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Zhang, Q.

Publications and source records attributed to Zhang, Q..

At least 19 recordsLinked to original sources

Scaling genetic discovery through automated post-GWAS interpretation

Genome-wide association studies (GWAS) have made association discovery systematic and cumulative, yet evidence for interpreting associated loci remains selective, heterogeneous and difficult to accumulate across studies. Here we present Post-GWAS Intelligence (PGI), which couples context-dependent post-GWAS analysis to a common representation of analysis-derived evidence. Its multi-agent engine, VariantAgent, executes workflows, records tool-derived results in standardized variant-centred units and generates trait-level interpretations linked to their analytical basis and evidential status. Complementary benchmarks showed that VariantAgent outperformed tested general-purpose and biomedical agents, with its largest advantage when success required both a correct answer and a valid analytical trace. Across 34 published GWAS, VariantAgent achieved a mean per-study recovery of 82.1% for directly comparable findings. The largest gains lay downstream of association mapping, where uniform analysis extended published interpretations and identified regulatory relationships at established risk loci not previously reported in the source studies. Analysis of 1,041 GWAS yielded 530,949 evidence units and corresponding reports, which PGI organized into a cross-trait catalogue. Catalogue interrogation resolved an apparent cross-trait overlap into distinct signals and trait-specific biological hypotheses. This work demonstrates how PGI can extend genetic discovery beyond association mapping by enabling biological evidence to accumulate across studies.

genetics↗

Decoding Cell-level Circadian Phase and Rhythm-state Remodeling in Single-cell Transcriptomes With scRhythmBayes

Current circadian omics analysis tools have been developed primarily for bulk time-series. However, heterogeneous single-cell datasets require analysis and interpretation of rhythmic programs across different cells, cell types, and biological conditions. In these datasets, cellular circadian phase is usually unobserved, and sample-level zeitgeber time provides temporal information but does not define the exact phase of every cell. Existing methods address only selected aspects of this problem and do not model how cell-level phase evidence informs gene-level rhythmic parameters and rhythm-state remodeling. Here we present scRhythmBayes, a new probabilistic algorithm for phase-resolved circadian analysis in heterogeneous single-cell transcriptomes. scRhythmBayes combines a seed-gene count likelihood with a stratum-aware circular prior to infer cell-level circadian phase, incorporates phase support into gene-level rhythmic modeling, and assigns posterior probabilities to interpretable rhythm-state categories, including rhythm gain, rhythm loss, amplitude remodeling and phase shifts. In heterogeneous simulations, scRhythmBayes achieved higher phase-recovery accuracy than existing phase-inference methods and maintained balanced performance across rhythmic gene detection, parameter recovery, and rhythm-state classification. Stress tests defined practical requirements for seed-gene support, zeitgeber anchoring, temporal sampling density, and cell number. In mouse liver, scRhythmBayes revealed distinct circadian phase occupancy and rhythm-state organization between periportal and pericentral hepatocytes, supporting a temporal dimension of liver zonation. It also identified cell-type-specific rhythmic markers that were largely distinct from conventional static markers, indicating that cellular identity includes temporal programs not captured by average expression. In human heart disease data, phase-coordination and phase-distribution analyses identified cell-type-selective temporal disorganization rather than uniform circadian disruption across the tissue. scRhythmBayes therefore provides a statistically grounded approach for resolving circadian organization across cells, genes, cell types and biological conditions.

bioinformatics↗

Low-order MLKL oligomers induce irregular membrane defects in necroptosis

MLKL is a key executioner of necroptosis, a lytic form of regulated cell death associated with inflammatory pathologies. Upon activation by RIPK3-mediated phosphorylation, MLKL oligomerizes and translocates to the plasma membrane to induce permeabilization through yet unknown mechanisms. By imaging single particles of MLKL at the plasma membrane of necroptotic cells, here we show that, upon activation, MLKL transitions from a diffuse cytosolic distribution to discrete, low-mobility assemblies before cell bursting. This is accompanied by oligomerization into a mixture of species ranging from monomers up to hexamers. We directly visualize MLKL-induced membrane damage using atomic force microscopy imaging in supported bilayers, which reveals the formation of irregular membrane openings whose edges are only partially decorated with protein. Our findings support a mechanism in which low-order self-assemblies of MLKL disrupt plasma membrane integrity and stabilize membrane defect edges without organizing into defined pore structures, in contrast to membrane permeabilization in other forms of cell death. This reframes necroptosis execution and dissects membrane insertion of MLKL oligomers for potential targeting.

biophysics↗

LongVerse: verified AI agents for Nanopore and PacBio DNA methylation analysis

AI agents now run sequencing pipelines, but their outputs are checked by proxy, with rubrics or language-model judges. LongVerse turns one English sentence into haplotype-resolved DNA methylation from Nanopore or PacBio reads, through agents that read the data, supply arguments to a pinned pipeline and never write code. On HG002, a healthy reference genome, agent and human expert produced byte-identical calls (r = 1.000) that agreed with bisulfite sequencing (r = 0.943, 0.884), at $0.12 to $0.54 of conversation per run. A consensus agent that admits any per-site caller reduced error 10.7 percent against the best published caller.

bioinformatics↗

Valine Engages a Prefrontal mGluR5 Allosteric Site to Preserve Social Behavior

Branched-chain amino acid metabolism is linked to neurodevelopmental disorders, but whether a defined amino acid directly modulates cortical receptor signaling to control behavior is unknown. Here, we show that valine is uniquely required for sociability through medial prefrontal cortex (mPFC) circuitry. Partial dietary valine restriction, but not comparable leucine or isoleucine restriction, or mPFC-restricted acceleration of BCAA catabolism through Bckdk loss, disrupts social interaction, and local valine supplementation rescues behavior. LiP-SMap, surface plasmon resonance, cryo-EM, and mutagenesis identify metabotropic glutamate receptor 5 (mGluR5) as a direct valine target and reveal a valine-selective allosteric pocket distinct from the glutamate site. Valine is signaling-biased: it drives {beta}-arrestin-linked output yet has little standalone Gq efficacy, instead potentiating glutamate-evoked mGluR5 signaling to recruit endocannabinoid/CB1 feedback, gate inhibitory transmission, and preserve mPFC-dependent social behavior. Together, these findings identify valine as a diet-derived allosteric ligand that links amino acid availability to cortical circuit control of social behavior.

neuroscience↗

Short-Term Plasticity of Contrast Surround Suppression During Repeated Psychophysical Measurement

Perceptual surround suppression is commonly treated as stable within a testing session, but recent evidence for normalization reweighting suggests that suppressive interactions can be modified by recent visual input. Here, we asked whether surround suppression changes over minutes of repeated measurement and whether such change varies with stimulus and sampling conditions. Across four psychophysical experiments, we tracked contrast and motion surround suppression while varying center-surround orientation and stimulus spatiotemporal properties. With static gratings, contrast surround suppression increased early and then approached a plateau, with no evidence that this change depended on center-surround orientation. With dynamic gratings, an increase emerged only under finer temporal sampling and more sustained stimulation, and model-based analyses suggested a faster increase than with static stimulation. Motion surround suppression measured with drifting gratings showed no systematic change. Together, these findings show that contrast surround suppression can change within a single psychophysical session without a separate adaptation phase, and that its measured time course varies with stimulus and sampling conditions. The results are consistent with rapid, experience-dependent reweighting of contextual normalization.

neuroscience↗

Pan-cancer remodeling of reactive oxygen species homeostasis: distinct roles of the ROS-generation and antioxidant arms in immune context and outcome

Reactive oxygen species (ROS) are double-edged in cancer: tumor cells sustain high ROS generation while up-regulating antioxidant defense, so that the net balance between the two arms, rather than absolute ROS abundance, is the biologically meaningful quantity. Whether that balance defines a conserved tumor state across cancer types has been unclear. Here an experimentally measured ROS phenotype, obtained from a three-channel gold-nanocluster array, was generalized to public transcriptomes through a 107-gene ROS program scored in a large cell-line panel and in pan-cancer tumor and adjacent-normal tissue. The optical readout was reproducible and fully discriminative between cell lines, establishing the fingerprint as a quantitative starting point. In tumors, the generation and antioxidant arms were coordinately up-regulated relative to normal tissue, yet the two arms dissociated in their biology. The composite balance score tracked reduced immune infiltration consistently across cohorts, but arm-level decomposition showed that it was carried by low generation-arm activity in immune-cold tumors and followed arithmetically from how the balance score is defined; the association does not implicate antioxidant defense in immune infiltration. The clinical association pointed the other way: adverse outcome was attributable to the antioxidant arm, whereas generation was null. In cohort-held-out validation, tumors were separated from adjacent normal tissue, with redox programs outperforming generic phenotypic programs, indicating that the same program transfers across tumor types. Tumor remodeling of ROS homeostasis is therefore widespread, but its two arms carry distinct meanings: the balance score, driven by its generation term, reports immune context, whereas the antioxidant arm reports clinical risk.

cancer biology↗

Transcriptional perturbation effects remain structured beyond the leading global trend

Prior work has described a global transcriptional response to gene perturbation, with many perturbations moving cells along a common direction. We find that perturbations contribute differently to this leading global trend and that substantial organization remains beyond it, including transferable perturbation-specific effects across datasets and a common hierarchy of responsive genes across perturbations. These patterns make simple strategies surprisingly competitive even in predicting effects beyond the leading global component.

genomics↗

Porcine epidemic diarrhea virus Whole-Genome Diversity Reveals Divergence of Contemporary Populations in the United States

Porcine epidemic diarrhea virus (PEDV) was introduced into the United States (USA) in 2013 and has circulated ever since. PEDV causes significant losses in the swine industry, with the greatest impact observed during the first years following its introduction into the country. However, the evolutionary dynamics of the virus remain underexplored. Recently, analyses of the Spike protein subunit S1 suggested that the PEDV population currently circulating in the USA represents a fraction of its historical diversity and comprises only two clades restricted to specific geographic regions. These observations were based on a limited set of S1 sequences. Furthermore, our understanding of evolutionary dynamics at the genomic level is even more limited, as complete PEDV genomes have not been generated since 2017. Here, we characterized the evolutionary dynamics of PEDV populations in the USA using complete genomes and assessed the contribution of different evolutionary mechanisms to shaping population structure. We analyzed 338 complete genomes, of which 25 were obtained in this study, together with 623 Spike S1 sequences, of which 41 were obtained in this study. All sequences obtained in this study were assigned to the non-S-INDEL strain. No S-INDEL sequences were recovered in this study, although these viruses continue to circulate in the country. Our analyses revealed that most sequences sampled after 2023 formed a monophyletic clade divergent from sequences collected in previous years, suggesting an ongoing temporal population structuring. Population structure analyses further supported evidence of population differentiation over time. Recombination analyses indicated that sequences belonging to the contemporary clade shared a single recombination event, suggesting that this clade may have emerged through recombination. Time-scaled S1 phylogenies were consistent with previous findings, supporting the circulation of only two contemporary clades; however, one of these clades was detected for the first time outside its previously reported geographic range. Our findings document temporal genetic differentiation in US PEDV populations, consistent with continued viral evolution, and broaden the known geographic distribution of contemporary S1 clades. These results highlight the value of continued genomic surveillance to monitor changes in PEDV diversity and distribution and provide sequence data to inform vaccine research.

microbiology↗

Information regimes in population-graph neuroimaging: cohort context and implementation-dependent evaluation sensitivity

Population-graph models aggregate a query subject with an unlabeled cohort, so their reported performance can depend on the evaluation regime rather than on predictive information alone. Evaluation context has two separable axes: the visibility of the cohort during training, and the context available at inference. We study the training-time axis under a protocol fixed before analysis, on an ABIDE-I cohort of 871 subjects (20 acquisition sites). Two population-graph implementations are compared: a learned/adaptive graph (EV-GCN) and a static weighted graph (Parisot-GCN). Within each implementation the cohort, connectivity features, outer splits and training and selection rules are held fixed, and only the information available to the graph varies. The primary estimand is the implementation-by-training-context interaction: the difference between the P0-to-P1 AUC changes of the two implementations ({Delta}{Delta}AUC). Across three preselected outer splits (666, 1024, 2024) the direction is consistent and positive, with observed interaction range 0.06285-0.13090; uncertainty is assessed separately within each split by paired subject and site-cluster bootstrap resampling and is reported per split. Splits are replication units: they are not pooled and no cross-split average is reported, so the result is not a claim of invariance to arbitrary splits. These findings tie reported population-graph performance to the evaluation information regime and motivate explicit reporting of cohort visibility. The contribution is methodological: a two-axis taxonomy of information regimes, preselected cross-split evidence that the sensitivity of reported performance to cohort context depends on the implementation, and a reusable reporting checklist.

neuroscience↗

Cross-Tissue Inflammatory Convergence Supports a Putative Muscle-Brain Axis in Alzheimer's Disease

Background Reduced muscle strength is associated with cognitive decline and Alzheimer's disease (AD), suggesting that skeletal-muscle aging and AD-related neurodegeneration may share biological mechanisms. Vascular dysfunction, immune activation, and extracellular-matrix remodeling have been implicated in both tissues, but the extent of shared cellular and molecular programs remains unclear. We investigated cross-tissue transcriptional programs in aging skeletal muscle and AD-related brain tissue and evaluated whether related signals are detectable in peripheral blood. Methods We analyzed independent, unpaired human transcriptomic datasets from skeletal muscle (23 donors), the SEA-AD middle temporal gyrus atlas (84 donors), and peripheral blood. Donor-aware models identified age-associated changes in muscle and transcriptomic changes across continuous pseudoprogression score (CPS) in brain. Cross-tissue comparisons focused on 30 brain donors within an early CPS window. Shared tissue-derived programs were assessed in peripheral blood mononuclear cells and independently tested in frailty bulk-blood and Alzheimer's Disease Neuroimaging Initiative (ADNI) whole-blood datasets. CellOracle simulations were used to prioritize candidate transcriptional regulators. Results Aging muscle showed increased inflammatory, extracellular-matrix, and stromal communication programs. During early AD-related brain progression, vascular and immune populations showed increasing inflammatory and matrix-associated activity together with shifts in intercellular communication. Cross-tissue analysis identified six concordantly enriched pathways comprising 109 leading-edge genes in endothelial populations and four pathways comprising 51 genes in myeloid populations. Related peripheral-blood signatures varied across cohorts and cell types. However, in independent frailty and ADNI mild cognitive impairment and AD datasets, 23 of 30 predefined tissue-derived pathway-subset tests showed significant positive enrichment after false-discovery-rate correction, whereas individual gene-level bulk-blood associations did not survive correction. CellOracle analyses prioritized ETS2 and BHLHE40 as exploratory endothelial regulators whose simulated perturbation consistently reduced selected shared program scores across tissues. Conclusions Aging skeletal muscle and AD-related brain tissue exhibit convergent endothelial and myeloid inflammatory programs, with related pathway-level signals detectable in peripheral blood. These cross-tissue programs and candidate endothelial regulators provide testable hypotheses for mechanisms linking systemic aging, muscle dysfunction, and AD-related neurodegeneration.

neuroscience↗

Cell type-resolved connectomic reconstruction of the spinal dorsal horn reveals sensory neuron synaptic organization and modality-specific inhibition

Touch, temperature, chemical, and noxious stimuli are detected by more than a dozen peripheral sensory neuron subtypes that together mediate somatosensation. Somatosensory neurons form synapses in the spinal cord dorsal horn, the first site of central somatosensory processing, to engage the downstream circuitry that drives both spinal reflexes and ascending pathways for perception. Yet how sensory neuron synapses are organized into the spinal cord circuitry remains poorly understood. Here we present two connectomic tools for studying somatosensory circuits of the spinal cord dorsal horn: dSC1, a large-scale, synapse-resolved electron microscopy (EM) dataset of the mouse dorsal horn, and SPINE (SPinal cord Integrated NEuron classifier), a deep-learning pipeline that identifies major sensory neuron subtypes directly from EM images. Applying SPINE to the dSC1 dataset, we found marked heterogeneity in synaptic architecture across physiologically distinct sensory neuron subtypes: low-threshold mechanoreceptors form substantially more synapses and receive far more GABAergic presynaptic inhibition than high-threshold mechanoreceptors, thermoreceptors and nociceptors. Cell-type-resolved circuit reconstruction further identified five populations of spinal inhibitory neurons (IhNs) that form synapses onto sensory neuron axons, each preferentially driven by and presynaptically inhibiting a single sensory subtype - a homotypic, modality-specific inhibitory motif. Most of these IhNs also feedforwardly inhibit other sensory pathways - a heterotypic, cross modality motif. Together, dSC1 and SPINE enable cell-type-resolved spinal cord circuit reconstruction and reveal a dual-mode logic of sensory-evoked inhibition that shapes modality-specific signaling at the earliest stage of somatosensory processing.

neuroscience↗

A synaptic wiring diagram underlying modality-specific ascending pathways of the superficial spinal dorsal horn

The spinal cord dorsal horn transforms tactile, thermal, pruritic, and nociceptive signals from peripheral somatosensory neurons into outputs to the brain, but how dorsal horn circuits organize diverse inputs into modality-selective ascending pathways is poorly understood. Using dSC1, a large-scale electron microscopy dataset of the mouse dorsal horn, we mapped the circuitry connecting primary sensory afferents to projection neurons of the anterolateral tract (ALT), a principal pathway conveying somatosensory information to the brain. We find that afferents encoding distinct modalities are routed onto connectivity-defined populations of ALT projection neurons. Nociceptors and cold thermoreceptors directly innervate distinct lamina I ALT projection neuron populations specialized for conveying noxious and cooling signals, respectively, whereas tactile and pruritic signals instead pass through a recurrent, multilayered network involving interneurons called vertical cells before reaching a separate ALT projection population. This distinction, direct versus polysynaptic wiring, underlies marked differences in sensory convergence and establishes segregated ascending channels for conveying signals encoding the principal somatosensory modalities. Together, our findings define an architectural logic by which the dorsal horn integrates and routes tactile, thermal, pruritic, and nociceptive information into distinct ALT output pathways to the brain.

neuroscience↗

A 3D in vitro follicle-endometrial organoid co-culture model recapitulates ovarian control of human endometrial decidualization

Female infertility remains a major women's reproductive health challenge, with impaired endometrial receptivity contributing to recurrent implantation failure, early pregnancy loss, and pregnancy complications. Mechanistic studies of human early pregnancy are limited by the lack of physiologically relevant models, and existing in vitro endometrial models rely on exogenous hormone treatment and do not recapitulate the dynamic ovarian control of endometrial growth and differentiation. Here, as a proof of concept, we developed a 3D in vitro ovarian follicle-endometrial organoid co-culture model. Immature murine follicles were cultured in vitro to obtain preovulatory follicles, followed by human chorionic gonadotropin (hCG) stimulation to induce ovulation and luteinization. Luteinized follicles were co-cultured with human endometrial stroma organoids for 6 days. The results showed that luteinized follicles generated a dynamic endocrine environment characterized by progressively increasing progesterone levels and declining estradiol levels. Follicle co-culture induced morphological differentiation and expression of decidualization markers in human endo-organoids. Transcriptomic analysis revealed strong genome-wide concordance between follicle co-culture and conventional decidualization induction using estradiol/progesterone/cAMP (EPC), with significant enrichment of decidualization-associated genes and pathways under both conditions. Nevertheless, the two conditions also exhibited distinct transcriptional states, with EPC preferentially enriched for more mature decidualization-associated programs, whereas luteinized follicle co-culture retained features of the early stage of decidualization. Functional perturbation testing revealed that the progesterone receptor (PR) antagonist RU486 attenuated co-culture-induced endometrial decidualization. Together, these results establish a new in vitro new approach methodology (NAM) that recapitulates ovarian control of endometrial decidualization. This platform provides a human-relevant NAM model to investigate the physiology and pathophysiology of endometrial decidualization, and to evaluate how drugs and environmental chemicals perturb uterine receptivity and early pregnancy.

pharmacology and toxicology↗

Development and characterization of a miniaturized focused ultrasound extraction (FUSE) system to enable rapid, point-of-care genetic testing

Point-of-care (POC) molecular detection has enabled rapid and accessible testing at or near the site of need, facilitating timely decision-making in clinical, field, and industrial settings. Despite these advancements, molecular detection platforms remain limited in their ability to prepare DNA from complex tissue matrices, restricting their applicability to a narrow range of sample types. To address sample preparation inefficiencies, focused ultrasound extraction (FUSE) has been developed for the rapid release of DNA from robust tissue matrices. FUSE disintegrates target tissue and releases DNA by delivering high pressure ultrasound pulses that generate a cavitation bubble cloud. In prior work, FUSE was performed using large, laboratory-based equipment. Here, a miniaturized FUSE device was designed and tested to enable POC DNA sample preparation. Design constraints were defined based on prior FUSE experiments, and based on these metrics, a 750 kHz cylindrical transducer was fabricated. The pressure output was measured, and high-speed optical imaging demonstrated that the transducer generated sustained cavitation within the defined focal region. The feasibility of DNA extraction from piscine and timber tissues was evaluated, and results show that the device can release DNA with quality suitable for PCR-based detection from both tissue types. These results demonstrate the potential of this device to rapidly extract DNA from complex samples and expand the applicability of molecular detection platforms.

bioengineering↗

Single-Cell Analysis of Non-Functioning Gonadotroph Tumors Identifies Lineage Infidelity and Tumor Growth Programs

Non-functioning gonadotroph (NFG) tumors are the most common type of non-functioning pituitary adenomas and can cause significant symptoms due to mass effect. However, the molecular programs underlying NFG tumor growth and their relationship to the normal anterior pituitary gland (APG) are poorly understood. To gain a deeper understanding of NFG tumor biology in the context of the normal APG, we performed single-cell/nucleus RNA-sequencing on 21 NFG tumors and 8 APG samples in human, generating the largest transcriptomic dataset of its kind. Single-cell/nucleus sequencing yielded 77,342, cells from APG samples and 152,649 cells from NFG tumor samples. Differential expression analysis within the APG identified novel marker genes of each neuroendocrine cell type and defined distinct transcriptional signatures of anterior and posterior pituitary stem cell populations. Comparison with tumor transcriptomes revealed that NFG tumor cells most closely resemble gonadotrophs, while also showing significant enrichment of thyrotroph and somatotroph markers. Notably, there was no significant overlap with stem cell markers, suggesting that NFG tumors most likely originate from differentiated gonadotrophs but can take on transcriptional characteristics of other neuroendocrine cell types. Pseudobulk profiling of NFG tumor cells demonstrated that tumor volume is significantly positively correlated with 83 genes including known oncogenes CAD, BRF2, and SOX12, along with 16 zinc finger transcription factors. Lastly, analysis of the tumor microenvironment revealed proportional increases in myeloid, endothelial, and mural cell populations in tumor samples compared to APG samples and highlighted cross talk between tumor and endothelial, mesenchymal, and immune populations via VEGF, PDGF, and MIF signaling pathways respectively. This study identifies novel transcriptomic signatures determining cell type identity in both APG neuroendocrine and NFG tumor cells. Our findings elucidate molecular programs driving NFG lineage infidelity and tumor growth, highlighting candidate predictors of patient outcomes and potential targets for therapeutic intervention.

cancer biology↗

Tracing high transductive cohort AUC to same-site supervision in a site-aware population GNN for multisite fMRI

Population-graph models can use cohort-level context, complicating interpretation of strong multisite neuroimaging performance. We investigated which information pathways accounted for high transductive cohort discrimination in a site-aware heterogeneous population graph neural network for autism classification. Using a frozen ABIDE-I cohort (871 participants, 20 sites) and evaluation protocol, we applied controlled graph, feature, supervision, and architecture interventions to a clean-room re-implementation across C-PAC and NIAK preprocessing pipelines. Cohort out-of-fold AUC was approximately 0.94. A site-only graph retained similarly high discrimination (0.948 versus 0.941 for the full model), with no statistically significant difference detected. Removing same-site supervision under a site-wise intervention reduced AUC to 0.480, whereas a supervision-budget control retaining same-site supervision achieved 0.921. Among the alternative heads evaluated, the high cohort AUC was observed only with the sex-heterogeneous dual-channel head; a canonical topology-only Parisot-GCN did not reproduce it. A demographic classifier using site, sex, and their interaction achieved a best pooled AUC of 0.51. Under leave-one-site-out evaluation, AUC was 0.522 for C-PAC and 0.532 for NIAK, with confidence intervals including 0.5 and no clear evidence of unseen-site discrimination; an imaging-only reference achieved 0.653 and 0.588. Because leave-one-site-out evaluation jointly changes supervision availability, target-site topology, and training-domain composition, the decrease cannot be attributed to a single factor. These findings distinguish cohort-visible transductive performance from unseen-site generalization and motivate explicit site-only, no-graph, and site-held-out controls.

neuroscience↗

Exposure to a mixture of long-chain PFAS disrupts ovarian follicle development, ovulation, and luteinization in female mice

Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants associated with adverse female reproductive outcomes. However, most prior studies examined individual PFAS, despite that humans are exposed to complex PFAS mixtures. Here, we investigated the ovarian effects of a mixture of five commonly detected long-chain PFAS, including PFOS, PFOA, PFNA, PFDA, and PFHxS, using complementary in vivo mouse and in vitro 3D ovarian follicle culture models. Young adult female mice were exposed to a range of concentrations of the PFAS mixture (PFASmix) through daily drinking water for 8 weeks. PFASmix at high concentrations (PFASmix-high) disrupted mouse estrous cyclicity, decreased circulating estradiol, increased testosterone, and promoted follicular atresia. Antral follicles from exposed mice showed decreased Lhcgr and increased Amh expression. Although the number of ovulated oocytes was unchanged following superovulation, PFASmix-high reduced oocyte size and polar body extrusion and altered oocyte cytoskeletal characteristics. In the 3D follicle culture model, direct PFASmix exposure impaired follicle growth, ovulation, and oocyte maturation in a concentration-dependent manner. PFASmix-high suppressed the periovulatory expression of Areg, Ereg, Tnfaip6, and Adamts1 and impaired corpus luteal (CL) development and function, as evidenced by reduced CL spheroid growth and cell survival, decreased expression of Star, Cyp11a1, Hsd3b1, and Lhcgr, and reduced progesterone secretion. PFASmix-high exposure additionally altered the expression of multiple PPAR{gamma}-associated genes in cultured ovarian follicles. Benchmark dose modeling identified estrous cyclicity and follicle rupture as sensitive in vivo and in vitro endpoints, respectively. Together, these findings demonstrated that exposure to the mixture of long-chain PFAS disrupts multiple stages of ovarian function, from follicular development, oocyte maturation to ovulation and luteal development and function and provided mechanistic and quantitative evidence for the reproductive toxicity of PFAS mixtures.

pharmacology and toxicology↗