Probing High-order Protein Complexes Using Native Mass Spectrometry and Hydrogen/Deuterium Exchange Mass Spectrometry: A Case Study Using Fresh and Commercial Hemoglobin Samples.

Native mass spectrometry (MS) analysis of protein complexes is highly susceptible to matrix effect, and addressing this predicament using buffer exchange is a common approach. Nevertheless, optimization of the buffer exchange protocol is not trivial. With the use of hemoglobin (Hb) as the model entity, it was discovered that the native mass spectrum of protein assembly is highly dependent on the buffer-exchange protocol. Given the dependence of native MS on the purification protocol, this work attempts to use hydrogen/deuterium exchange mass spectrometry (HDX-MS) for comparative studies of hemoglobin complexes in untreated fresh and commercial samples. The information obtained from the HDX study was found to correlate well with the native mass spectrometry analysis of the properly buffer-exchanged Hb samples. Both native MS and HDX-MS showed that the fresh Hb sample has retained the expected tetrameric structure, whereas the commercial Hb has largely been denatured to the dimeric form. These findings prove the complementarity of native MS and HDX-MS in the analysis of high-order protein complexes and stress the necessity to validate the integrity of the high-order structures of the proteins prior to the use of the protein samples for other biomedical studies.

Non-linear association between air pollutants and secondary sensitive skin in acne patients.

BACKGROUND: There is a growing number of patients suffering from sensitive skin secondary to acne, but its prevalence and influencing factors are not yet well-understood. OBJECTIVE: The aim of this study is to investigate the nonlinear relationship between air pollutants and secondary sensitive skin in acne patients. METHODS: A cross-sectional study comprising 4325 acne outpatients in China was carried out between September 2021 and December 2022, employing a simple random sampling approach. Air pollutants data was derived from the nearest air quality monitoring station corresponding to the subjects' residential locations. Furthermore, socio-economic characteristics, biological attributes, and lifestyle data of patients were acquired via questionnaire surveys. The data were subsequently analyzed utilizing the XGBoost machine learning model. RESULTS: A nonlinear relationship has been observed between secondary sensitive skin in acne patients and various factors, including particulate matter (PM2.5), inhalable particulate matter (PM10), ozone (O3), sulfur dioxide (SO2), nitrogen dioxide (NO2), carbon monoxide (CO), the severity of depression, different levels of exercise intensity, acne grading, frequency of sunscreen application, gender, and age. CONCLUSION: The occurrence of secondary sensitive skin in acne patients be mitigated through the implementation of measures such as the control of air pollutant emissions, regulation of negative emotions, and improvement of personal lifestyle.

CWF19L2 is Essential for Male Fertility and Spermatogenesis by Regulating Alternative Splicing.

The progression of spermatogenesis along specific developmental trajectories depends on the coordinated regulation of pre-mRNA alternative splicing (AS) at the post-transcriptional level. However, the fundamental mechanism of AS in spermatogenesis remains to be investigated. Here, it is demonstrated that CWF19L2 plays a pivotal role in spermatogenesis and male fertility. In germline conditional Cwf19l2 knockout mice exhibiting male sterility, impaired spermatogenesis characterized by increased apoptosis and decreased differentiated spermatogonia and spermatocytes is observed. That CWF19L2 interacted with several spliceosome proteins to participate in the proper assembly and stability of the spliceosome is discovered. By integrating RNA-seq and LACE-seq data, it is further confirmed CWF19L2 directly bound and regulated the splicing of genes related to spermatogenesis (Znhit1, Btrc, and Fbxw7) and RNA splicing (Rbfox1, Celf1, and Rbm10). Additionally, CWF19L2 can indirectly amplify its effect on splicing regulation through modulating RBFOX1. Collectively, this research establishes that CWF19L2 orchestrates a splicing factor network to ensure accurate pre-mRNA splicing during the early steps of spermatogenesis.

Analyzing noncovalent interactions between notoginseng saponins and lysozyme by deposition scanning intensity fading MALDI-TOF mass spectrometry.

Analysis of noncovalent interactions between natural products and proteins is important for rapid screening of active ingredients and understanding their pharmacological activities. In this work, the intensity fading MALDI-TOF mass spectrometry (IF-MALDI-MS) method with improved reproducibility was implemented to investigate the binding interactions between saponins from Panax notoginseng and lysozyme. The benchmark IF-MALDI-MS experiment was established using N,N',N″-triacetylchitotriose-lysozyme as a model system. The reproducibility of ion intensities in IF-MALDI-MS was improved by scanning the whole sample deposition with a focused laser beam. The relative standard deviation (RSD) of deposition scanning IF-MALDI-MS is 5.7%. Similar decay trends of the relative intensities of notoginseng saponins against increasing amounts of lysozyme were observed for all six notoginseng saponins. The half-maximal fading concentration (FC50) was calculated to quantitatively characterize the binding affinity of each ligand based on the decay curve. According to the FC50 values obtained, the binding affinities of the six notoginseng saponins were evaluated in the following order: notoginsenoside S > notoginsenoside Fc > ginsenoside Rb1 > ginsenoside Rd > notoginsenoside Ft1 > ginsenoside Rg1. The binding order was in accordance with molecular docking studies, which showed hydrogen bonding might play a key role in stabilizing the binding interaction. Our results demonstrated that deposition scanning IF-MALDI-MS can provide valuable information on the noncovalent interactions between ligands and proteins.

MiRNA-224-5p regulates the defective permeability barrier in sensitive skin by targeting claudin-5.

BACKGROUND: Sensitive skin is hypersensitive to various external stimuli and a defective epidermal permeability barrier is an important clinical feature of sensitive skin. Claudin-5 (CLDN5) expression levels decrease in sensitive skin. This study aimed to explore the impact of CLDN5 deficiency on the permeability barrier in sensitive skin and the regulatory role of miRNAs in CLDN5 expression. MATERIALS AND METHODS: A total of 26 patients were retrospectively enrolled, and the CLDN5 expression and permeability barrier dysfunction in vitro were assessed. Then miRNA-224-5p expression was also assessed in sensitive skin. RESULTS: Immunofluorescence and electron microscopy revealed reduced CLDN5 expression, increased miR-224-5p expression, and disrupted intercellular junctions in sensitive skin. CLDN5 knockdown was associated with lower transepithelial electrical resistance (TEER) and Lucifer yellow penetration in keratinocytes and organotypic skin models. The RNA-seq and qRT-PCR results indicated elevated miR-224-5p expression in sensitive skin; MiR-224-5p directly interacted with the 3`UTR of CLDN5, resulting in CLDN5 deficiency in the luciferase reporter assay. Finally, miR-224-5p reduced TEER in keratinocyte cultures. CONCLUSION: These results suggest that the miR-224-5p-induced reduction in CLDN5 expression leads to impaired permeability barrier function, and that miR-224-5p could be a potential therapeutic target for sensitive skin.

The deubiquitinase cofactor UAF1 interacts with USP1 and plays an essential role in spermiogenesis.

Spermiogenesis defines the final phase of male germ cell differentiation. While multiple deubiquitinating enzymes have been linked to spermiogenesis, the impacts of deubiquitination on spermiogenesis remain poorly characterized. Here, we investigated the function of UAF1 in mouse spermiogenesis. We selectively deleted Uaf1 in premeiotic germ cells using the Stra8-Cre knock-in mouse strain (Uaf1 sKO), and found that Uaf1 is essential for spermiogenesis and male fertility. Further, UAF1 interacts and colocalizes with USP1 in the testes. Conditional knockout of Uaf1 in testes results in disturbed protein levels and localization of USP1, suggesting that UAF1 regulates spermiogenesis through the function of the deubiquitinating enzyme USP1. Using tandem mass tag-based proteomics, we identified that conditional knockout of Uaf1 in the testes results in reduced levels of proteins that are essential for spermiogenesis. Thus, we conclude that the UAF1/USP1 deubiquitinase complex is essential for normal spermiogenesis by regulating the levels of spermiogenesis-related proteins.

[Identification and visual analysis of ginsenosides in multi-steamed roots of Panax quinquefolium based on UPLC-Q-TOF-MS/MS and MALDI-MSI].

This study aims to investigate the component variations and spatial distribution of ginsenosides in Panax quinquefolium roots during repeated steaming and drying. Ultra performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry(UPLC-Q-TOF-MS/MS) was employed to identify the ginsenosides in the root extract. Matrix-assisted laser desorption/ionization mass spectrometry imaging(MALDI-MSI) was employed to visualize the spatial distribution and spatiotemporal changes of prototype ginsenosides and metabolites in P. quinquefolium roots. The UPLC results showed that 90 ginsenosides were identified during the steaming process of the roots, and polar ginsenosides were converted into low polar or non-polar ginsenosides. The content of prototype ginsenosides decreased, while that of rare ginsenosides increased, which included 20(S/R)-ginsenoside Rg_3, 20(S/R)-ginsenoside Rh_2, and ginsenosides Rk_1, Rg_5, Rs_5, and Rs_4. MALDI-MSI results showed that ginsenosides were mainly distributed in the epidermis and phloem. As the steaming times increased, ginsenosides were transported to the xylem and medulla. This study provides fundamental information for revealing the changes of biological activity and pharmacological effect of P. quinquefolium roots that are caused by repeated steaming and drying and gives a reference for expanding the application scope of this herbal medicine.

Lactobacillus paracasei ZFM54 alters the metabolomic profiles of yogurt and the co-fermented yogurt improves the gut microecology of human adults.

Gut microbiota imbalance could lead to various diseases, making it important to optimize the structure of the gut flora in adults. Lactobacillus paracasei ZFM54 is a bacteriocin- and folic acid-producing Lactobacillus strain. Herein, L. paracasei ZFM54 was used as the potentially probiotic bacterium to ferment milk together with a yogurt starter. We optimized the fermentation conditions, and the obtained yogurts were then subjected to volatile and nonvolatile metabolome analysis, showing that L. paracasei ZFM54 can not only improve the acidity, water holding capacity and live lactic acid bacteria counts, but also improve many volatile acid contents and increase some beneficial nonvolatile metabolites, such as N-ethyl glycine and l-lysine, endowing the yogurt with more flavor and better function. The regulatory effects of the co-fermented yogurt on the intestinal microecology of volunteers were investigated by 16S rRNA sequencing and short-chain fatty acid (SCFA) analysis after consuming the yogurt for a 2-wk period, showing a better effect to increase the relative abundance of beneficial bacteria such as Ruminococcus and Alistipes, decrease harmful bacteria (Escherichia-Shigella and Enterobacter), and enhance the production of SCFA (acetate, propionate, and butyric acid) compared with the control yogurt. We found that L. paracasei ZFM54 can significantly improve the health benefits of yogurt, laying the foundation for its commercial application in improving gut microbiota.

A millimeter water-in-oil droplet as an alternative back exchange prevention strategy for hydrogen/deuterium exchange mass spectrometry of peptides/proteins.

Hydrogen/deuterium exchange mass spectrometry (HDX-MS) is a versatile bioanalytical technique for protein analysis. Since the reliability of HDX-MS analysis considerably depends on the retention of deuterium labels in the post-labeling workflow, deuterium/hydrogen (D/H) back exchange prevention strategies, including decreasing the pH, temperature, and exposure time to protic sources of the deuterated samples, are widely adopted in the conventional HDX-MS protocol. Herein, an alternative and effective back exchange prevention strategy based on the encapsulation of a millimeter droplet of a labeled peptide solution in a water-immiscible organic solvent (cyclohexane) is proposed. Cyclohexane was used to prevent the undesirable uptake of water by the droplet from the atmospheric vapor through the air-water interface. Using the pepsin digest of deuterated myoglobin, our results show that back exchange kinetics of deuterated peptides is retarded in a millimeter droplet as compared to that in the bulk solution. Performing pepsin digestion directly in a water-in-oil droplet at room temperature (18-21 °C) was found to preserve more deuterium labels than that in the bulk digestion with an ice-water bath. Based on the present findings, it is proposed that keeping deuterated peptides in the form of water-in-oil droplets during the post-labelling workflow will facilitate the preservation of deuterium labels on the peptide backbone and thereby enhance the reliability of the H/D exchange data.

The hidden diet: Synthetic antioxidants in packaged food and their impact on human exposure and health.

Synthetic antioxidants (AOs) are commonly used in everyday items and industrial products to inhibit oxidative deterioration. However, the presence of AOs in food packaging and packaged foods has not been thoroughly documented. Moreover, studies on human exposure to AOs through skin contact with packaging or ingesting packaged foods are limited. In this study, we analyzed twenty-three AOs-including synthetic phenolic antioxidants (SPAs) and organophosphite antioxidants (OPAs)-along with six transformation products in various food samples and their packaging materials. We found AOs in food products at concentrations ranging from 1.30 × 103 to 1.77 × 105 ng/g, which exceeded the levels in both outer packaging (6.05 × 102-3.07 × 104 ng/g) and inner packaging (2.27 × 102-1.09 × 105 ng/g). The most common AOs detected in foodstuffs were tris(2,4-di-tert-butylphenyl) phosphate (AO168O), butylated hydroxytoluene (BHT), and octadecyl-3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (AO1076), together constituting 95.7 % of the total AOs found. Our preliminary exposure assessment revealed that dietary exposure-estimated at a median of 2.55 × 104 ng/kg body weight/day for children and 1.24 × 104 ng/kg body weight/day for adults-is a more significant exposure route than dermal contact with packaging. Notably, four AOs were identified in food for the first time, with BHT making up 76.8 % and 67.6 % of the total BHT intake for children and adults, respectively. These findings suggest that food consumption is a significant source of BHT exposure. The estimated daily intakes of AOs via consumption of foodstuffs were compared with the recommended acceptable daily intake to assess the risks. This systematic investigation into AOs contributes to understanding potential exposure and health risks associated with AOs in packaged foods. It emphasizes the need for further evaluation of human exposure to these substances.

Facile synthesis of hydroxylated triazine-based magnetic microporous organic network for ultrahigh adsorption of phenylurea herbicides: An experimental and density-functional theory study.

Microporous organic networks (MONs) are highly porous materials that are particularly useful in analytical chemistry. However, the use of these materials is often limited by the functional groups available on their surface. Here, we described the polymerization of a sea urchin-like structure material at ambient temperature, that was functionalized with hydroxyl, carboxyl, and triazine groups and denoted as OH-COOH-MON-TEPT. A substantial proportion of OH-COOH-MON-TEPT was intricately decorated EDA-Fe3O4, creating a well-designed configuration (EDA-Fe3O4 @OH-COOH-MON-TEPT-EDC) for superior adsorption of the target analytes phenylurea herbicides (PUHs) via magnetic solid-phase extraction (MSPE). The proposed method showed remarkably low limits of detection ranging from 0.03 to 0.22 ng·L-1. Experimental investigations and theoretical analyses unveiled the adsorption mode between EDA-Fe3O4 @OH-COOH-MON-TEPT-EDC and PUHs. These findings establish a robust foundation for potential applications of EDA-Fe3O4 @OH-COOH-MON-TEPT-EDC in the analysis of various polar contaminants.

A review of factors influencing sensitive skin: an emphasis on built environment characteristics.

Background: Sensitive skin (SS) is a condition characterized by hyperreactivity. Impacting around 37 percent of the worldwide population and exerting an influence on the quality of life for affected individuals. Its prevalence rate has increased due to factors such as elevating stress levels and deteriorating environmental conditions. The exposome factors influencing SS have extended from demographic, biological attributes, and lifestyle to external environments. Built environments (BEs) have demonstrated as root drivers for changes in behaviors and environmental exposure which have the potential to trigger SS, but the review of the associations between BEs and SS is currently lacking. Objective: This review aims to achieve two primary objectives: (1) Examine exposome factors that exert influence on SS at the individual and environmental levels. (2) Develop a theoretical framework that establishes a connection between BEs and SS, thereby offering valuable insights into the impact of the built environment on this condition. Methods: An extensive literature search was carried out across multiple fields, including sociology, epidemiology, basic medicine, clinical medicine, and environmental research, with a focus on SS. To identify pertinent references, renowned databases such as PubMed, Web of Science, and CNKI were utilized. Results: SS is the outcome of interactions between individual attributes and environmental factors. These influencing factors can be categorized into five distinct classes: (1) demographic and socioeconomic characteristics including age, gender, and race; (2) physiological and biological attributes such as emotional changes, skin types, sleep disorders, and menstrual cycles in women; (3) behavioral factors, such as spicy diet, cosmetic use, alcohol consumption, and physical exercise; (4) natural environmental features, including climate conditions and air pollution; (5) built environmental features such as population density, green space availability, road network density, and access to public transportation, also have the potential to affect the condition. Conclusion: The importance of interdisciplinary integration lies in its ability to ascertain whether and how BEs are impacting SS. By elucidating the role of BEs in conjunction with other factors in the onset of SS, we can provide guidance for future research endeavors and the formulation of interventions aimed at mitigating the prevalence of SS.

A Surface Matrix of Au NPs Decorated Graphdiyne for Multifunctional Laser Desorption/Ionization Mass Spectrometry.

The development of the valid strategy to enhance laser desorption/ionization efficiency gives rise to widespread concern in surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) technology. Herein, a hybrid of Au NP-decorated graphdiyne (Au/GDY) was fabricated and employed as the SALDI-MS matrix for the first time, and a mechanism based on photothermal and photochemical energy conversions was proposed to understand LDI processes. Given theoretical simulations and microstructure characterizations, it was revealed that the formation of a coupled thermal field and internal electric field endow the as-prepared Au/GDY matrix with superior desorption and ionization efficiency, respectively. Moreover, laser-induced matrix ablation introduced strain and defect level into the Au/GDY hybrid, suppressing the recombination of charge carriers and thereby facilitating analyte ionization. The optimized Au/GDY matrix allowed for reliable detection of trace sulfacetamide and visualization of exogenous/endogenous components in biological tissues. This work offers an integrated solution to promote LDI efficiency based on collaborative photothermal conversion and internal electric field, and may inspire the design of novel semiconductor-based surface matrices.

Current Knowledge of Individual and Combined Toxicities of Aflatoxin B1 and Fumonisin B1 In Vitro.

Mycotoxins are considered the most threating natural contaminants in food. Among these mycotoxins, aflatoxin B1 (AFB1) and fumonisin B1 (FB1) are the most prominent fungal metabolites that represent high food safety risks, due to their widespread co-occurrence in several food commodities, and their profound toxic effects on humans. Considering the ethical and more humane animal research, the 3Rs (replacement, reduction, and refinement) principle has been promoted in the last few years. Therefore, this review aims to summarize the research studies conducted up to date on the toxicological effects that AFB1 and FB1 can induce on human health, through the examination of a selected number of in vitro studies. Although the impact of both toxins, as well as their combination, were investigated in different cell lines, the majority of the work was carried out in hepatic cell lines, especially HepG2, owing to the contaminants' liver toxicity. In all the reviewed studies, AFB1 and FB1 could invoke, after short-term exposure, cell apoptosis, by inducing several pathways (oxidative stress, the mitochondrial pathway, ER stress, the Fas/FasL signaling pathway, and the TNF-α signal pathway). Among these pathways, mitochondria are the primary target of both toxins. The interaction of AFB1 and FB1, whether additive, synergistic, or antagonistic, depends to great extent on FB1/AFB1 ratio. However, it is generally manifested synergistically, via the induction of oxidative stress and mitochondria dysfunction, through the expression of the Bcl-2 family and p53 proteins. Therefore, AFB1 and FB1 mixture may enhance more in vitro toxic effects, and carry a higher significant risk factor, than the individual presence of each toxin.

Identification of raffinose family oligosaccharides in processed Rehmannia glutinosa Libosch using matrix-assisted laser desorption/ionization mass spectrometry image combined with machine learning.

RATIONALE: Currently, research on oligosaccharides primarily focuses on the physiological activity and function, with a few studies elaborating on the spatial distribution characterization and variation in the processing of Rehmannia glutinosa Libosch. Thus, imaging the spatial distributions and dynamic changes in oligosaccharides during the steaming process is significant for characterizing the metabolic networks of R. glutinosa. It will be beneficial to characterize the impact of steaming on the active ingredients and distribution patterns in different parts of the plant. METHODS: A highly sensitive matrix-assisted laser desorption/ionization mass spectrometry image (MALDI-MSI) method was used to visualize the spatial distribution of oligosaccharides in processed R. glutinosa. Furthermore, machine learning was used to distinguish the processed R. glutinosa samples obtained under different steaming conditions. RESULTS: Imaging results showed that the oligosaccharides in the fresh R. glutinosa were mainly distributed in the cortex and xylem. As steaming progressed, the tetra- and pentasaccharides were hydrolyzed and diffused gradually into the tissue section. MALDI-MS profiling combined with machine learning was used to identify the processed R. glutinosa samples accurately at different steaming intervals. Eight algorithms were used to build classification machine learning models, which were evaluated for accuracy, precision, recall, and F1 score. The linear discriminant analysis and random forest models performed the best, with prediction accuracies of 0.98 and 0.97, respectively, and thus can be considered for identifying the steaming durations of R. glutinosa. CONCLUSIONS: MALDI-MSI combined with machine learning can be used to visualize the distribution of oligosaccharides and identify the processed samples after steaming for different durations. This can enhance our understanding of the metabolic changes that occur during the steaming process of R. glutinosa; meanwhile, it is expected to provide a theoretical reference for the standardization and modernization of processing in the field of medicinal plants.

Spatially Resolved Metabolomics Combined with the 3D Tumor-Immune Cell Coculture Spheroid Highlights Metabolic Alterations during Antitumor Immune Response.

The metabolic cross-talk between tumor and immune cells plays key roles in immune cell function and immune checkpoint blockade therapy. However, the characterization of tumor immunometabolism and its spatiotemporal alterations during immune response in a complex tumor microenvironment is challenging. Here, a 3D tumor-immune cell coculture spheroid model was developed to mimic tumor-immune interactions, combined with mass spectrometry imaging-based spatially resolved metabolomics to visualize tumor immunometabolic alterations during immune response. The inhibition of T cells was simulated by coculturing breast tumor spheroids with Jurkat T cells, and the reactivation of T cells can be monitored through diminishing cancer PD-L1 expressions by berberine. This system enables simultaneously screening and imaging discriminatory metabolites that are altered during T cell-mediated antitumor immune response and characterizing the distributions of berberine and its metabolites in tumor spheroids. We discovered that the transport and catabolism of glutamine were significantly reprogrammed during the antitumor immune response at both metabolite and enzyme levels, corresponding to its indispensable roles in energy metabolism and building new biomass. The combination of spatially resolved metabolomics with the 3D tumor-immune cell coculture spheroid visually reveals metabolic interactions between tumor and immune cells and possibly helps decipher the role of immunometabolic alterations in tumor immunotherapy.

Aspergillus flavus and Fusarium verticillioides and Their Main Mycotoxins: Global Distribution and Scenarios of Interactions in Maize.

Maize is frequently contaminated with multiple mycotoxins, especially those produced by Aspergillus flavus and Fusarium verticillioides. As mycotoxin contamination is a critical factor that destabilizes global food safety, the current review provides an updated overview of the (co-)occurrence of A. flavus and F. verticillioides and (co-)contamination of aflatoxin B1 (AFB1) and fumonisin B1 (FB1) in maize. Furthermore, it summarizes their interactions in maize. The gathered data predict the (co-)occurrence and virulence of A. flavus and F. verticillioides would increase worldwide, especially in European cold climate countries. Studies on the interaction of both fungi regarding their growth mainly showed antagonistic interactions in vitro or in planta conditions. However, the (co-)contamination of AFB1 and FB1 has risen worldwide in the last decade. Primarily, this co-contamination increased by 32% in Europe (2010-2020 vs. 1992-2009). This implies that fungi and mycotoxins would severely threaten European-grown maize.

Environmental exposure to per- and perfluoroalkyl substances in early pregnancy and newborn anogenital distance: A prospective cohort study.

Per- and polyfluoroalkyl substances (PFAS) are persistent, ubiquitous pollutants, and the current epidemiological evidence regarding the impact of in utero exposure to PFAS on anogenital distance (AGD) is limited and inconclusive. The primary aim of this study was to investigate the potential associations between maternal exposure to PFAS during pregnancy and AGD in newborns. A total of 2273 mother-child pairs were recruited for this study, and both PFAS levels and AGD were measured. Multiple linear regression models were utilized to explore the relationships between individual PFAS and AGD. Additionally, quantile-based g-computation (QGC) was employed to assess the joint effects of mixtures of PFAS on AGD. Our findings showed that maternal exposure to PFOS (ß = 0.518, 95% CI: 0.093, 0.942), PFNA (ß = 0.487, 95% CI: 0.037, 0.937), PFDA (ß = 0.443, 95% CI: 0.048, 0.838), PFUA (ß = 0.434, 95% CI: 0.031, 0.838), and PFBS (ß = 0.444, 95% CI: 0.124, 0.763) during early pregnancy had a significant positive association with AGD in boys. Similarly, in girls, maternal exposure to PFOS (ß = 0.423, 95% CI: 0.006, 0.841), PFNA (ß = 0.641, 95% CI: 0.207, 1.074), PFDA (ß = 0.670, 95% CI: 0.306, 1.033), PFUA (ß = 0.895, 95% CI: 0.509, 1.281), and PFBS (ß = 0.474, 95% CI: 0.178, 0.770) had a positive association with AGD, while PFOA (ß = -1.254, 95% CI: -1.786, -0.723) had a negative association. QGC models further confirmed that PFAS mixtures were positively associated with AGD. Moreover, PFBS was the primary contributor to the joint effects of PFAS mixtures on AGD. In summary, our study has provided further corroboration for the possibility that PFAS exposure can have an impact on AGD in both boys and girls. The use of AGD as a promising biomarker for endocrine disruption highlights the significance of our findings, which may have valuable clinical implications for reproductive diseases.