Single-cell DNA methylome and 3D multi-omic atlas of the adult mouse brain.

Cytosine DNA methylation is essential in brain development and is implicated in various neurological disorders. Understanding DNA methylation diversity across the entire brain in a spatial context is fundamental for a complete molecular atlas of brain cell types and their gene regulatory landscapes. Here we used single-nucleus methylome sequencing (snmC-seq3) and multi-omic sequencing (snm3C-seq)1 technologies to generate 301,626 methylomes and 176,003 chromatin conformation-methylome joint profiles from 117 dissected regions throughout the adult mouse brain. Using iterative clustering and integrating with companion whole-brain transcriptome and chromatin accessibility datasets, we constructed a methylation-based cell taxonomy with 4,673 cell groups and 274 cross-modality-annotated subclasses. We identified 2.6 million differentially methylated regions across the genome that represent potential gene regulation elements. Notably, we observed spatial cytosine methylation patterns on both genes and regulatory elements in cell types within and across brain regions. Brain-wide spatial transcriptomics data validated the association of spatial epigenetic diversity with transcription and improved the anatomical mapping of our epigenetic datasets. Furthermore, chromatin conformation diversities occurred in important neuronal genes and were highly associated with DNA methylation and transcription changes. Brain-wide cell-type comparisons enabled the construction of regulatory networks that incorporate transcription factors, regulatory elements and their potential downstream gene targets. Finally, intragenic DNA methylation and chromatin conformation patterns predicted alternative gene isoform expression observed in a whole-brain SMART-seq2 dataset. Our study establishes a brain-wide, single-cell DNA methylome and 3D multi-omic atlas and provides a valuable resource for comprehending the cellular-spatial and regulatory genome diversity of the mouse brain.

Conserved and divergent gene regulatory programs of the mammalian neocortex.

Divergence of cis-regulatory elements drives species-specific traits1, but how this manifests in the evolution of the neocortex at the molecular and cellular level remains unclear. Here we investigated the gene regulatory programs in the primary motor cortex of human, macaque, marmoset and mouse using single-cell multiomics assays, generating gene expression, chromatin accessibility, DNA methylome and chromosomal conformation profiles from a total of over 200,000 cells. From these data, we show evidence that divergence of transcription factor expression corresponds to species-specific epigenome landscapes. We find that conserved and divergent gene regulatory features are reflected in the evolution of the three-dimensional genome. Transposable elements contribute to nearly 80% of the human-specific candidate cis-regulatory elements in cortical cells. Through machine learning, we develop sequence-based predictors of candidate cis-regulatory elements in different species and demonstrate that the genomic regulatory syntax is highly preserved from rodents to primates. Finally, we show that epigenetic conservation combined with sequence similarity helps to uncover functional cis-regulatory elements and enhances our ability to interpret genetic variants contributing to neurological disease and traits.

Brain-wide correspondence of neuronal epigenomics and distant projections.

Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.

DNA methylation atlas of the mouse brain at single-cell resolution.

Mammalian brain cells show remarkable diversity in gene expression, anatomy and function, yet the regulatory DNA landscape underlying this extensive heterogeneity is poorly understood. Here we carry out a comprehensive assessment of the epigenomes of mouse brain cell types by applying single-nucleus DNA methylation sequencing1,2 to profile 103,982 nuclei (including 95,815 neurons and 8,167 non-neuronal cells) from 45 regions of the mouse cortex, hippocampus, striatum, pallidum and olfactory areas. We identified 161 cell clusters with distinct spatial locations and projection targets. We constructed taxonomies of these epigenetic types, annotated with signature genes, regulatory elements and transcription factors. These features indicate the potential regulatory landscape supporting the assignment of putative cell types and reveal repetitive usage of regulators in excitatory and inhibitory cells for determining subtypes. The DNA methylation landscape of excitatory neurons in the cortex and hippocampus varied continuously along spatial gradients. Using this deep dataset, we constructed an artificial neural network model that precisely predicts single neuron cell-type identity and brain area spatial location. Integration of high-resolution DNA methylomes with single-nucleus chromatin accessibility data3 enabled prediction of high-confidence enhancer-gene interactions for all identified cell types, which were subsequently validated by cell-type-specific chromatin conformation capture experiments4. By combining multi-omic datasets (DNA methylation, chromatin contacts, and open chromatin) from single nuclei and annotating the regulatory genome of hundreds of cell types in the mouse brain, our DNA methylation atlas establishes the epigenetic basis for neuronal diversity and spatial organization throughout the mouse cerebrum.

Epigenomic diversity of cortical projection neurons in the mouse brain.

Neuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

Comparative cellular analysis of motor cortex in human, marmoset and mouse.

The primary motor cortex (M1) is essential for voluntary fine-motor control and is functionally conserved across mammals1. Here, using high-throughput transcriptomic and epigenomic profiling of more than 450,000 single nuclei in humans, marmoset monkeys and mice, we demonstrate a broadly conserved cellular makeup of this region, with similarities that mirror evolutionary distance and are consistent between the transcriptome and epigenome. The core conserved molecular identities of neuronal and non-neuronal cell types allow us to generate a cross-species consensus classification of cell types, and to infer conserved properties of cell types across species. Despite the overall conservation, however, many species-dependent specializations are apparent, including differences in cell-type proportions, gene expression, DNA methylation and chromatin state. Few cell-type marker genes are conserved across species, revealing a short list of candidate genes and regulatory mechanisms that are responsible for conserved features of homologous cell types, such as the GABAergic chandelier cells. This consensus transcriptomic classification allows us to use patch-seq (a combination of whole-cell patch-clamp recordings, RNA sequencing and morphological characterization) to identify corticospinal Betz cells from layer 5 in non-human primates and humans, and to characterize their highly specialized physiology and anatomy. These findings highlight the robust molecular underpinnings of cell-type diversity in M1 across mammals, and point to the genes and regulatory pathways responsible for the functional identity of cell types and their species-specific adaptations.

Spatiotemporal DNA methylome dynamics of the developing mouse fetus.

Cytosine DNA methylation is essential for mammalian development but understanding of its spatiotemporal distribution in the developing embryo remains limited1,2. Here, as part of the mouse Encyclopedia of DNA Elements (ENCODE) project, we profiled 168 methylomes from 12 mouse tissues or organs at 9 developmental stages from embryogenesis to adulthood. We identified 1,808,810 genomic regions that showed variations in CG methylation by comparing the methylomes of different tissues or organs from different developmental stages. These DNA elements predominantly lose CG methylation during fetal development, whereas the trend is reversed after birth. During late stages of fetal development, non-CG methylation accumulated within the bodies of key developmental transcription factor genes, coinciding with their transcriptional repression. Integration of genome-wide DNA methylation, histone modification and chromatin accessibility data enabled us to predict 461,141 putative developmental tissue-specific enhancers, the human orthologues of which were enriched for disease-associated genetic variants. These spatiotemporal epigenome maps provide a resource for studies of gene regulation during tissue or organ progression, and a starting point for investigating regulatory elements that are involved in human developmental disorders.

C4d, rather than C3d and C5b-9, is associated with graft loss in recurrent IgA deposition after kidney transplantation.

INTRODUCTION: Recurrent IgA deposition is common after kidney transplantation. However, it is difficult to define whether IgA deposition is innocuous or contributes to organ damage. Next, although complement is known to be involved in the pathogenesis of IgA nephropathy (IgAN), its involvement has not been studied systematically in kidney transplant recipients (KTR). METHODS: KTR with biopsy-proven native IgAN who underwent kidney biopsy after transplantation between 1995 and 2020 were included. Recurrent IgA deposition was defined as IgA deposit in the glomerulus. Staining of complement factors C4d, C3d, and C5b-9 were quantitatively evaluated using ImageScope. RESULTS: Sixty-seven KTR (85% male, 46±13 years old, 12 [6-24] months after transplantation, 58% with indication biopsy) were included in the analyses. Of them, 25 (37%) had recurrent IgA deposition. There were no clinical differences between KTR with and without recurrent IgA deposition. C3d and C5b-9 were always present in biopsies with IgA deposition, while C4d was present in 48% of the biopsies. During a median follow-up of 9.6 [4.8-14] years, 18 (27%) KTR developed death-censored graft failure. Recurrent IgA deposition was not associated with graft failure. Of the evaluated complement factors, only C4d staining was associated with graft failure in KTR with recurrent IgA deposition (Hazard ratio = 2.55, 95% confidence interval = 1.07-6.03, p = 0.034). CONCLUSIONS: Recurrent IgA deposition was not associated with graft failure in itself. C4d, when present, is strongly associated with graft loss in KTR with recurrent IgA deposition, suggesting a pathogenic role for the lectin pathway in recurrent IgAN.

Autonomic cardiac function in children and adolescents with long COVID: a case-controlled study.

Although the mechanisms underlying the pathophysiology of long COVID condition are still debated, there is growing evidence that autonomic dysfunction may play a role in the long-term complications or persisting symptoms observed in a significant proportion of patients after SARS-CoV-2 infection. However, studies focused on autonomic dysfunction have primarily been conducted in adults, while autonomic function has not yet been investigated in pediatric subjects. In this study, for the first time, we assessed whether pediatric patients with long COVID present abnormalities in autonomic cardiac function. Fifty-six long COVID pediatric patients (mean age 10.3 ± 3.8 y) and 27 age-, sex-, and body surface area-matched healthy controls (mean age 10.4 ± 4.5y) underwent a standard 12-lead electrocardiography (ECG) and 24-h ECG Holter monitoring. Autonomic cardiac function was assessed by time-domain and frequency-domain heart rate variability parameters. A comprehensive echocardiographic study was also obtained by two-dimensional echocardiography and tissue Doppler imaging. Data analysis showed that pediatric patients with long COVID had significant changes in HRV variables compared to healthy controls: significantly lower r-MSSD (root mean square of successive RR interval differences, 47.4 ± 16.9 versus 60.4 ± 29.1, p = 0.02), significant higher values VLF (very low frequency, 2077.8 ± 1023.3 versus 494.3 ± 1015.5 ms, p = 0.000), LF (low frequency, 1340.3 ± 635.6 versus 354.6 ± 816.8 ms, p = 0.000), and HF (high frequency, 895.7 ± 575.8 versus 278.9 ± 616.7 ms, p = 0.000). No significant differences were observed between the two groups both in systolic and diastolic parameters by echocardiography.  Conclusion: These findings suggest that pediatric patients with long COVID have an imbalance of cardiac autonomic function toward a relative predominance of parasympathetic tone, as already reported in adult patients with long COVID. Further studies are needed to clarify the clinical significance of this autonomic dysfunction and demonstrate its role as a pathophysiological mechanism of long COVID, paving the way for effective therapeutic and preventive strategies. What is Known: • Long Covid in children has been described globally, but studies have mostly focused on collecting the temporal evolution of persisting symptoms. What is New: • Cardiac autonomic imbalance toward a relative predominance of parasympathetic tone is a mechanism underlying Long Covid in children, as also described in adults.

Evaluating differences in milk production, reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows.

The objective was to assess differences in productive and reproductive performance, and survival associated with vaginal discharge characteristics and fever in postpartum dairy cows located in western and southern states of the United States. This retrospective cohort study included data from 3 experiments conducted in 9 dairies. Vaginal discharge was evaluated twice within 12 DIM and scored on a 5-point scale. The highest vaginal discharge score observed for each cow was used to allocate them into 1 of 5 possible groups (VD group) as follows: VD 1 and 2 (VD 1/2; n = 1,174) = clear mucus or lochia with or without flecks of pus; VD 3 (n = 1,802) = mucopurulent with <50% pus; VD 4 (n = 1,643) = mucopurulent with ≥50% of pus or nonfetid reddish-brownish mucus, n = 1,643; VD 5 = fetid, watery, and reddish-brownish, n = 1,800. All VD 5 cows received treatment according to each herd's protocol. Rectal temperature was assessed in a subset of VD 5 cows, and subsequently divided into fever (rectal temperature ≥39.5°C; n = 334) and no fever (n = 558) groups. A smaller proportion of cows with VD 5 (67.6%) resumed ovarian cyclicity compared with VD 1/2 (76.2%) and VD 4 (72.9%) cows; however, a similar proportion of VD 5 and VD 3 (72.6%) cows resumed ovarian cyclicity. A smaller proportion of VD 5 (85.8%) cows received at least one AI compared with VD 1/2 (91.5%), VD 3 (91.0%), or VD 4 (91.6%) cows. Although we did not detect differences in pregnancy at first AI according to VD, fewer cows with VD 5 (64.4%) were pregnant at 300 DIM than cows with VD 1/2 (76.5%), VD 3 (76.2%), or VD 4 (74.7%). Hazard of pregnancy by 300 DIM was smaller for VD 5 compared with VD 1/2, VD 3, or VD 4 cows. A greater proportion of VD 5 cows were removed from the herd within 300 DIM compared with other VD groups. Milk production was 760 kg lower within 300 DIM for VD 5 compared with VD 2, VD 3, and VD 4, whereas VD 2, VD 3, and VD 4 had similar milk production. We did not detect an association between fever at diagnosis of VD 5 and reproductive performance or milk production. A greater proportion of VD 5 cows without fever were removed from the herd by 300 DIM compared with VD 5 cows with fever. Differences in productive and reproductive performance, and removal of the herd were restricted to fetid, watery, and reddish-brownish vaginal discharge, which was independent of fever.

Search for Dark-Matter-Nucleon Interactions via Migdal Effect with DarkSide-50.

Dark matter elastic scattering off nuclei can result in the excitation and ionization of the recoiling atom through the so-called Migdal effect. The energy deposition from the ionization electron adds to the energy deposited by the recoiling nuclear system and allows for the detection of interactions of sub-GeV/c^{2} mass dark matter. We present new constraints for sub-GeV/c^{2} dark matter using the dual-phase liquid argon time projection chamber of the DarkSide-50 experiment with an exposure of (12 306±184) kg d. The analysis is based on the ionization signal alone and significantly enhances the sensitivity of DarkSide-50, enabling sensitivity to dark matter with masses down to 40 MeV/c^{2}. Furthermore, it sets the most stringent upper limit on the spin independent dark matter nucleon cross section for masses below 3.6 GeV/c^{2}.

Search for Dark Matter Particle Interactions with Electron Final States with DarkSide-50.

We present a search for dark matter particles with sub-GeV/c^{2} masses whose interactions have final state electrons using the DarkSide-50 experiment's (12 306±184) kg d low-radioactivity liquid argon exposure. By analyzing the ionization signals, we exclude new parameter space for the dark matter-electron cross section σ[over ¯]_{e}, the axioelectric coupling constant g_{Ae}, and the dark photon kinetic mixing parameter κ. We also set the first dark matter direct-detection constraints on the mixing angle |U_{e4}|^{2} for keV/c^{2} sterile neutrinos.

Patients with laboratory criteria of anti-phospholipid syndrome and 'non-criteria' manifestations: a multicenter cohort.

OBJECTIVES: Patients with laboratory criteria for anti-phospholipid syndrome (APS) but presenting only 'non-criteria' clinical manifestations are scarcely characterized in the literature. We aimed to analyse a cohort of these patients regarding the most prevalent manifestations, antibody profile, and treatments, while establishing a comparison with definite APS patients. METHOD: A retrospective analysis was conducted of individuals fulfilling APS laboratory criteria assessed in two tertiary European hospitals between 2005 and 2020. Patients without clinical criteria but with non-criteria manifestations (termed 'clinical non-criteria') and age-/gender-matched controls were included. RESULTS: Altogether, 75 clinical non-criteria patients were analysed, with haematological (thrombocytopenia, haemolytic anaemia) and 'mild' neurological manifestations (white-matter lesions, migraine) as the most prevalent non-obstetric involvements. These patients displayed more thrombocytopenia [odds ratio (OR) = 3.6, 95% confidence interval (CI) 1.7-7.6; p = 0.001] than controls with APS, but severe manifestations, such as valvular heart disease (p < 0.001), livedoid vasculopathy, seizures, chorea, transverse myelitis, bone necrosis, and alveolar haemorrhage, occurred only in definite APS patients. Corticosteroids were required by 40% of patients with thrombocytopenia. Manifestations in anticoagulated patients included white-matter lesions, nephropathy, superficial vein thrombosis, amaurosis fugax, and livedoid vasculopathy. Suspicion of progression towards systemic lupus erythematosus (SLE) occurred in 19% of non-SLE individuals. CONCLUSION: 'Clinical non-criteria' patients displayed significant treatment use, predominantly haematological involvement, and less severe manifestations than definite APS controls. Some patients may additionally progress to future SLE. The impact of certain manifestations flags them as potential future contributors to classifying individuals as definite APS.

Pneumococcal vaccination status among cirrhotic patients in Italy: a neglected topic.

To date, few reports have evaluated the pneumococcal vaccination status in cirrhotic patients. No data are available for European countries. We have explored this topic and the potential independent predictors motivating lack of vaccination in Italy. Between January 1st and June 30th 2022, 1419 cirrhotic patients of any etiology were consecutively enrolled in an observational, prospective study at 8 referral centers in Italy. Adjusted odds ratios (ORs) for the association with lack of vaccination were evaluated by multiple logistic regression analysis. Overall vaccine coverage was 17.9% (8.9% in patients < 65 years of age and 27.1% in those aged ≥ 65 years; p < 0.001). Among the 1165 unvaccinated patients, 1068 (91.7%) reported lack of information regarding vaccination as the reason for not having undergone vaccination. Independent predictors associated with lack of vaccination were age < 65 years (OR 3.39, CI 95% 2.41-4.76) and a higher number of schooling years (OR 2.14, CI 95% 1.58-2.91); alcoholic etiology resulted only marginally associated (OR 1.91, CI 95% 1.03-3.52). These findings establish evidence on how pneumococcal vaccination status in Italy is largely suboptimal among cirrhotic patients. These results raise concern, considering the severe outcomes of pneumococcal infection in patients with chronic liver diseases.

Exploring adenovirus in water environments: a systematic review and meta-analysis.

Adenoviruses (AdVs) have a significant impact in both medical and environmental contexts. The objective of this study was to investigate the prevalence of AdV in different water types, such as untreated and treated wastewater, surface water, groundwater, drinking water, and other water matrices. A total of 239 articles were included in this meta-analysis. Adenoviruses were detected in various waters worldwide. The overall prevalence in water was found to be 59.2%, with the highest prevalence in untreated wastewater (83.1%) and treated wastewater (75.3%), followed by "other water matrices" (53.4%), surface water (49.5%) drinking water (22.7%), and groundwater (18.5%). Most of the studies did not assess the viability of the viruses, leading to weak links between water contamination and risk. Both human and animal AdV were found in water environments. The findings suggest that water, including drinking water, could be a significant route of AdV transmission in both developed and developing economies.

Estimating genetic parameters of reproductive, carcass, and meat quality traits in Polled Nellore cattle.

Considering the economic and commercial efficiency of the beef production chain, the yield and quality of the meat produced must also be included in breeding programs. For the Nellore breed, including the polled herd, these aspects have not been much studied. The aim of this study was to estimate genetic parameters for scrotal circumference adjusted to 365 (SC365) and 450 (SC450) days of age, age at first calving (AFC), accumulated productivity (AP), stayability (STAY), longissimus muscle area (LMA), thickness of subcutaneous fat over the 12th-13th ribs (BF), thickness of subcutaneous fat over the rump (RF), and shear force measured by Warner-Bratzler shear force (WBSF) of polled Nellore cattle. Bayesian analyses were performed by adopting a linear animal model, whereas STAY analyses used the linear threshold model. Heritability estimates were 0.31 (SC365), 0.37 (SC450), 0.16 (AFC), 0.25 (AP), 0.16 (STAY), 0.30 (LMA), 0.13 (BF), 0.24 (RF), and 0.15 (WBSF), indicating moderate response to selection. Genetic and residual correlations between SC365 and SC450 were high (0.91 and 0.74, respectively), as well as the genetic correlations of AP with SC365, SC450, AFC, and STAY (0.61, 0.62, - 0.69, and 0.83, respectively). Genetic and residual correlations of WBSF with reproductive and carcass characteristics exhibited high standard deviations, however favorable. Based on the results, it is expected that in the medium term, animals with greater sexual precocity will also have greater accumulated productivity and longer permanence of females in the herd, along with superior carcass traits. However, due to the low heritabilities and small genetic associations with reproductive traits, fat thickness characteristics (BF and RF) will still require direct selection.

Investigating the effect of temporal, geographic, and management factors on US Holstein lactation curve parameters.

We fit the Wood's lactation model to an extensive database of test-day milk production records of US Holstein cows to obtain lactation-specific parameter estimates and investigated the effects of temporal, spatial, and management factors on lactation curve parameters and 305-d milk yield. Our approach included 2 steps as follows: (1) individual animal-parity parameter estimation with nonlinear least-squares optimization of the Wood's lactation curve parameters, and (2) mixed-effects model analysis of 8,595,413 sets of parameter estimates from individual lactation curves. Further, we conducted an analysis that included all parities and a separate analysis for first lactation heifers. Results showed that parity had the most significant effect on the scale (parameter a), the rate of decay (parameter c), and the 305-d milk yield. The month of calving had the largest effect on the rate of increase (parameter b) for models fit with data from all lactations. The calving month had the most significant effect on all lactation curve parameters for first lactation models. However, age at first calving, year, and milking frequency accounted for a higher proportion of the variance than month for first lactation 305-d milk yield. All parameter estimates and 305-d milk yield increased as parity increased; parameter a and 305-d milk yield rose, and parameters b and c decreased as year and milking frequency increased. Calving month estimates parameters a, b, c, and 305-d milk yield were the lowest values for September, May, June, and July, respectively. The results also indicated the random effects of herd and cow improved model fit. Lactation curve parameter estimates from the mixed-model analysis of individual lactation curve fits describe well US Holstein lactation curves according to temporal, spatial, and management factors.

Freezing effect on the oleuropein content of olive leaves extracts obtained from microwave-assisted extraction.

This work aimed to investigate the effect of freezing on the oleuropein content obtained from olive leaves extracts. The extracts were obtained by microwave-assisted extraction using different solvents, pH, temperatures and microwave irradiation time. Afterward, HPLC was used to identify and quantify the amount of oleuropein in the extracts. A part of the extracts was immediately analyzed, and another was frozen for a week. The experimental results highlighted that the storage condition has a significant (p < 0.05) effect on the oleuropein content. Regardless of the extraction condition, the frozen storage was responsible for a decrease in the oleuropein content, ranging from 5.38 to 70.09%. These results indicate that it is important to consider the degradation of oleuropein in frozen olive leaf extracts so that subsequent applications are suitable.