. RESULTS: A total of 463 subjects were enrolled; of the subjects, 22 (4.8%) had no stress, 407 (87.9%) had mild stress, 34 (7.3%) had moderate stress. Generalized linear regression analysis revealed significant factors (P<0.05) related to pregnancy stress, including family financial burden, subjective support, fertility desire, gender of the first child, and gender preference. Additionally, 16 subjects were interviewed, and through analysis, three major themes emerged, each comprising 12 sub-themes associated with pregnancy stress. These themes were identified as fertility factors (worry about maternal and child health, birth experience, and parenting stress), family factors ( financial burden, second child care problems, first child's acceptance of the second child, family concerns, fertility desire, and gender preference) and social factors (involving life events, career development and workload). CONCLUSION: The diver factors contribute to pregnancy stress among pregnant women under China's two-child policy. Our study could be used to develop appropriate interventions to reduce pregnancy stress and to enhance the mental health of women pregnant with a second child.