Lead contamination in human milk affects infants' language trajectory: results from a prospective cohort study.

Infants growing up in low- and middle-income countries are at increased risk of suffering adverse childhood experiences, including exposure to environmental pollution and lack of cognitive stimulation. In this study, we aimed to examine the levels of metals in the human milk of women living in São Paulo City, Brazil, and determine the effects on infants' neurodevelopment. For such, a total of 185 human milk samples were analyzed for arsenic (As), lead (Pb), mercury (Hg), and cadmium (Cd) using inductively coupled plasma mass spectrometry (ICP-MS). We applied the Bayley scales of infant and toddler development Third Edition (Bayley-III) to assess developmental milestones. In our analysis, we found a mean (standard deviation) concentration of As in human milk equal to 2.76 (4.09) µg L-1, followed by Pb 2.09 (5.36) and Hg 1.96 (6.68). Cd was not detected. We observed that infants exposed to Pb presented language trajectories lower than non-exposed infants (ß = -0.413; 95% CI -0.653, -0.173) after adjustment for infant age, maternal education, socioeconomic status, infant sex, and sample weights. Our results report As, Pb, and Hg contamination in human milk, and that infant exposure to Pb decreased infants' language development. These results evidence maternal-child environmental exposure and its detrimental impact on infants' health.

Cavity approach for the approximation of spectral density of graphs with heterogeneous structures.

Graphs have become widely used to represent and study social, biological, and technological systems. Statistical methods to analyze empirical graphs were proposed based on the graph's spectral density. However, their running time is cubic in the number of vertices, precluding direct application to large instances. Thus, efficient algorithms to calculate the spectral density become necessary. For sparse graphs, the cavity method can efficiently approximate the spectral density of locally treelike undirected and directed graphs. However, it does not apply to most empirical graphs because they have heterogeneous structures. Thus, we propose methods for undirected and directed graphs with heterogeneous structures using a new vertex's neighborhood definition and the cavity approach. Our methods' time and space complexities are O(|E|h_{max}^{3}t) and O(|E|h_{max}^{2}t), respectively, where |E| is the number of edges, h_{max} is the size of the largest local neighborhood of a vertex, and t is the number of iterations required for convergence. We demonstrate the practical efficacy by estimating the spectral density of simulated and real-world undirected and directed graphs.

A model-based clustering algorithm with covariates adjustment and its application to lung cancer stratification.

Usually, the clustering process is the first step in several data analyses. Clustering allows identify patterns we did not note before and helps raise new hypotheses. However, one challenge when analyzing empirical data is the presence of covariates, which may mask the obtained clustering structure. For example, suppose we are interested in clustering a set of individuals into controls and cancer patients. A clustering algorithm could group subjects into young and elderly in this case. It may happen because the age at diagnosis is associated with cancer. Thus, we developed CEM-Co, a model-based clustering algorithm that removes/minimizes undesirable covariates' effects during the clustering process. We applied CEM-Co on a gene expression dataset composed of 129 stage I non-small cell lung cancer patients. As a result, we identified a subgroup with a poorer prognosis, while standard clustering algorithms failed.

Spectral density-based clustering algorithms for complex networks.

Introduction: Clustering is usually the first exploratory analysis step in empirical data. When the data set comprises graphs, the most common approaches focus on clustering its vertices. In this work, we are interested in grouping networks with similar connectivity structures together instead of grouping vertices of the graph. We could apply this approach to functional brain networks (FBNs) for identifying subgroups of people presenting similar functional connectivity, such as studying a mental disorder. The main problem is that real-world networks present natural fluctuations, which we should consider. Methods: In this context, spectral density is an exciting feature because graphs generated by different models present distinct spectral densities, thus presenting different connectivity structures. We introduce two clustering methods: k-means for graphs of the same size and gCEM, a model-based approach for graphs of different sizes. We evaluated their performance in toy models. Finally, we applied them to FBNs of monkeys under anesthesia and a dataset of chemical compounds. Results: We show that our methods work well in both toy models and real-world data. They present good results for clustering graphs presenting different connectivity structures even when they present the same number of edges, vertices, and degree of centrality. Discussion: We recommend using k-means-based clustering for graphs when graphs present the same number of vertices and the gCEM method when graphs present a different number of vertices.

Autoregulation of blood flow drives early hypotension in a rat model of systemic inflammation induced by bacterial lipopolysaccharide.

Uncontrolled vasodilation is known to account for hypotension in the advanced stages of sepsis and other systemic inflammatory conditions, but the mechanisms of hypotension in earlier stages of such conditions are not clear. By monitoring hemodynamics with the highest temporal resolution in unanesthetized rats, in combination with ex-vivo assessment of vascular function, we found that early development of hypotension following injection of bacterial lipopolysaccharide is brought about by a fall in vascular resistance when arterioles are still fully responsive to vasoactive agents. This approach further uncovered that the early development of hypotension stabilized blood flow. We thus hypothesized that prioritization of the local mechanisms of blood flow regulation (tissue autoregulation) over the brain-driven mechanisms of pressure regulation (baroreflex) underscored the early development of hypotension in this model. Consistent with this hypothesis, an assessment of squared coherence and partial-directed coherence revealed that, at the onset of hypotension, the flow-pressure relationship was strengthened at frequencies (<0.2 Hz) known to be associated with autoregulation. The autoregulatory escape to phenylephrine-induced vasoconstriction, another proxy of autoregulation, was also strengthened in this phase. The competitive demand that drives prioritization of flow over pressure regulation could be edema-associated hypovolemia, as this became detectable at the onset of hypotension. Accordingly, blood transfusion aimed at preventing hypovolemia brought the autoregulation proxies back to normal and prevented the fall in vascular resistance. This novel hypothesis opens a new avenue of investigation into the mechanisms that can drive hypotension in systemic inflammation.

Predicting survival in metastatic non-small cell lung cancer patients with poor ECOG-PS: A single-arm prospective study.

BACKGROUND: Patients with advanced non-small cell lung cancer (NSCLC) are a heterogeneous population with short lifespan. We aimed to develop methods to better differentiate patients whose survival was >90 days. METHODS: We evaluated 83 characteristics of 106 treatment-naïve, stage IV NSCLC patients with Eastern Cooperative Oncology Group Performance Status (ECOG-PS) >1. Automated machine learning was used to select a model and optimize hyperparameters. 100-fold bootstrapping was performed for dimensionality reduction for a second ("lite") model. Performance was measured by C-statistic and accuracy metrics in an out-of-sample validation cohort. The "lite" model was validated on a second independent, prospective cohort (N = 42). Network analysis (NA) was performed to evaluate the differences in centrality and connectivity of features. RESULTS: The selected method was ExtraTrees Classifier, with C-statistic of 0.82 (p < 0.01) and accuracy of 0.81 (p = 0.01). The "lite" model had 16 variables and obtained C-statistic of 0.84 (p < 0.01) and accuracy of 0.75 (p = 0.039) in the first cohort, and C-statistic of 0.706 (p < 0.01) and accuracy of 0.714 (p < 0.01) in the second cohort. The networks of patients with lower survival were more interconnected. Features related to cachexia, inflammation, and quality of life had statistically different prestige scores in NA. CONCLUSIONS: Machine learning can assist in the prognostic evaluation of advanced NSCLC. The model generated with a reduced number of features showed high accessibility and reasonable metrics. Features related to quality of life, cachexia, and performance status had increased correlation and importance scores, suggesting that they play a role at later disease stages, in line with the biological rationale already described.

Applications of graph theory to the analysis of fNIRS data in hyperscanning paradigms.

Hyperscanning is a promising tool for investigating the neurobiological underpinning of social interactions and affective bonds. Recently, graph theory measures, such as modularity, have been proposed for estimating the global synchronization between brains. This paper proposes the bootstrap modularity test as a way of determining whether a pair of brains is coactivated. This test is illustrated as a screening tool in an application to fNIRS data collected from the prefrontal cortex and temporoparietal junction of five dyads composed of a teacher and a preschooler while performing an interaction task. In this application, graph hub centrality measures identify that the dyad's synchronization is critically explained by the relation between teacher's language and number processing and the child's phonological processing. The analysis of these metrics may provide further insights into the neurobiological underpinnings of interaction, such as in educational contexts.

Genome-wide promoter methylation profiling in a cellular model of melanoma progression reveals markers of malignancy and metastasis that predict melanoma survival.

The epigenetic changes associated with melanoma progression to advanced and metastatic stages are still poorly understood. To shed light on the CpG methylation dynamics during melanoma development, we analyzed the methylome profiles of a four-stage cell line model of melanoma progression: non-tumorigenic melanocytes (melan-a), premalignant melanocytes (4C), non-metastatic melanoma cells (4C11-), and metastatic melanoma cells (4C11+). We identified 540 hypo- and 37 hypermethylated gene promoters that together characterized a malignancy signature, and 646 hypo- and 520 hypermethylated promoters that distinguished a metastasis signature. Differentially methylated genes from these signatures were correlated with overall survival using TCGA-SKCM methylation data. Moreover, multivariate Cox analyses with LASSO regularization identified panels of 33 and 31 CpGs, respectively, from the malignancy and metastasis signatures that predicted poor survival. We found a concordant relationship between DNA methylation and transcriptional levels for genes from the malignancy (Pyroxd2 and Ptgfrn) and metastasis (Arnt2, Igfbp4 and Ptprf) signatures, which were both also correlated with melanoma prognosis. Altogether, this study reveals novel CpGs methylation markers associated with malignancy and metastasis that collectively could improve the survival prediction of melanoma patients.

The intracellular bacterium Rickettsia rickettsii exerts an inhibitory effect on the apoptosis of tick cells.

BACKGROUND: Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector-pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26. METHODS: The proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined. RESULTS: Tick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth. CONCLUSIONS: Taken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.

Noninvasive prenatal paternity determination using microhaplotypes: a pilot study.

BACKGROUND: The use of noninvasive techniques to determine paternity prenatally is increasing because it reduces the risks associated with invasive procedures. Current methods, based on SNPs, use the analysis of at least 148 markers, on average. METHODS: To reduce the number of regions, we used microhaplotypes, which are chromosomal segments smaller than 200 bp containing two or more SNPs. Our method employs massively parallel sequencing and analysis of microhaplotypes as genetic markers. We tested 20 microhaplotypes and ascertained that 19 obey Hardy-Weinberg equilibrium and are independent, and data from the 1000 Genomes Project were used for population frequency and simulations. RESULTS: We performed simulations of true and false paternity, using the 1000 Genomes Project data, to confirm if the microhaplotypes could be used as genetic markers. We observed that at least 13 microhaplotypes should be used to decrease the chances of false positives. Then, we applied the method in 31 trios, and it was able to correctly assign the fatherhood in cases where the alleged father was the real father, excluding the inconclusive results. We also cross evaluated the mother-plasma duos with the alleged fathers for false inclusions within our data, and we observed that the use of at least 15 microhaplotypes in real data also decreases the false inclusions. CONCLUSIONS: In this work, we demonstrated that microhaplotypes can be used to determine prenatal paternity by using only 15 regions and with admixtures of DNA.

Fluorescence-based method is more accurate than counting-based methods for plotting growth curves of adherent cells.

OBJECTIVE: Cell growth curves constitute one of the primary assays employed to analyze cell proliferation dynamics of in vitro cultured cells under specific culture conditions. From the cell growth curve, it is possible to assess the behavior of proliferating cells under different conditions, such as drug treatment and genomic editions. Traditionally, growth curves for adherent cells are obtained by seeding the cells in multiple-well plates and counting the total number of cells at different time points. Here, we compare this traditional method to the fluorescence-based method, which is based on the CFSE fluorescence decay over time. RESULTS: The fluorescence-based method is not dependent on the determination of the total number of cells, but rather is approached by assessing the fluorescence of a sample of single cells from a cell population at different time points after plating. Therefore, this method is not biased due to either cell loss during harvesting or to the presence of cellular debris and cell clumps. Moreover, the fluorescence-based method displays lower variation among different measurements of the same time point, which increases the reliability on the determination of lag, log and stationary phase transitions.

Large miRNA survival analysis reveals a prognostic four-biomarker signature for triple negative breast cancer.

Triple negative breast cancer (TNBC) is currently the only major breast tumor subtype without effective targeted therapy and, as a consequence, usually presents a poor outcome. Due to its more aggressive phenotype, there is an urgent clinical need to identify novel biomarkers that discriminate individuals with poor prognosis. We hypothesize that miRNAs can be used to this end because they are involved in the initiation and progression of tumors by altering the expression of their target genes. To identify a prognostic biomarker in TNBC, we analyzed the miRNA expression of a cohort composed of 185 patients diagnosed with TNBC using penalized Cox regression models. We identified a four-biomarker signature based on miR-221, miR-1305, miR-4708, and RMDN2 expression levels that allowed for the subdivision of TNBC into high- or low-risk groups (Hazard Ratio - HR = 0.32; 95% Confidence Interval - CI = 0.11-0.91; p = 0.03) and are also statistically associated with survival outcome in subgroups of postmenopausal status (HR = 0.19; 95% CI = 0.04-0.90; p= 0.016), node negative status (HR = 0.12; 95% CI = 0.01-1.04; p = 0.026), and tumors larger than 2cm (HR = 0.21; 95% CI = 0.05-0.81; p = 0.021). This four-biomarker signature was significantly associated with TNBC as an independent prognostic factor for survival.

Fluorescence-based method is more accurate than counting-based methods for plotting growth curves of adherent cells

Objective: Cell growth curves constitute one of the primary assays employed to analyze cell proliferation dynamics of in vitro cultured cells under specific culture conditions. From the cell growth curve, it is possible to assess the behavior of proliferating cells under different conditions, such as drug treatment and genomic editions. Traditionally, growth curves for adherent cells are obtained by seeding the cells in multiple-well plates and counting the total number of cells at different time points. Here, we compare this traditional method to the fluorescence-based method, which is based on the CFSE fluorescence decay over time. Results: The fluorescence-based method is not dependent on the determination of the total number of cells, but rather is approached by assessing the fluorescence of a sample of single cells from a cell population at different time points after plating. Therefore, this method is not biased due to either cell loss during harvesting or to the presence of cellular debris and cell clumps. Moreover, the fluorescence-based method displays lower variation among different measurements of the same time point, which increases the reliability on the determination of lag, log and stationary phase transitions.

Potential Confounders in the Analysis of Brazilian Adolescent's Health: A Combination of Machine Learning and Graph Theory.

The prevalence of health problems during childhood and adolescence is high in developing countries such as Brazil. Social inequality, violence, and malnutrition have strong impact on youth health. To better understand these issues we propose to combine machine-learning methods and graph analysis to build predictive networks applied to the Brazilian National Student Health Survey (PenSE 2015) data, a large dataset that consists of questionnaires filled by the students. By using a combination of gradient boosting machines and centrality hub metric, it was possible to identify potential confounders to be considered when conducting association analyses among variables. The variables were ranked according to their hub centrality to predict the other variables from a directed weighted-graph perspective. The top five ranked confounder variables were "gender", "oral health care", "intended education level", and two variables associated with nutrition habits-"eat while watching TV" and "never eat fast-food". In conclusion, although causal effects cannot be inferred from the data, we believe that the proposed approach might be a useful tool to obtain novel insights on the association between variables and to identify general factors related to health conditions.

BioNetStat: A Tool for Biological Networks Differential Analysis.

The study of interactions among biological components can be carried out by using methods grounded on network theory. Most of these methods focus on the comparison of two biological networks (e.g., control vs. disease). However, biological systems often present more than two biological states (e.g., tumor grades). To compare two or more networks simultaneously, we developed BioNetStat, a Bioconductor package with a user-friendly graphical interface. BioNetStat compares correlation networks based on the probability distribution of a feature of the graph (e.g., centrality measures). The analysis of the structural alterations on the network reveals significant modifications in the system. For example, the analysis of centrality measures provides information about how the relevance of the nodes changes among the biological states. We evaluated the performance of BioNetStat in both, toy models and two case studies. The latter related to gene expression of tumor cells and plant metabolism. Results based on simulated scenarios suggest that the statistical power of BioNetStat is less sensitive to the increase of the number of networks than Gene Set Coexpression Analysis (GSCA). Also, besides being able to identify nodes with modified centralities, BioNetStat identified altered networks associated with signaling pathways that were not identified by other methods.

Dynamic Gene Network Analysis of Caco-2 Cell Response to Shiga Toxin-Producing Escherichia coli-Associated Hemolytic-Uremic Syndrome.

Shiga toxin-producing Escherichia coli (STEC) O113:H21 strains are associated with human diarrhea and some strains may cause hemolytic-uremic syndrome (HUS). In Brazil, these strains are commonly found in cattle but, so far, were not isolated from HUS patients. Here, a system biology approach was used to investigate the differential transcriptomic and phenotypic responses of enterocyte-like Caco-2 cells to two STEC O113:H21 strains with similar virulence factor profiles (i.e. expressing stx2, ehxA, epeA, espA, iha, saa, sab, and subA): EH41 (Caco-2/EH41), isolated from a HUS patient in Australia, and Ec472/01 (Caco-2/Ec472), isolated from bovine feces in Brazil, during a 3 h period of bacteria-enterocyte interaction. Gene co-expression network analysis for Caco-2/EH41 revealed a quite abrupt pattern of topological variation along 3 h of enterocyte-bacteria interaction when compared with networks obtained for Caco-2/Ec472. Transcriptional module characterization revealed that EH41 induces inflammatory and apoptotic responses in Caco-2 cells just after the first hour of enterocyte-bacteria interaction, whereas the response to Ec472/01 is associated with cytoskeleton organization at the first hour, followed by the expression of immune response modulators. Scanning electron microscopy showed more intense microvilli destruction in Caco-2 cells exposed to EH41 when compared to those exposed to Ec472/01. Altogether, these results show that EH41 expresses virulence genes, inducing a distinctive host cell response, and is likely associated with severe pathogenicity.

The Transcriptome of the Salivary Glands of Amblyomma aureolatum Reveals the Antimicrobial Peptide Microplusin as an Important Factor for the Tick Protection Against Rickettsia rickettsii Infection.

The salivary glands (SG) of ixodid ticks play a pivotal role in blood feeding, producing both the cement and the saliva. The cement is an adhesive substance that helps the attachment of the tick to the host skin, while the saliva contains a rich mixture of antihemostatic, anti-inflammatory, and immunomodulatory substances that allow ticks to properly acquire the blood meal. The tick saliva is also a vehicle used by several pathogens to be transmitted to the vertebrate host, including various bacterial species from the genus Rickettsia. Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes the severe Rocky Mountain spotted fever. In Brazil, the dog yellow tick Amblyomma aureolatum is a vector of R. rickettsii. In the current study, the effects of an experimental infection with R. rickettsii on the global gene expression profile of A. aureolatum SG was determined by next-generation RNA sequencing. A total of 260 coding sequences (CDSs) were modulated by infection, among which 161 were upregulated and 99 were downregulated. Regarding CDSs in the immunity category, we highlight one sequence encoding one microplusin-like antimicrobial peptide (AMP) (Ambaur-69859). AMPs are important effectors of the arthropod immune system, which lack the adaptive response of the immune system of vertebrates. The expression of microplusin was confirmed to be significantly upregulated in the SG as well as in the midgut (MG) of infected A. aureolatum by a quantitative polymerase chain reaction preceded by reverse transcription. The knockdown of the microplusin expression by RNA interference caused a significant increase in the prevalence of infected ticks in relation to the control. In addition, a higher rickettsial load of one order of magnitude was recorded in both the MG and SG of ticks that received microplusin-specific dsRNA. No effect of microplusin knockdown was observed on the R. rickettsii transmission to rabbits. Moreover, no significant differences in tick engorgement and oviposition were recorded in ticks that received dsMicroplusin, demonstrating that microplusin knockdown has no effect on tick fitness. Further studies must be performed to determine the mechanism of action of this AMP against R. rickettsii.

Gene expression and promoter methylation of angiogenic and lymphangiogenic factors as prognostic markers in melanoma.

The high mortality rate of melanoma is broadly associated with its metastatic potential. Tumor cell dissemination is strictly dependent on vascularization; therefore, angiogenesis and lymphangiogenesis play an essential role in metastasis. Hence, a better understanding of the players of tumor vascularization and establishing them as new molecular biomarkers might help to overcome the poor prognosis of melanoma patients. Here, we further characterized a linear murine model of melanoma progression and showed that the aggressiveness of melanoma cells is closely associated with high expression of angiogenic factors, such as Vegfc, Angpt2, and Six1, and that blockade of the vascular endothelial growth factor pathway by the inhibitor axitinib abrogates their tumorigenic potential in vitro and in the in vivo chicken chorioallantoic membrane assay. Furthermore, analysis of The Cancer Genome Atlas data revealed that the expression of the angiogenic factor ANGPT2 (P-value = 0.044) and the lymphangiogenic receptor VEGFR-3 (P-value = 0.002) were independent prognostic factors of overall survival in melanoma patients. Enhanced reduced representation bisulfite sequencing-based methylome profiling revealed for the first time a link between abnormal VEGFC, ANGPT2, and SIX1 gene expression and promoter hypomethylation in melanoma cells. In patients, VEGFC (P-value = 0.031), ANGPT2 (P-value < 0.001), and SIX1 (P-value = 0.009) promoter hypomethylation were independent prognostic factors of shorter overall survival. Hence, our data suggest that these angio- and lymphangiogenesis factors are potential biomarkers of melanoma prognosis. Moreover, these findings strongly support the applicability of our melanoma progression model to unravel new biomarkers for this aggressive human disease.

CHD7 promotes glioblastoma cell motility and invasiveness through transcriptional modulation of an invasion signature.

Chromatin remodeler proteins exert an important function in promoting dynamic modifications in the chromatin architecture, performing a central role in regulating gene transcription. Deregulation of these molecular machines may lead to striking perturbations in normal cell function. The CHD7 gene is a member of the chromodomain helicase DNA-binding family and, when mutated, has been shown to be the cause of the CHARGE syndrome, a severe developmental human disorder. Moreover, CHD7 has been described to be essential for neural stem cells and it is also highly expressed or mutated in a number of human cancers. However, its potential role in glioblastoma has not yet been tested. Here, we show that CHD7 is up-regulated in human glioma tissues and we demonstrate that CHD7 knockout (KO) in LN-229 glioblastoma cells suppresses anchorage-independent growth and spheroid invasion in vitro. Additionally, CHD7 KO impairs tumor growth and increases overall survival in an orthotopic mouse xenograft model. Conversely, ectopic overexpression of CHD7 in LN-428 and A172 glioblastoma cell lines increases cell motility and invasiveness in vitro and promotes LN-428 tumor growth in vivo. Finally, RNA-seq analysis revealed that CHD7 modulates a specific transcriptional signature of invasion-related target genes. Further studies should explore clinical-translational implications for glioblastoma treatment.

HLA-G is upregulated in advanced endometriosis.

OBJECTIVE: To assess whether the HLA-G immunomodulatory protein is potentially involved in the pathophysiology of endometriosis or disease progression. STUDY DESIGN: Cross-sectional observational study of 227 women who underwent laparoscopy, being 146 for endometriosis excision and 81 for elective tubal ligation (control group). Soluble HLA-G (sHLA-G) levels in the serum and peritoneal fluid (PF), as well as the HLA-G protein expression in matched eutopic and ectopic endometrium of women with and without endometriosis were evaluated by ELISA and immunohistochemistry assays, respectively. Women with endometriosis were separated into groups according to the initial (I/II, n = 60) and advanced (III/IV, n = 86) stages of disease. sHLA-G measurement was performed only in women with matched serum and PF samples in both the control (CTRL; n = 77) and endometriosis (EDT; I-II, n = 60; III-IV, n = 83) groups. HLA-G protein expression was evaluated in 26 women with deep endometriosis (I-II, n = 12; III-IV, n = 14) and 22 controls. RESULTS: Higher concentrations of sHLA-G (P = 0.013) in the serum but not in the PF were observed in women with advanced endometriosis compared to the control group. In situ expression of HLA-G protein was also higher in ectopic (P = 0.018) but not in eutopic endometrium of women with advanced endometriosis compared to control group. CONCLUSION: Our findings suggest that HLA-G upregulation in advanced stages may contribute to the state of immunosuppression in endometriosis as disease progresses.