SOX9 reprograms endothelial cells by altering the chromatin landscape.

The transcription factor SOX9 is activated at the onset of endothelial-to-mesenchymal transition (EndMT) during embryonic development and in pathological conditions. Its roles in regulating these processes, however, are not clear. Using human umbilical vein endothelial cells (HUVECs) as an EndMT model, we show that SOX9 expression alone is sufficient to activate mesenchymal genes and steer endothelial cells towards a mesenchymal fate. By genome-wide mapping of the chromatin landscape, we show that SOX9 displays features of a pioneer transcription factor, such as opening of chromatin and leading to deposition of active histone modifications at silent chromatin regions, guided by SOX dimer motifs and H2A.Z enrichment. We further observe highly transient and dynamic SOX9 binding, possibly promoted through its eviction by histone phosphorylation. However, while SOX9 binding is dynamic, changes in the chromatin landscape and cell fate induced by SOX9 are persistent. Finally, our analysis of single-cell chromatin accessibility indicates that SOX9 opens chromatin to drive EndMT in atherosclerotic lesions in vivo. This study provides new insight into key molecular functions of SOX9 and mechanisms of EndMT and highlights the crucial developmental role of SOX9 and relevance to human disease.

Current noise of a protein-selective biological nanopore.

1/f current noise is ubiquitous in protein pores, porins, and channels. We have previously shown that a protein-selective biological nanopore with an external protein receptor can function as a 1/f noise generator when a high-affinity protein ligand is reversibly captured by the receptor. Here, we demonstrate that the binding affinity and concentration of the ligand are key determinants for the nature of current noise. For example, 1/f was absent when a protein ligand was reversibly captured at a much lower concentration than its equilibrium dissociation constant against the receptor. Furthermore, we also analyzed the composite current noise that resulted from mixtures of low-affinity and high-affinity ligands against the same receptor. This study highlights the significance of protein recognition events in the current noise fluctuations across biological membranes.

Hepatocyte Nuclear Factor 4-Alpha Is Essential for the Active Epigenetic State at Enhancers in Mouse Liver.

Cell-fate determination is influenced by interactions between master transcription factors (TFs) and cis-regulatory elements. Hepatocyte nuclear factor 4 alpha (HNF4A), a liver-enriched TF, acts as a master controller in specification of hepatic progenitor cells by regulating a network of TFs to control onset of hepatocyte cell fate. Using analysis of genome-wide histone modifications, DNA methylation, and hydroxymethylation in mouse hepatocytes, we show that HNF4A occupies active enhancers in hepatocytes and is essential for active histone and DNA signatures, especially acetylation of lysine 27 of histone 3 (H3K27ac) and 5-hydroxymethylcytosine (5hmC). In mice lacking HNF4A protein in hepatocytes, we observed a decrease in both H3K27ac and hydroxymethylation at regions bound by HNF4A. Mechanistically, HNF4A-associated hydroxymethylation (5hmC) requires its interaction with ten-eleven translocation methylcytosine dioxygenase 3 (TET3), a protein responsible for oxidation from 5mC to 5hmC. Furthermore, HNF4A regulates TET3 expression in liver by directly binding to an enhancer region. Conclusion: In conclusion, we identified that HNF4A is required for the active epigenetic state at enhancers that amplifies transcription of genes in hepatocytes.

Protein Ligand-Induced Amplification in the 1/f Noise of a Protein-Selective Nanopore.

Previous studies of transmembrane protein channels have employed noise analysis to examine their statistical current fluctuations. In general, these explorations determined a substrate-induced amplification in the Gaussian white noise of these systems at a low-frequency regime. This outcome implies a lack of slowly appearing fluctuations in the number and local mobility of diffusing charges in the presence of channel substrates. Such parameters are among the key factors in generating a low-frequency 1/f noise. Here, we show that a protein-selective biological nanopore exhibits a substrate-induced amplification in the 1/f noise. The modular composition of this biological nanopore includes a hydrophilic transmembrane protein pore fused to a water-soluble binding protein on its extramembranous side. In addition, this protein nanopore shows an open substate populated by a high-frequency current noise because of the flickering of an engineered polypeptide adaptor at the tip of the pore. However, the physical association of the protein ligand with the binding domain reversibly switches the protein nanopore from a high-frequency noise substate into a quiet substate. In the absence of the protein ligand, our nanopore shows a low-frequency white noise. Remarkably, in the presence of the protein ligand, an amplified low-frequency 1/f noise was detected in a ligand concentration-dependent fashion. This finding suggests slowly occurring equilibrium fluctuations in the density and local mobility of charge carriers under these conditions. Furthermore, we report that the excess in 1/f noise is generated by reversible switches between the noisy ligand-released substate and the quiet ligand-captured substate. Finally, quantitative aspects of the low-frequency 1/f noise are in accord with theoretical predictions of the current noise analysis of protein channel-ligand interactions.

Imprint stability and plasticity during development.

There have been a number of recent insights in the area of genomic imprinting, the phenomenon whereby one of two autosomal alleles is selected for expression based on the parent of origin. This is due in part to a proliferation of new techniques for interrogating the genome that are leading researchers working on organisms other than mouse and human, where imprinting has been most studied, to become interested in looking for potential imprinting effects. Here, we recap what is known about the importance of imprints for growth and body size, as well as the main types of locus control. Interestingly, work from a number of labs has now shown that maintenance of the imprint post implantation appears to be a more crucial step than previously appreciated. We ask whether imprints can be reprogrammed somatically, how many loci there are and how conserved imprinted regions are in other species. Finally, we survey some of the methods available for examining DNA methylation genome-wide and look to the future of this burgeoning field.

Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores.

There have been only a few studies reporting on the impact of polyhistidine affinity tags on the structure, function, and dynamics of proteins. Because of the relatively short size of the tags, they are often thought to have little or no effect on the conformation or activity of a protein. Here, using membrane protein design and single-molecule electrophysiology, we determined that the presence of a hexahistidine arm at the N-terminus of a truncated FhuA-based protein nanopore, leaving the C-terminus untagged, produces an unusual increase in the unitary conductance to ∼8 nS in 1 M KCl. To the best of our knowledge, this is the largest single-channel conductance ever recorded with a monomeric ß-barrel outer membrane protein. The hexahistidine arm was captured by an anti-polyhistidine tag monoclonal antibody added to the side of the channel-forming protein addition, but not to the opposite side, documenting that this truncated FhuA-based protein nanopore inserts into a planar lipid bilayer with a preferred orientation. This finding is in agreement with the protein insertion in vivo, in which the large loops face the extracellular side of the membrane. The aberrantly large single-channel conductance, likely induced by a greater cross-sectional area of the pore lumen, along with the vectorial insertion into a lipid membrane, will have profound implications for further developments of engineered protein nanopores.

Global redesign of a native ß-barrel scaffold.

One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded ß-barrel protein into a large-conductance protein pore. Here, we show that this redesigned ß-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric ß-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.

Interrogating Detergent Desolvation of Nanopore-Forming Proteins by Fluorescence Polarization Spectroscopy.

Understanding how membrane proteins interact with detergents is of fundamental and practical significance in structural and chemical biology as well as in nanobiotechnology. Current methods for inspecting protein-detergent complex (PDC) interfaces require high concentrations of protein and are of low throughput. Here, we describe a scalable, spectroscopic approach that uses nanomolar protein concentrations in native solutions. This approach, which is based on steady-state fluorescence polarization (FP) spectroscopy, kinetically resolves the dissociation of detergents from membrane proteins and protein unfolding. For satisfactorily solubilizing detergents, at concentrations much greater than the critical micelle concentration (CMC), the fluorescence anisotropy was independent of detergent concentration. In contrast, at detergent concentrations comparable with or below the CMC, the anisotropy readout underwent a time-dependent decrease, showing a specific and sensitive protein unfolding signature. Functionally reconstituted membrane proteins into a bilayer membrane confirmed predictions made by these FP-based determinations with respect to varying refolding conditions. From a practical point of view, this 96-well analytical approach will facilitate a massively parallel assessment of the PDC interfacial interactions under a fairly broad range of micellar and environmental conditions. We expect that these studies will potentially accelerate research in membrane proteins pertaining to their extraction, solubilization, stabilization, and crystallization, as well as reconstitution into bilayer membranes.

Ontogeny, conservation and functional significance of maternally inherited DNA methylation at two classes of non-imprinted genes.

A functional role for DNA methylation has been well-established at imprinted loci, which inherit methylation uniparentally, most commonly from the mother via the oocyte. Many CpG islands not associated with imprinting also inherit methylation from the oocyte, although the functional significance of this, and the common features of the genes affected, are unclear. We identify two major subclasses of genes associated with these gametic differentially methylated regions (gDMRs), namely those important for brain and for testis function. The gDMRs at these genes retain the methylation acquired in the oocyte through preimplantation development, but become fully methylated postimplantation by de novo methylation of the paternal allele. Each gene class displays unique features, with the gDMR located at the promoter of the testis genes but intragenically for the brain genes. Significantly, demethylation using knockout, knockdown or pharmacological approaches in mouse stem cells and fibroblasts resulted in transcriptional derepression of the testis genes, indicating that they may be affected by environmental exposures, in either mother or offspring, that cause demethylation. Features of the brain gene group suggest that they might represent a pool from which many imprinted genes have evolved. The locations of the gDMRs, as well as methylation levels and repression effects, were also conserved in human cells.

Synthesis of CNS active thyrotropin-releasing hormone (TRH)-like peptides: Biological evaluation and effect on cognitive impairment induced by cerebral ischemia in mice.

Thyrotropin-releasing hormone (TRH)-like peptides were synthesized by replacing critical histidine and pGlu residues in the native peptide. The peptides were evaluated in vitro for receptor binding activity assay and in the cell functional assay; the peptides exhibit selective basal signaling agonist behavior toward TRH-R2. For example, peptides 8a, 8b, 8c, 8 f, 8 h, 8 l and 12 d activated TRH-R2 with potency (EC50) of 0.53 µM, 0.048 µM, 0.05 µM, 0.006 µM, 0.31 µM, 0.034 µM and 0.004 µM, respectively. In contrast for signaling activation of TRH-R1, the same peptide required higher concentration of 19.35 µM, 3.98 µM, 2.54 µM, 0.287 µM, 11.28 µM, 0.986 µM and 0.944 µM, respectively. The results showed that peptides were 36.5, 82.9, 50.8, 47.8, 36.3, 32.6 and 235-fold selective to TRH-R2 receptor subtype. The peptides were investigated for CNS activity at 10 µmol/kg in pentobarbital-induced sleep assay study. Peptides 8c (16.5 ± 1.4 min) and 8l (16.5 ± 2.1 min) displayed excellent CNS activity. In an in vivo study, peptide 8c did not cause significant change in the rat plasma TSH levels. The peptide 8c was further investigated for neuroprotective potential, and significantly reduced infracts volume and neurological score in the focal cerebral ischemia model in mice. Peptide 8c also significantly lowered MDA levels, indicating reduction of oxidative and enhanced percentage cell survival in CA1 region, when compared to ischemic brain.

5-Hydroxymethylation marks a class of neuronal gene regulated by intragenic methylcytosine levels.

We recently identified a class of neuronal gene inheriting high levels of intragenic methylation from the mother and maintaining this through later development. We show here that these genes are implicated in basic neuronal functions such as post-synaptic signalling, rather than neuronal development and inherit high levels of 5mC, but not 5hmC, from the mother. 5mC is distributed across the gene body and appears to facilitate transcription, as transcription is reduced in DNA methyltransferase I (Dnmt1) knockout embryonic stem cells as well as in fibroblasts treated with a methyltransferase inhibitor. However in adult brain, transcription is more closely associated with a gain in 5hmC, which occurs without a measurable loss of 5mC. These findings add to growing evidence that there may be a role for 5mC in promoting transcription as well as its classical role in gene silencing.

HNF4A guides the MLL4 complex to establish and maintain H3K4me1 at gene regulatory elements.

Hepatocyte nuclear factor 4A (HNF4A/NR2a1), a transcriptional regulator of hepatocyte identity, controls genes that are crucial for liver functions, primarily through binding to enhancers. In mammalian cells, active and primed enhancers are marked by monomethylation of histone 3 (H3) at lysine 4 (K4) (H3K4me1) in a cell type-specific manner. How this modification is established and maintained at enhancers in connection with transcription factors (TFs) remains unknown. Using analysis of genome-wide histone modifications, TF binding, chromatin accessibility and gene expression, we show that HNF4A is essential for an active chromatin state. Using HNF4A loss and gain of function experiments in vivo and in cell lines in vitro, we show that HNF4A affects H3K4me1, H3K27ac and chromatin accessibility, highlighting its contribution to the establishment and maintenance of a transcriptionally permissive epigenetic state. Mechanistically, HNF4A interacts with the mixed-lineage leukaemia 4 (MLL4) complex facilitating recruitment to HNF4A-bound regions. Our findings indicate that HNF4A enriches H3K4me1, H3K27ac and establishes chromatin opening at transcriptional regulatory regions.

Biovalorization of mango byproduct through enzymatic extraction of dietary fiber.

Mango is considered one of the most important tropical fruits worldwide in terms of its consumption and consumer acceptability. Its processing generates huge quantities of mango byproducts, which is often discarded unscrupulously into the environment and, therefore, needs effective waste management practices. The extraction of mango peels' dietary fiber using enzymatic method can be a useful valorization strategy for management of mango by-products. In the present investigation, dietary fiber (soluble and insoluble fraction) was extracted by enzymatic hydrolysis using α-amylase, protease, and amyloglucosidase. Highest yield of dietary fiber (67.5%, w/w) was obtained at 60 °C temperature using recommended enzyme concentrations including α-amylase (40 µL), protease (110 µL), and amyloglucosidase (200 µL) after a treatment time of 60 min. SEM analysis indicated the increased porosity of dietary fiber samples caused due to the hydrolytic effect of enzymes on its surface structure, whereas FTIR analysis confirmed the functional groups present in dietary fiber. The coexistence of crystalline and amorphous nature of polymers present in soluble and insoluble fractions of dietary fiber was assessed by XRD analysis. Further, the analysis of functional properties including WHC, OHC, and SC revealed the suitability of using extracted mango peel's dietary fiber in the food systems.

Determination of Sexual Dimorphism of the Human Sacrum Based on Receiver Operating Characteristic Curve Analysis of Morphometric Parameters.

Background Sex estimation of unidentified incomplete skeletons poses a challenge to paleoanthropologists and forensic experts. The sacrum is a part of the axial skeleton and contributes to the formation of the pelvic girdle. It is a significant bone for the identification of the sex in the human skeletal system due to associated functional differences of the pelvic bones in males and females. However, there is a lack of cognizance of different morphometric parameters of the sacrum which may be crucial for determining sex, particularly when a part of the bone is available. This study aimed to recognize the best morphometric parameters for the identification of the sex of the sacrum even when fragmented bones were available and compare the various parameters for sexual dimorphism in different populations. Methodology The study was conducted on 110 dry adult human sacra in the anatomy department. Out of these, 42 sacra were female and 68 were male. Morphometric measurements were performed with the help of a digital vernier caliper. Statistical analysis was performed using SPSS version 17.0 (SPSS Inc., Chicago, IL, USA). Morphometric measurements of male and female sacra were compared using Student's t-test. The receiver operating characteristic (ROC) curve analysis was performed to establish the most appropriate cut-off values for each parameter. Results The mean sacral length measured from the promontory to the apex of the sacrum was higher in males compared to females (p < 0.001), whereas the sacral index was higher in female sacra in comparison to male sacra (p < 0.001). Furthermore, the mean height of the first posterior sacral foramina (PSF) was higher in male sacra bilaterally (p < 0.05). On ROC analysis, the area under the curve was 0.994 for the sacral index and 0.862 for the sacral length. Conclusions In this study, the sacral index was noted to be the most important morphometric parameter for the identification of the sex of the sacra. Additionally, the height of the S2 body, the height of the first anterior sacral foramina, and the height of the first PSF can be contemplated with an accuracy of 60-70% if only a part of the sacrum is available for determining the sex. Hence, this study emphasizes the significance of morphometric parameters of the sacrum in the determination of sex, especially in forensic cases when the skull and pelvis are fragmented or unavailable.

Synthetic Multivalent Disulfide-Constrained Peptide Agonists Potentiate Wnt1/ß-Catenin Signaling via LRP6 Coreceptor Clustering.

Wnt ligands are critical for tissue homeostasis and form a complex with LRP6 and frizzled coreceptors to initiate Wnt/ß-catenin signaling. Yet, how different Wnts achieve various levels of signaling activation through distinct domains on LRP6 remains elusive. Developing tool ligands that target individual LRP6 domains could help elucidate the mechanism of Wnt signaling regulation and uncover pharmacological approaches for pathway modulation. We employed directed evolution of a disulfide constrained peptide (DCP) to identify molecules that bind to the third ß-propeller domain of LRP6. The DCPs antagonize Wnt3a while sparing Wnt1 signaling. Using PEG linkers with different geometries, we converted the Wnt3a antagonist DCPs to multivalent molecules that potentiated Wnt1 signaling by clustering the LRP6 coreceptor. The mechanism of potentiation is unique as it occurred only in the presence of extracellular secreted Wnt1 ligand. While all DCPs recognized a similar binding interface on LRP6, they displayed different spatial orientations that influenced their cellular activities. Moreover, structural analyses revealed that the DCPs exhibited new folds that were distinct from the parent DCP framework they were evolved from. The multivalent ligand design principles highlighted in this study provide a path for developing peptide agonists that modulate different branches of cellular Wnt signaling.

Cell diversity and plasticity during atrioventricular heart valve EMTs.

Epithelial-to-mesenchymal transitions (EMTs) of both endocardium and epicardium guide atrioventricular heart valve formation, but the cellular complexity and small scale of this tissue have restricted analyses. To circumvent these issues, we analyzed over 50,000 murine single-cell transcriptomes from embryonic day (E)7.75 hearts to E12.5 atrioventricular canals. We delineate mesenchymal and endocardial bifurcation during endocardial EMT, identify a distinct, transdifferentiating epicardial population during epicardial EMT, and reveal the activation of epithelial-mesenchymal plasticity during both processes. In Sox9-deficient valves, we observe increased epithelial-mesenchymal plasticity, indicating a role for SOX9 in promoting endothelial and mesenchymal cell fate decisions. Lastly, we deconvolve cell interactions guiding the initiation and progression of cardiac valve EMTs. Overall, these data reveal mechanisms of emergence of mesenchyme from endocardium or epicardium at single-cell resolution and will serve as an atlas of EMT initiation and progression with broad implications in regenerative medicine and cancer biology.

DNA methylation plays an important role in promoter choice and protein production at the mouse Dnmt3L locus.

The DNA methyltransferase 3-like (Dnmt3L) protein is a crucial cofactor in the germ line for the de novo methyltransferase Dnmt3a, which establishes imprints and represses transposable elements. We have previously shown that Dnmt3L transcription is regulated via three different promoters in mice, producing transcripts we term Dnmt3L(s) (stem cell), Dnmt3L(o) (oocyte) and Dnmt3L(at) (adult testis). Here we show that both Dnmt3L(s) and Dnmt3L(o) produce full-length proteins but that the Dnmt3L(at) transcripts are not translated. Although not a canonical CpG island, the Dnmt3L(s) promoter is silenced by methylation during somatic differentiation in parallel with germ-cell-specific genes. During oocyte growth, Dnmt3L(s) also becomes heavily methylated and silenced and this requires its own gene product, since there is complete loss of methylation and derepression of transcription from this promoter in oocytes derived from Dnmt3L(-/-) mice. Methylation of the Dnmt3L(s) promoter is established prior to the completion of imprinting and explains the requirement in mouse oocytes for the Dnmt3L(o) promoter, located in an intron of the neighboring unmethylated Aire gene. Overall these results give insight into how and why promoter switching at the mouse Dnmt3L locus occurs and provide one of the first examples of a non-imprinted locus where methylation plays a role in promoter choice. The derepression of the Dnmt3L(s) promoter in the knockout oocytes also suggests that other non-imprinted loci may be dysregulated in these cells and contribute to the phenotype of the resultant mice.

Role of inflammatory markers and their trends in predicting the outcome of medical expulsive therapy for distal ureteric calculus.

OBJECTIVE: Symptomatic ureteric stones cause surrounding inflammation-promoting obstruction. C-reactive protein (CRP), white blood cell count (WC), and neutrophil percentage (NP) tend to rise after inflammatory response. Monitoring response during the course of medical expulsive therapy (MET) may help in deciding early intervention, thereby decreasing morbidity. We assessed the role and trends of these markers in predicting the outcome of MET. MATERIALS AND METHODS: One hundred and ninety-two patients with distal ureteric calculus of size >5 mm were included in this prospective study from April 2017 to March 2018 after ethical committee approval. CRP, NP, and WC were measured on day 1, 7, and 14 of MET, and analysis was done. RESULTS: On univariate analysis, stone size and mean values of CRP, WC, and NP on day 1, 7 and 14 in stone nonpassers were significantly higher compared to stone passers (P < 0.05). Receiver operator curve analysis showed area under the curve value of 0.798 (P = 0.001) for CRP and cut off value determined was 1.35 mg/dL. Multivariate analysis of different variables showed significant association of higher CRP (>1.35 mg/dL) and larger stone size (>7 mm) with MET failure. Decreasing trend of CRP was seen in both groups, but values were higher in stone nonpassers. WC and NP showed decreasing trend in stone passers but persistently high in stone nonpassers. CONCLUSIONS: Higher CRP and larger stone size were associated with failure of MET. WC and NP showed decreasing trend in stone passers and persistently higher in nonpassers, which may potentially predict failure of MET, however, their role need to be further studied.

Tumor-associated antigen PRAME exhibits dualistic functions that are targetable in diffuse large B cell lymphoma.

PRAME is a prominent member of the cancer testis antigen family of proteins, which triggers autologous T cell-mediated immune responses. Integrative genomic analysis in diffuse large B cell lymphoma (DLBCL) uncovered recurrent and highly focal deletions of 22q11.22, including the PRAME gene, which were associated with poor outcome. PRAME-deleted tumors showed cytotoxic T cell immune escape and were associated with cold tumor microenvironments. In addition, PRAME downmodulation was strongly associated with somatic EZH2 Y641 mutations in DLBCL. In turn, PRC2-regulated genes were repressed in isogenic PRAME-KO lymphoma cell lines, and PRAME was found to directly interact with EZH2 as a negative regulator. EZH2 inhibition with EPZ-6438 abrogated these extrinsic and intrinsic effects, leading to PRAME expression and microenvironment restoration in vivo. Our data highlight multiple functions of PRAME during lymphomagenesis and provide a preclinical rationale for synergistic therapies combining epigenetic reprogramming with PRAME-targeted therapies.

Association of circulating monocyte chemoattractant protein-1 levels with polycystic ovary syndrome: A meta-analysis.

PROBLEM: Polycystic ovary syndrome (PCOS) is a common hormonal disorder that has a huge impact on the human infertility. Increased levels of various circulating inflammatory cytokines have been observed in PCOS patients, which can contribute to the pathogenesis of PCOS. Monocyte chemoattractant protein-1 (MCP-1), a secretory chemokine, is a potent chemotactic factor that recruits monocytes/macrophages to inflammatory foci. Several previous studies comparing the circulating MCP-1 levels between non-PCOS and PCOS patients have yielded contradictory results. Therefore, the aim of this meta-analysis was to investigate whether circulating MCP-1 levels vary between non-PCOS and PCOS patients. METHODS: Research articles published before November 11, 2020, were screened to identify eligible studies. Heterogeneity, sensitivity, and publication bias were analyzed using STATA software. Standardized mean difference (SMD) with a 95% confidence interval (CI) was calculated by the STATA software using a random-effects model. RESULTS: 11 studies were included in this meta-analysis involving 897 individuals: 368 non-PCOS patient and 529 PCOS patients. Our pooled meta-analysis results show that circulating MCP-1 levels were significantly higher in PCOS patients than in non-PCOS patients (SMD = 0.84, 95% CI = [0.37, 1.31], Z = 3.50, p < 0.01). However, due to the limited number of studies included in this meta-analysis, subgroup analysis determined that circulating MCP-1 levels were not significantly varied between obese non-PCOS and obese PCOS patients (SMD = 0.42, 95% CI = [-0.65, 1.49], Z = 0.77, p = 0.442) as well as between non-PCOS and PCOS patients without obesity (SMD = 2.04, 95% CI = [-0.84, 4.93], Z = 1.39, p = 0.166). In addition, circulating MCP-1 levels were also not significantly different between obese and non-obese PCOS patients (SMD = -0.04, 95% CI = [-0.68, 0.60], Z = 0.11, p = 0.909). CONCLUSION: Our findings reveal that circulating MCP-1 levels are upregulated in women with PCOS and are associated with an increased risk of PCOS.