Several studies have linked maternal asthma, excess BMI, and low vitamin D status with increased risk of Preeclampsia (PE) development. Given prior evidence in the literature and our observations from the subjects in the Vitamin D Antenatal Asthma Reduction Trial (VDAART), we hypothesized that PE, maternal asthma, vitamin D insufficiency, and excess body mass index (BMI) might share both peripheral blood and placental gene signatures that link these conditions together. We used samples collected in the VDAART to investigate relationships between these four conditions and gene expression patterns in peripheral blood obtained at early pregnancy. We identified a core set of differentially expressed genes in all comparisons between women with and without these four conditions and confirmed them in two separate sets of samples. We confirmed the differential expression of the shared gene signatures in the placenta from an independent study of preeclampsia cases and controls and constructed the preeclampsia module using protein–protein interaction networks. CXC chemokine genes showed the highest degrees of connectivity and betweenness centrality in the peripheral blood and placental modules. The shared gene signatures demonstrate the biological pathways involved in preeclampsia at the pre-clinical stage and may be used for the prediction of preeclampsia. This pregnancy-specific complication accounts for a quarter of all maternal mortality1 and causes substantial maternal and prenatal morbidity4. Because the definitive etiopathogenesis of PE is not yet understood, preventive measures are scarce 5, and the only definitive treatment is delivery of the fetus. Previous investigations have identified multiple risk factors for PE, a closer exploration of which might aid in identifying the responsible biological pathways and help us identify novel strategies for its primary and secondary prevention. Several studies and systematic reviews of these studies demonstrated that maternal asthma6, vitamin D insufficiency7, and excess body mass index (BMI)9 during pregnancy are risk factors for PE. Results from other investigations suggest these risk factors might interact and share pathobiological pathways. Excess BMI has been linked to asthma12, a pro-inflammatory environment during pregnancy, both systemically and in the placenta13, and modulation of bioavailability of vitamin D14. Finally, vitamin D status also affects maternal asthma activity during pregnancy15 and contributes directly to PE risk17.Preeclampsia (PE) is a complex pregnancy disorder that can affect up to 8% of pregnant women20. Findings from these studies also demonstrated the interplay between maternal asthma, BMI, and vitamin D levels during pregnancy, particularly in early pregnancy 21. Abnormal placentation and immune interactions at the maternal–fetal interface have been recognized as the root cause of PE22; however, the onset, severity, and progression of the disease are significantly affected by maternal risk factors and response to molecules released from the placenta. Direct exploration of dysregulation in the biological processes related to PE is not easily studied at early gestational time points owing to limited access to placental tissue in development. However, maternal peripheral blood is accessible at early pregnancy, and blood transcriptional changes could mirror placental processes, providing additional insight into molecular mechanisms occurring in women who develop PE and resulting in an improved predictive capability.The Vitamin D Antenatal Asthma Reduction Trial (VDAART) was a randomized, double-blind, placebo-controlled clinical trial of vitamin D supplementation  in pregnant women to prevent the development of pregnancy complications such as PE and asthma or atopy in their children by the age of 3 years old. Ancillary studies conducted using data from this trial corroborate the evidence that maternal asthma, particularly lack of asthma control, vitamin D (25-hydroxyvitamin D [25OHD]) deficiency, and excess BMI are risk factors for PEGiven prior evidence in the literature and from the VDAART cohort, we hypothesized that PE, maternal asthma, vitamin D insufficiency, and excess BMI in early pregnancy might share dysregulated biological pathways in peripheral blood and placenta. To investigate this hypothesis, we conducted a microarray differential gene expression study in a nested case–control subset of pregnant women participating in the VDAART trial. We identified a core set of differentially expressed gene signatures associated with each study condition that was confirmed in the early pregnancy of separate sets of pregnant women Fig. . To demoP < 0.001). Vitamin D insufficiency (25OHD < 30 ng/ml) at 10–18 wga was also more frequent among pregnant women with a PE diagnosis compared with controls with no complication during their pregnancies . Among 104 pregnant women who had a BMI ≥ 25 kg/m2 at early pregnancy, 34 women (33%) had PE during their pregnancies. Among 63 pregnant women with asthma, 20 had PE (32%). However, the distribution of pregnant women with a BMI ≥ 25 kg/m2 at early pregnancy and those with asthma were not different between pregnancies with and without PE  had a mean gestational age of 14 ± 2.66 weeks . Among other baseline characteristics of this cohort , who were used for the replication of differentially expressed (DE) PE gene signatures identified in the discovery VDAART cohort, were mainly white pregnant women  with the mean of gestational ages 16 ± 2.0 weeks (11–18 wga). Further details of the OMEGA study population’s characteristics by PE status are publishedP = 0.025).The VDAART replication cohort N = 24) was selected among asthmatic pregnant women who did not have PE or gestational hypertension during pregnancy. The baseline characteristics by asthma status are provided in Table 4 was selFigures 2 was also associated with differential expression of 1,107 genes (608 upregulated) relative to those with a BMI < 25 kg/m2 (FDR < 0.05). Vitamin D status of these subjects dichotomized at 30 ng/mL resulted in the differential expression of 789 genes (369 upregulated) associated with early-pregnancy vitamin D insufficiency .Differential expression analysis of peripheral blood transcriptomes at early pregnancy (10–18 wga) identified 965 genes with differential expressions (463 upregulated) in pregnant women who later developed PE relative to their controls with uncomplicated pregnancies in the discovery cohort (FDR < 0.05). In the same cohort and time point, there were 1,214 (603 upregulated) genes with differential expression between pregnant women with and without asthma (FDR < 0.05). An early-pregnancy BMI ≥ 25 kg/mP < 0.05) in an extra set of pregnant women from a second cohort. Genes with differential expressions identified in the discovery stage in association with maternal asthma, excess BMI, and vitamin D insufficiency at early pregnancy were confirmed in the peripheral blood of pregnant women at 10–18 wga with no PE complication during their pregnancies. Accordingly, 625 (323 upregulated) out of 1214 DE genes (51%) in association with maternal asthma status were confirmed in the VDAART replication cohort (P < 0.05); 664 (363 upregulated) out of 1107 genes (60%) with differential expression related to BMI status were also confirmed in the VDAART replication cohort (P < 0.05). Similarly, 494 (215 upregulated) out of 789 of the DE genes (62%) related to vitamin D status were confirmed in the VDAART replication cohort (P < 0.05).Of 965 genes with differential expression in association with PE status in the discovery cohort, 507 genes (53%) (244 upregulated) were confirmed in the peripheral blood of pregnant women at 11–18 wga who later developed PE relative to their controls  comprised the overlapping gene signatures ; innate and adaptive immune response ; inflammatory response (N = 33); neutrophil-mediated immunity (N = 37); regulation of cytokine and T-cell activation (N = 28 and 14 respectively); cytokine-mediated signaling pathway (N = 23); interleukin-10 (IL-10) production; and reactive oxygen species metabolic process (N = 13), all with a corrected P-value < 0.05; Fig. Functional terms that were enriched for asthma, vitamin D and BMI gene signature sets demonstrated high GO semantic similarity measures with PE gene signature sets , suggesting the core similarity function of enriched biological and molecular pathways in the four sets of gene signatures. Two hundred and seven of 238 overlapping gene signatures (87%) of the four sets of replicated DE genes  were mapped to their known GO functional terms. GO enrichment analysis linked the 238 overlapping gene signatures to several immunologic functions including immune system process, response, and regulation . Accordingly, among the PE gene signatures, 474 (93.49% [474/507]) were mapped onto the PPI network. The number of mapped genes for the gene signatures associated with maternal asthma, BMI and vitamin D status were 551 (88.16% [551/625]), 579 (87.20% [579/664]), and 436 (88.26% [436/494]), respectively.After replication, each of the four sets of gene signatures was individually mapped onto the interactome and demonstrated enrichment and local clustering in the PPI network . The members (nodes) of the module demonstrated an internal average degree of 5 (529 direct interactions) with an average local clustering coefficient of 0.43 in the PPI network, as a measure of how complete the neighborhood of a module node was.Of the 238 overlapping gene signatures, 223 (93.70%) genes were mapped onto the interactome and comprised the overlapping gene module , HP, CD274, ITGA2B, ORM1, CD24, BPI, NLRP3, FCGR2B, and OLFM4 showed high degrees of connectivity and betweenness centrality , namely, the “observable overlapping module” Fig. . The LCCity Fig. .We ran a sensitivity analysis to further validate our results by running differential expression analysis on subjects with all four study conditions (N = 15) versus subjects with none of the study conditions (N = 13). 202 of the genes revealed in this analysis appeared in the list of 238 replicate overlapping gene signatures (85% [202/238]) , we also evaluated their closeness to each other in the PPI network. All modules were significantly close to one another, with all Z-scores < − 1.65, corresponding to a N = 238 genes with an expression value in all samples at each trimester). One hundred twenty-one genes out of 238 overlapping signatures in the peripheral blood (50.84%) were differentially expressed between the first and second trimesters; of which 119 were mapped onto the interactome with 67 genes having direct interactions comprising the LCC , of which, 105 were mapped onto the interactome with an LCC of 50 nodes. Of note, 74 genes that were differentially expressed in the first trimester relative to the second trimester were also differentially expressed in the third trimester relative to the second trimester  from healthy pregnancies, all of the genes in the overlapping signature module were expressed in the placenta samples from the first, second, and third trimesters , IL10, IL4, and IL6, genes in the neighborhood of the LCC genes. Of note, CYP27A1 was a member of the replicated DE overlapping gene signatures and was also downregulated in peripheral blood of those pregnant women who later developed PE during their pregnancies.N = 238 genes with an expression value in term-placenta of healthy and preeclamptic pregnancies). Ninety-eight genes out of 238 genes (41%) in the overlapping signatures in peripheral blood were differentially expressed in the term-placenta of pregnancies with PE relative to those from healthy pregnancies , the genes in the overlapping signature module were expressed in term-placenta samples of healthy pregnancies and those from pregnancies with PE  and visualize their connections. We further explored the tissue specificity of these gene signatures in the placenta from preeclamptic and normal pregnancies and ultimately identified the shared signatures (submodule) in peripheral blood and placenta.18. Given these results, we felt that the transcriptomic-level analyses on these four study conditions was warranted to investigate biological pathways related to PE at the pre-clinical stage. Blood may be a medium for surrogate information on the proteins expressed in the placenta. Previous investigations conducted gene expression analyses on pregnancies with PE versus those with uncomplicated pregnancies in either peripheral blood or placental tissue25 mostly at term and delivery; however, few studies have sought to identify gene networks related to PE and its maternal risk factors, i.e., asthma, BMI, and vitamin D status in peripheral blood and placental tissue at early pregnancy26.Previous investigations conducted in the VDAART cohort (N = 816), as well as external cohorts, revealed that maternal asthma, early-pregnancy excess BMI and vitamin D insufficiency are risk factors for PE developmentOur analysis revealed a common whole-blood gene signature associated with PE and the risk of factors of maternal asthma, excess BMI, and vitamin D insufficiency. Sensitivity analysis proved the robustness of this gene signature when compared to genes differentially expressed between subjects with all conditions versus those with no conditions. Furthermore, we found modular closeness between the four gene sets as well as a gene network representing the intersection of our study conditions. Through functional enrichment, we found that many of the genes in each get set and the overlapping network were related to immune system processes, thereby suggesting a functional closeness of the modules to one another and their biological interactions at early pregnancy.27. The genes that displayed a high level of betweenness among overlapping whole-blood gene signatures were ARG1, PPBP, and CXCL10. C-X-C motif chemokine 10 (CXCL10), a member of the chemokine family, has been previously linked in the literature to PE, with one study finding significantly higher levels of CXCL10 in PE patients versus controls28. Another investigation reported that vitamin D receptor agonists exert a suppression of inflammatory processes and reduce the release of CXCL1029. A few studies have shown an inverse relationship of serum CXCL10 and vitamin D concentrations in autoimmune diseases30 that could potentially be rectified with vitamin D supplementation32. CXCL10 is also a powerful chemokine inducing Th1-mediated responses (i.e. IFN-gamma and IL-2) whose suppressions are essential for a shift in TH1/TH2 balance for a normal pregnancy33; higher levels of CXCL10 have been shown in PE34. Relevantly, vitamin D receptor (VDR) agonists could switch the immune system cell balance from Th1 to Th2 dominance while counteracting CXCL10 production and release by different cell types29.Several genes in the overlapping module exhibited high centrality measures, i.e., connectivity degree with neighboring genes and betweenness centrality degree, defined as the extent to which a gene lies on the shortest path between two other genes. Genes with a high level of connectivity and betweenness centrality degrees can be leverage points in a network system owing to their control over the passage of information between network components ARG1), an anti-inflammatory-specific gene has been linked to allergic airway inflammation and asthma35 and vitamin D-mediated inflammation inhibition36. ARG1 is an important effector molecule for granulocytic myeloid-derived suppressor cells (G-MDSCs) which maintain maternal–fetal tolerance during pregnancy. ARG1 has found to be reduced in PE patients compared to healthy pregnant women resulting in inhibition of the development of G-MDSCs37. Additionally, one characteristic of hypertension, a major feature of PE, is a decreased endothelium-dependent nitric oxide (NO)-mediated vasodilation. The upregulation of vascular ARG1, which is involved in L-arginine/NO metabolism, could also contribute to decreased NO-mediated vasodilation and hypertension38.Increased expression of arginase 1 (PPBP), another highly connected gene in our network, has been identified as a potential placental biomarker for PE and asthma in the literature40. Other noteworthy genes include cysteine-rich secretory protein 3 (CRISP3), which has also been linked to the development of PE, asthma, and weight gain43 and metalloproteinase-8 (MMP8) which has been reported in association with pre and in-pregnancy excess BMI, PE, and asthma as well4445. Notably, our peripheral blood transcriptome analysis of spontaneous preterm birth (sPTB: 24–32 wga vs Term: > 37 wga) and vitamin D insufficiency status (< 30 ng/mL) in mothers from the VDAART revealed CRISP3, ARG1, and MMP8 as highly connected genes in the overlapping module48. These genes might play an important role in the pathobiology of both PE and sPTB at earlier stages of pregnancy and their function might be modified by vitamin D status during pregnancy as well as other maternal risk factors.Pro-Platelet Basic Protein  are recognized to be central in the pathogenesis of PEThe shared gene signature we identified across four study conditions provides evidence for tissue-specific gene signatures that not only link these conditions together but might allow us to identify high-risk women predisposed to PE in early pregnancy given a sample of peripheral blood. Furthermore, this gene signature provides insight into disease conditions that are associated with the later development of PE, a finding that can help direct early intervention for the prevention of PE. The shared gene signatures between these comparisons suggest that deviation of normal placenta biological process is partly trimester-dependent which could be influenced by maternal factors Fig. .A limitation of this study was the sample size of the replication cohorts in peripheral blood and placenta from normal pregnancy which might have affected the size of the corresponding modules. Nevertheless, this limitation was inevitable based on the currently available datasets relevant to this study by pregnancy time points and conditions. Additionally, maternal asthma status was determined based on the participant’s report of physician-diagnosed asthma.In conclusion, our investigation revealed a network that represents common gene signatures in peripheral encompassing PE, asthma, excess BMI and insufficient vitamin D status at early pregnancy. We found that these DE peripheral blood genes were also present in placental tissue, suggesting that the mechanisms in which maternal asthma, BMI, and insufficient vitamin D put women at risk for PE may come into effect during early pregnancy through affecting the immune responses or dysregulation of biological processes in the placenta. Based on these findings, further studies to distinguish the key placental intrinsic and extrinsic factors responsible for the establishment of normal pregnancy or derailment to PE are warranted.Figure I.Transcriptomic variations related to preeclampsia at early pregnancy59. DE genes were identified through permutation analysis, after setting the percentage of the false prediction threshold < 0.05. We identified genes associated with PE case status using the same procedure on the OMEGA cohort. At the replication step, those discovered genes associated with PE having a two-sided P-value < 0.05 in the replication sets were considered for determining the overlap with transcriptome signatures associated with maternal asthma, vitamin D insufficiency, and excess BMI.After a surrogate variable analysis and adjustment for expression heterogeneity, we carried out differential gene expression analysis of genes related to PE at the preclinical stage (10–18 wga) using the Bioconductor “RankProd” package. The analytical package implements the Rank Product (RP) method for identifying the DE genes that are consistently upregulated (or downregulated) in a number of experiment samples. Such an approach and the applied non-parametric statistic by the RP method allow for increased performance in the case of small sample size and heterogeneity of samplesII.Transcriptomic variations related to maternal asthma, BMI and vitamin D status2) at 10–18 wga in the discovery VDAART group (N = 157). Similar to the PE transcriptome analysis, DE (up- or downregulated) genes were identified through permutation analysis, after setting the percentage of the false prediction threshold < 0.05. We identified DE genes associated with each pregnancy condition, i.e., maternal asthma, vitamin D status, and BMI status, using the same procedure in an extra set of pregnant women from the VDAART (N = 24) without PE. At the replication step, those discovered genes having a two-tailed P-value < 0.05 obtained for each condition were considered for the identification of the overlapping transcriptome signatures.Using the same rank product method, we separately identified genes associated with vitamin D insufficiency status (< 30 ng/ml), maternal asthma status, and excess BMI  and those with none  in the VDAART cohort . We determined how many of these DE genes were also present in the replicated overlapping gene signatures.For a sensitivity analysis, we identified the DE genes between subjects with all conditions  was separately carried out with g: Profiler61. Accordingly, gene group functioning profiling was performed with default options limiting the output to significant results (multiple testing-corrected P < 0.05). Within each of the enriched GO terms, the involved genes of each condition were listed.To obtain biological knowledge of the replicated gene signatures and categorize them according to their known biological functions, we conducted Gene Ontology (GO) enrichment analysis (EA). GOEA returns the functional roles of a gene set organized by three orthogonal aspects: biological processes, molecular function, and cell component62. GOSemSim integrates multiple functional similarity algorithms to compute functional similarities between gene products based on the information content of GO terms and the similarity of their associated GO annotation. To compute semantic similarity among the sets of our gene products, we used the “Wang” method that scores the semantic similarity of two GO terms based on both the locations of these terms in the GO graph and their relations with their ancestor terms62. The semantic similarity scores of multiple GO terms associated with gene products were combined using a “Best Match Strategy (BMA)” to a score between 0 to 1, indicating the degree of the GO term and functional similarity of compared sets of gene signatures. The closer the numeric value is to one, the greater the similarity in GO terms and functions of the two sets of genes.To assess the pairwise functional similarity in humans for our 4 gene signatures and their overlap, we used the mclusterSim function in the R package GOSemSim to analyze the overall similarity of the lists of gene signatures with respect to their GO annotationsV.Direct interaction of transcriptome signatures in the interactome, the module closeness, and their overlap63. We used direct interactions between genes (proteins) with a combined confidence score > 0.4 which provides more than 50% confidence in the proposed PPIs63. The results were updated with the most recent STRING database, “v11.0,” available online (https://string-db.org/). We used the information from the PPI database and each of the four sets of DE genes to construct observable modules for each disease condition. We further measured the closeness of PE module to maternal asthma, BMI and vitamin D modules in comparison to unrelated conditions in the PPI .We examined the local clustering and physical interactions of the early-pregnancy replicated gene signatures for each gene set by mapping them to the human interactome (protein–protein interaction [PPI] network) using the R Bioconductor package “STRINGdb” which is the R interface for the STRING PPI database64, MetaCore from Clarivate Analytics65 and literature search (PubMed and Google Scholar).The intersection of the 4 sets of replicated DE gene signatures related to the study conditions  was determined. The overlap of gene signatures was used for further investigation of their direct interactions in the interactome and largest connected component (LCC), GOEA and pathway analyses, network visualization, literature curation, as well as tissue specificity in the placenta. We curated the overlapping gene signatures in association with PE using 3 resources of GeneCardsVI.Placental expression of peripheral blood overlapping gene signatureshttps://ncbi.nlm.nih.gov/geo). GEO is an online public repository for microarray and high-throughput expression data maintained by the National Center for Biotechnology Information (NCBI)66. The datasets were both on the platform of Affymetrix Human Gene 1.0 ST Array.To assess the expression variations in the overlap of peripheral blood gene signatures with the placenta during the studied period (10–18 wga) as well as under pregnancies with PE, we used two Gene Expression Omnibus (GEO) datasets  and term, four samples for each time point (GSE9984)68. The raw data were downloaded, and background adjustment, log2 transformation, and quantile normalization on the arrays were applied by “rma” function in R BioConductor’s “affy” library. We first examined whether the overlapping gene signatures were expressed in all samples across the 3 pregnancy time points. Next, we conducted SVA-adjusted differential gene expression analysis using the RankProd method on the 1st and 2nd-trimester samples, approximately corresponding to the gestational age of samples in our study (10–18 wga). We examined whether the overlapping gene signatures, at least in part, were also differentially expressed in the placenta of early to mid-healthy pregnancy (P < 0.05).Entrez ID was used to integrate expression data from multiple microarray platforms 69. Differential gene expression analysis was conducted using the same methodology as described above to identify a subset of overlapping gene signatures differentially expressed in the placenta from pregnancies with PE compared with those from healthy pregnancies.To assess whether the overlapping gene signatures were also differentially expressed in the placentae of pregnancies with PE compared to the placentae of healthy pregnancies, we used a second GEO database of human placental microarray data (GSE75010). This dataset contains term-placental gene expression profiles of 80 and 77 pregnancies with and without PE, respectivelySupplementary information 1.Supplementary information 2.Supplementary information 3.


P = 0.023). Importantly, CSF IL-6 levels differed significantly between cases with DCI and infarctions and patients with DCI and no infarction , while findings in the latter patient group were similar to cases with no vasospasm . Together, these data support a potential role for elevated CSF IL-6 levels as a biomarker for DCI with infarction rather than for DCI in general. This fits well with a growing body of evidence linking neuroinflammation to ischemia and infarction, but  limits the diagnostic usefulness of CSF IL-6 levels in SAH patients.Interleukin 6 (IL-6) is a prominent proinflammatory cytokine and has been discussed as a potential biomarker for delayed cerebral ischemia (DCI) following aneurysmal subarachnoid hemorrhage. In the present study we have analyzed the time course of serum and cerebrospinal fluid (CSF) IL-6 levels in 82 patients with severe aneurysmal subarachnoid hemorrhage (SAH) requiring external ventricular drains in correlation to angiographic vasospasm, delayed cerebral ischemia, secondary infarctions and other clinical parameters. We observed much higher daily mean IL-6 levels  in the CSF than the serum of the patients with a peak between days 4 and 14 including a maximum on day 5 after SAH. Individual CSF peak levels correlated significantly with DCI (mean day 4–14 peak, DCI: 26,291 ± 24,159 pg/ml vs. no DCI: 16,184 ± 13,163 pg/ml;  A major complication following SAH is the occurrence of delayed cerebral ischemia (DCI). The term is commonly used to describe a clinical syndrome consisting of secondary neurological impairment often responsive to induced hypertension and not attributable to aneurysm occlusion, (treatment of) hydrocephalus, seizures, septic complications or other competing causes. A very sizable number of patients with DCI develop cerebral infarcts11. Secondary cerebral infarction is a major cause of poor clinical outcome or even death following SAH12. The term vasospasm (VS) was originally coined to describe the somewhat mechanistic concept of major artery narrowing causing neurological deterioration due to territorial perfusion deficits which may or may not result in infarction. However, the correlation between VS, i.e. arterial narrowing demonstrated by angiography and DCI and infarction is not perfect, and a more recent view of the pathophysiology of DCI will acknowledge a role for additional factors such as involvement of small arteries, thromboembolism, spreading depressions and neuroinflammation20. While DCI is primarily seen in patients with aneurysmal SAH, it may occur occasionally in cases in which no aneurysm can be demonstrated and even complicate the treatment of completely unrelated conditions such as traumatic brain injury, brain tumor and CNS infections24.Spontaneous aneurysmal subarachnoid hemorrhage (SAH) is a common cerebrovascular disease and neurosurgical emergency associated with serious complications and a high mortality rate requiring timely aneurysm occlusion and constant neurosurgical and critical care observation during the acute phase26. The occurrence of imaging proven secondary infarctions might well be the most robust and relevant criterion in the context of a clinical study11. However, it can only be used in a post-hoc setting i.e. it is not suitable for intervention studies since it does not account for successful treatment. Therefore, identification of a diagnostic biomarker would be a major step forward in the management of DCI.Of note, defining and diagnosing DCI in the clinical context is not a trivial matter. Most would probably use a combination of clinical  and transcranial Doppler sonography and/or imaging findings 27. Interleukin (IL)-6 in the cerebrospinal fluid (CSF) and serum have been discussed in some detail as possible biomarkers of DCI. IL-6 is commonly regarded as a prominent mediator of the inflammatory response following SAH. Several investigators have therefore analyzed IL-6 levels in SAH patients, and some studies describe a correlation between elevated IL-6 levels and DCI33. IL-6 levels may correlate with the patients’ health related quality of life (HRQoL)34.Modern intensive care facilities allow for detailed monitoring of numerous clinical, technical as well as laboratory variables which have been associated with the risk for DCI and other SAH related complications and patient outcomeAt the authors’ institution, determination of serum and CSF IL-6 is performed as part of the routine monitoring and management of intensive care patients with aneurysmal SAH which made serial CSF and serum IL-6 determinations available from a relatively large patient series. In view of the possible correlation between increased IL-6 levels and DCI just outlined, we analyzed these data in order to evaluate IL-6 as a possible diagnostic (and prognostic) biomarker for DCI. The identification of a DCI biomarker might be particularly relevant for patients with severe SAH who are often sedated and ventilated and therefore cannot be monitored by serial neurological exams.The hospital’s database was searched to identify patients treated for SAH in the Department of Neurosurgery of the Bethel Clinic in Bielefeld, Germany from February 2016 through February 2019. Cases without aneurysms and patients with AVM associated aneurysms were excluded. In 82 consecutive cases requiring external ventricular drainage serial CSF IL-6 levels  could be made available. These patients were included in the final analysis.35, location of the aneurysm, time of occlusion and occlusion technique (clipping vs. coiling). We also documented the specifics of all additional surgical or neurointerventional measures  and medical complications . The modified Rankin Scale (mRS) was used to describe the patients’ clinical outcome at discharge. A good clinical outcome was defined as mRS 0–2 in accordance with the literature38. All clinical, radiological and laboratory data were analyzed retrospectively. No study-specific investigations or tests were performed. In particular, serum and CSF IL-6 levels were obtained as part of the routine monitoring of intensive care patients with aneurysmal SAH. The study was approved by the responsible ethics committee . All procedures involving human participants were in accordance with the ethical standards of the institutional and all national research committees and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Individualized informed consent was obtained from all patients or their legal guardians for hospital treatment including all diagnostic and treatment modalities as well as for statistical analyses of all clinical and laboratory data.Pertinent demographic , laboratory, radiological and treatment data were extracted from the patients’ medical records including time of bleeding, World Federation of Neurosurgical Societies (WFNS) grade, Fisher gradeAll medical records and radiological studies were reviewed and assessed for the occurrence and the time of onset of clinical symptoms of DCI and cerebral infarctions. Cerebral infarctions were classified as aneurysm treatment-related  or secondary infarction . All patients had a CT or MR scan within 48 h. of aneurysm occlusion.Per routine all SAH patients had daily TCD examinations from day ≤ 3 to 14 (if awake and without symptoms) or 21 (if sedated). Cases with a clinical diagnosis of DCI had TCD examinations until symptoms resolved and at least until day 21. CT angiography/perfusion studies were obtained as indicated in order to identify cases with angiographic VS and perfusion deficits. Patients had conventional angiographies for aneurysm diagnosis , for aneurysm coiling, for clip control (per routine on day 10–14) and for salvage spasmolysis (see Results).For the purpose of this study, DCI was defined as imaging evidence of secondary infarctions unrelated to the aneurysm occlusion procedure or other interventions; and/or secondary neurological impairment after exclusion of competing causes, responsive to induced hypertension with or without secondary infarcts. VS was defined by pathognomonic transcranial Doppler findings (mean MCA or ACA flow > 120 cm/s and Lindegaard index > 3) and/or angiographic vasospasm .39. For the purposes of our analysis we have therefore studied day 0–3 and 4–14 CSF IL-6 peak values separately in addition to overall CSF and serum peaks (day 0–28).IL-6 levels in CSF and serum [pg/ml] were obtained per routine  directly after a CSF drain was placed and daily thereafter until the drain was removed. VS is most often seen between days 4 and 14 following SAH, and several groups investigating a possible link between IL-6 levels and vasospasm have hence focused on day 4 to 14 IL-6 findingsStatistical analysis was performed using a commercially available software . Standard procedures  were used for univariate analyses as indicated. IL-6 levels were studied using mean and maximum values, and logistic regression analysis. Receiver operating characteristics (ROC) analysis was used to assess the potential of CSF IL-6 levels as a diagnostic marker.We enrolled 82 cases with aneurysmal subarachnoid hemorrhage which required CSF drainage in this analysis. The majority of the external ventricular drains (EVD) were placed within 24 h. of admission . Table Thirty-eight patients (46.3%) were admitted directly to our emergency room, while 44 cases were referred from outside hospitals with no neurosurgical department. Six patients were admitted > 24 h. (range 2–9 days) after SAH. Clinical presentation (WFNS grades), initial CT findings (Fisher grades), aneurysm location and treatment are detailed in Table DCI as defined in the Methods section was eventually diagnosed in 34 (41.5%) cases. Of note, this includes 22 (26.8%) patients with DCI related delayed infarctions as evidenced by CT scanning. DCI was diagnosed 8.3 ± 3.4 days following SAH.All patients with DCI and/or VS were treated with induced hypertension (MAD > 100 mmHg). Salvage spasmolysis with intraarterial nimodipine was performed in 13 vasospastic patients. Permanent CSF shunting was required in 26 cases (31.7%). Favorable outcomes (= mRS 0–2) at discharge were seen in 23 (28.0%) of our patients. mRS 0–3 outcomes at discharge were observed in 34 (41.5%) cases.Serial IL-6 determinations allowed for an assessment of IL-6 levels over time following SAH. Serum IL-6 levels were available in 76/82 (92.7%) cases. Daily IL-6 CSF and serum levels are shown in Fig. 40. We could not confirm such findings when studying peak CSF IL-6 day 0–3 levels  and clinical signs compatible with an infection. We will acknowledge that also many cases with aseptic meningitis will meet these criteria. After exclusion of these patients (N = 31) from the analysis we found again very significant correlations between IL-6 peak levels and DCI .CSF IL-6 levels have also been discussed as potential markers of meningitis and ventriculitis10. We therefore analyzed CSF IL-6 overall and day 4–14 levels separately in patients with secondary infarctions (Table P = 0.005 and P = 0.003). Indeed, we observed very similar CSF IL-6 levels in these latter patients when compared to cases without DCI. Hence our data point to an association specifically between high CSF IL-6 levels and vasospastic infarctions .The literature contains somewhat conflicting data with respect to an association between elevated IL-6 levels and DCI. We hypothesized that this might reflect to some degree the different criteria used to define DCI. Imaging evidence of secondary infarctions is a strict but also probably the most robust and relevant criterion with respect to patient outcomeP = 0.0024). There was no significant difference between cases with DCI but no infarcts and cases without DCI  towards higher IL-6 levels in Fisher grade 4 versus 3 bleedings and  increased CSF IL-6 levels were seen in WFNS grade 5 SAHs. These latter findings point to some association between IL-6 levels and the initial severity of the bleed.IL-6 is a proinflammatory cytokine and neuroinflammation may be an important pathomechanism underlying and modifying not only vasospastic ischemia, but also other aspects of SAH. We therefore explored our dataset also for correlations between CSF IL-6 levels and characteristics of the clinical course and treatment of SAH other than DCI Table . The onl42. The present study has two principal findings. Firstly, we provide data that confirm a correlation between elevated CSF IL-6 levels and DCI. Secondly, our analysis suggests that this correlation chiefly reflects high CSF IL-6 levels in patients who develop secondary infarctions.DCI and delayed infarcts are major causes of morbidity and mortality following SAH. The pathophysiology of DCI and secondary infarction remains unclear, but the inflammatory response elicited by a cerebral hemorrhage may play a significant pathogenetic role. This has prompted several groups to investigate IL-6 which is one of the more prominent proinflammatory cytokines as a biomarker for DCI41. However, the respective associations were often weak and the wide range of actually measured values appeared to hamper its diagnostic usefulness. Our study confirms these earlier findings including the considerable interindividual variation of actual measurements. CSF rather than serum, and intermediate (day 4 to 14) rather than early (day 0–3) IL-6 levels seem to correlate with DCI. Absolute levels are most important and not the relative increase over time or the ratio between CSF and serum determinations. Our data do not necessarily contradict studies attributing some diagnostic value to these latter parameters. It is quite possible that the retrospective character of our analysis precluded the delineation of more subtle associations.A number of studies have investigated a potential correlation between elevated IL-6 levels and DCI. Most groups have published positive findings33. Lenski et al. have recently explored the usefulness of CSF IL-6 in differentiating between DCI and meningitis33. We found little evidence that elevated CSF IL-6 levels in our cohort reflect CNS infections. Of note, Lenski and co-workers used CSF IL-6 levels at the time of first diagnosis of DCI or meningitis rather than peak values over several days. While their approach may have been more sensitive than ours if one assumes that CSF IL-6 levels rapidly increase and decrease with the clinical course of meningitis it may have limitations with respect to patients with both, DCI and CNS infections.CNS infections might also result in elevated CSF IL-6 levels45. Most investigators have analyzed serum but a few also CSF IL-6 levels42. Importantly, these data underline the role of neuroinflammation in the pathophysiology of DCI. A focus on neuroinflammation will likely result in very different concepts of DCI prevention and treatment than the current emphasis on imaging-based diagnosis and angiographic treatment .It is quite possible that some of the discrepancies between the various studies investigating IL-6 in SAH simply reflect the problem of properly defining and diagnosing VS and DCI. Very intriguingly, we found high CSF IL-6 levels predominantly in cases with secondary (vasospastic) infarctions rather than patients with clinical or apparative findings thought to be pathognomonic for DCI. Our findings fit very well with a substantial body of data linking IL-6 to ischemic stroke. Indeed, IL-6 levels have been prominently discussed as potential biomarkers for ischemic stroke risk, outcome after stroke and even infarct size46. Very similar findings have also been reported by other groups34. However, possible associations of IL-6 levels with the severity of the bleed and other outcome relevant parameters also warrant consideration. Therefore, CSF IL-6 might have potential as a prognostic rather than as a diagnostic biomarker in SAH. We were unable to confirm an association with shunt-dependency as reported by others39.As pointed out, our data suggest that high CSF IL-6 levels will not identify patients who benefit from DCI treatment but rather those who will ultimately develop cerebral infarcts. This explains why CSF IL-6 levels were higher in patients with an adverse outcome in our study, since the occurrence of secondary infarcts is a major negative outcome predictor40. We did not analyze other markers of (neuro)inflammation. However, the pertinent literature provides little evidence pointing to their relevance in SAH patients29. The exclusion of cases managed without ventricular drains might have introduced some selection bias even though our cohort appears representative, since the distribution of various clinical parameters such as WFNS grade, aneurysm location, incidence of meningitis and shunt dependency, and outcome is roughly similar to published series41.We will readily acknowledge several important limitations of our study. Of note, we conducted a retrospective analysis of a dataset which was not primarily obtained with the intention to monitor and diagnose vasospasm. Similar to some prospective studies we allowed for the inclusion of patients who were not assayed dailyIn summary, we observed a strong correlation between elevated CSF IL-6 levels and secondary vasospastic infarcts in a sizable series of SAH patients. CSF IL-6 is a prominent proinflammatory cytokine and therefore our study adds to the growing body of data that links neuroinflammation to DCI. However, CSF IL-6 levels were not significantly elevated in patients with DCI responsive to treatment . This and the wide range of measured values limit the usefulness of CSF IL-6 as a biomarker for the diagnosis of DCI.


Phlegmonous and gangrenous appendicitis represent independent pathophysiological entities with different clinical courses ranging from spontaneous resolution to septic disease. However, reliable predictive methods for these clinical phenotypes have not yet been established. In an attempt to provide pathophysiological insights into the matter, a genomewide gene expression analysis was undertaken in patients with acute appendicitis.Peripheral blood mononuclear cells were isolated and, after histological confirmation of PA or GA, analysed for genomewide gene expression profiling using RNA microarray technology and subsequent pathway analysis.Samples from 29 patients aged 7–17 years were included. Genomewide gene expression analysis was performed on 13 samples of phlegmonous and 16 of gangrenous appendicitis. From a total of 56 666 genes, 3594 were significantly differently expressed. Distinct interaction between T and B cells in the phlegmonous appendicitis group was suggested by overexpression of T cell receptor α and β subunits, CD2, CD3, MHC II, CD40L, and the B cell markers CD72 and CD79, indicating an antiviral mechanism. In the gangrenous appendicitis group, expression of genes delineating antibacterial mechanisms was found.These results provide evidence for different and independent gene expression in phlegmonous and gangrenous appendicitis in general, but also suggest distinct immunological patterns for the respective entities. In particular, the findings are compatible with previous evidence of spontaneous resolution in phlegmonous and progressive disease in gangrenous appendicitis. Reliable distinction of phlegmonous and gangrenous appendicitis is not possible by current means. The aim of this study was to test the applicability of gene expression data including a pathway analysis for differentiation of the entities. Surgeons know how to treat appendicitis: either surgically with appendicectomy and supporting measures like abscess drainage, or conservatively without operationversus 76.6 per cent). Further support for the hypothesis of independent inflammatory entities is provided by immunological evidence. PA and GA differ significantly in terms of distinct underlying immunological patterns; there are primarily Th2-helper cell-dependent mechanisms in those with PA and Th1/Th17-dependent mechanisms in patients with gangrenous inflammation,The presence of necrosis seems to be the determining factor: necrotizing gangrenous appendicitis (GA) is associated with a complicated clinical course including increased rates of peritonitis and abscess formation, even if perforation is not present, whereas non-necrotizing, phlegmonous appendicitis (PA) usually seems to be self-limitingAs the inflammatory entity seemingly determines the clinical course, these types of appendicitis require different therapeutic courses of action. Therefore, decisive diagnostics are mandatory to reliably differentiate complicated from uncomplicated appendicitis.Clinical investigation and laboratory values are basic methods used in the diagnosis of acute appendicitisCurrently, imaging techniques such as CT seem to provide good capacity for differentiation between complicated and uncomplicated appendicitisThis comprehensive prospective whole-genome gene expression study essentially encompasses two objectives. First, the aim was to investigate the general extent of gene expression differences in children with PA and GA. Second, the entities were examined at a basic level of gene expression that would allow description of specific pathophysiological and, particularly, immunological differences in terms of pathways. Interesting differences between PA and GA have already been described at the genomic levelThis was a single-centre, prospective study of patients aged 7–17 years who had undergone surgery for acute appendicitis at the Department of Paediatric Surgery of Charité – Universitätsmedizin Berlin, between April and August 2019. The specific age group in this paediatric surgical population was determined as a necessary level of ability to consent and reading comprehension was required in order to be able to give informed consent to participate in the study. The study was approved for the limited number of patients (30 + expected exclusion rate of 10 per cent) by the institutional ethics committee (reference number ES2/130/16), with the perspective of a subsequent sample size calculation for a larger study.Inclusion criteria for the study were: patients aged 7–17 years with a sonographically suspected diagnosis of acute appendicitis and scheduled for appendicectomyExclusion criteria were: missing consent; concomitant disease; previous or present specific treatment for acute appendicitis ; antibiotic treatment within the past 2 weeks; interval between blood sample collection and isolation of peripheral blood mononuclear cells (PBMCs) more than 1 h; insufficient RNA quality (RNA integrity number (RIN) below 7); and appendicectomy not performed .® PM400  at room temperature for 30 min at 400g. After isolation of the monocyte layer, the cells were resuspended in PBS and centrifuged twice for 5 min at 400g. After final resuspension in 1 ml PBS, centrifugation and removal of the supernatant, the native cells were frozen at –20°C using a sample freezing container  and finally stored at –80°C in liquid nitrogen.In strict accordance with the ethics committee’s approval, blood samples (at least 5 ml) were drawn during routine medical laboratory investigations in the paediatric emergency department. The isolation of PBMCs was performed within 1 h after blood sampling. The blood from each individual was suspended in phosphate-buffered saline (PBS) at a 1 : 1 ratio. Density gradient centrifugation was performed using FicollFig. 1 shows the major histopathological features for group assignment (phlegmonous versus gangrenous) in comparison to normal appendix.After appendicectomy, the resected appendixes were classified histopathologically according to CarrAfter the primary histopathological examination, a second blinded evaluation was undertaken by a specialized paediatric pathologist.Total RNA was isolated from PBMCs using a NucleoSpin™ RNA Plus kit . RNA quality control was achieved using a 2100 Bioanalyzer  and the RNA 6000 Pico Kit for total RNA samples. For quantitative control, photometric measurement was performed using a NanoDrop™ 2000 spectrophotometer . The quality of each sample was evaluated based on the Bioanalyzer RIN. Samples with a RIN above 7 fulfilled quality control and were subjected to labelling.in vitro transcription for synthesis of cRNA labelled with cyanine 3-CTP was performed.For labelling, a Low Input QuickAmp Labeling Kit  was used to generate fluorescent complementary RNA (cRNA). A random primer–oligo-dT primer mixture was used for first-strand synthesis. After second-strand synthesis, Some 600 ng of each Cy3-labelled cRNA was hybridized at 65°C for 17 h on an OakLabs 8x60K ArrayXS Human Agilent microarray (design ID 79407) containing probes for 56 666 genes, using an Agilent Gene Expression Hybridization Kit , followed by microarray wash and scanning on a SureScan Microarray Scanner  at a resolution of 3 μm to generate 20-bit TIF files according to the manufacturer’s protocols.TIF files were extracted using Feature Extraction Software version 11  and the GE1_1105_Oct12 protocol. The resulting raw data were analysed using DirectArray Software . The signal distributions of the raw data were visualized using box plots to identify potential issues for individual samples.versus GA) by applying Welch’s test and calculating log2 fold changes for each gene. P < 0·050 was considered statistically significant.Samples were quantile normalized and subjected to statistical analysis  signal, whereas a signal above the mean Z-value indicates a positive (red) signal. Heatmap visualization was combined with hierarchical clustering, whereby samples with the most similar expression profiles—based on normalized expression values—were clustered together. Additionally, clusters were visualised as dendrograms, complementing the heatmaps.The significantly differentially expressed genes from the relevant pathways were visualized in a heatmap. For the colour representation, Fig. 2). No statistically significant differences were found with regard to age, sex distribution, and time from onset of symptoms to blood sample collection (Table 1).Thirty-three otherwise healthy patients with sonographic suspicion of acute appendicitis were initially included. RNA quality control led to exclusion of four samples that had signs of degradation: three from patients with GA and one from a patient with PA. After primary routine histopathological examination, 15 specimens were classified as phlegmonous and 14 as gangrenous. After blind histopathological re-evaluation by a specialized paediatric pathologist, two samples from patients with a primary diagnosis of PA were classified as gangrenous inflammation. The PA group eventually consisted of 13 samples and the GA group of 16 .Box plots of raw gene expression signals of the samples finally included and classified showed a similar distribution, with no obviously problematic samples (P < 0·050). The distribution of expression patterns (log2 fold change versus –log (P)) with respect to significant upregulation and downregulation of genes is illustrated in a volcano plot .From a total of 56 666 protein-coding and non-coding RNAs analysed, 3594 (6.3 per cent) were significantly differentially expressed between the groups .Eleven of the significantly increased expressed genes in the PA group concerning α or β subunits of the T cell receptor were found within the top 100 expressed genes. In the GA group, two significantly increased mRNAs were found among the top 100: SOCS3 and neutrophilic CD64. Based on statistically significant Diagnostic methods provide the framework for the surgical options for treatment of acute appendicitis. The authors’ motivation beyond clarification of the pathophysiologies was to show what could be expected from gene expression data in the diagnosis of acute appendicitis in the future, especially with regard to one of the most affected age groups. The medium-term goal is to obtain the simplest possible molecular marker providing the most valid information possible for rapid therapeutic decision-making. Such a marker should be safe, objective (without need for interpretation), easy to determine and, in terms of prospective informative value, superior to other diagnostic tools such as imaging, clinical examination, standard laboratory parameters, and combinatorial scores. The latter are especially useful, but they represent complicated constructs with many variables, which have to be interpreted partiallyOverall, 6.3 per cent of the genes analysed in this study were expressed significantly differently between the groups, demonstrating substantial differences among inflammatory entities. For complementary data analysis, the Kyoto Encyclopedia of Genes and Genomes was instrumentalized to map and link the disparately expressed genes to relevant molecular pathways. The results of this complex data analysis gave rise to the hypothesis that PA represents an immunological response to viral infections, whereas GA results in an expression pattern related to an antibacterial response.Most theories generally designate luminal obstruction as the primary origin of acute appendicitis,,,,,In the present study, PA was characterized by lymphoid-directed gene expression patterns. Particularly, overexpression of α and β subunits of the T cell receptor, MHC class II and CD40L, T cell markers CD2 and CD3, and B-cell markers CD72 and CD79 in patients with PA suggested a specific immunological synapse between T and B cellsTable S1.Interestingly, the susceptibility of humans with the functional IL-17A rs2275913 polymorphism for appendicitis has been associated with advanced inflammation,The study results are in accordance with previous immunological and infectiological investigations regarding acute appendicitis, and the authors’ hypothesis that PA and GA represent independent entities. Mean symptom duration and distribution of temporal patterns were comparable in the two groups. These findings are consistent with previous evidence of independent inflammatory courses in patients with PA and GA, without transition from one form to the other,Studies have shown that fetal development of the lymphatic tissue in the appendix is already characterized by persistence of T and B lymphocytesMicroarray-based comparative investigation for pathway analysis has been shown previously to be a highly effective toolThe results presented here offer valuable information regarding the general description of differential gene expression and identification of specific yet independent immunological gene expression patterns in light of current research. They provide proof of concept for a comprehensive follow-up study with an adequate sample size calculation focusing on gene expression profiling, possibly coupled with high-performance algorithms, to allow successful development of a simple and reliable biomarker to distinguish inflammatory entities within acute appendicitis in childhood. The methods described here could therefore provide a fundamental framework for reliable diagnostics in acute appendicitis at a simple, low-invasive, low-risk, and cost-effective level.HRH Crown Princess Lovisa’s Foundation, Stockholm, Sweden.zraa045_Supplementary_DataClick here for additional data file.


AATF (P = 8.95 × 10−9) and rs10738556 near chromatin-remodeling SMARCA2 (P = 2.83 × 10−8). In immunohistochemical analysis we observed that HT patients with homozygous AATF risk genotypes have decreased AATF expression  and increased apoptosis  in comparison to controls. HT patients with heterozygous SMARCA2 genotypes have decreased SMARCA2 expression, albeit without reaching statistical significance , and significantly increased apoptosis . By two lines of evidence we show that two highly plausible genetic loci, AATF and SMARCA2, may be involved in determining the thyroid volume of HT patients. The results of our study significantly add to the current knowledge of disturbed biological mechanisms in thyroid gland of HT patients.Thyroid volume of Hashimoto’s thyroiditis (HT) patients varies in size over the course of disease and it may reflect changes in biological function of thyroid gland. Patients with subclinical hypothyroidism predominantly have increased thyroid volume whereas patients with more pronounced hypothyroidism have smaller thyroid volumes. Suggested mechanism for thyroid atrophy is thyrocyte death due to apoptosis. We performed the first genome-wide association study (GWAS) of thyroid volume in two groups of HT patients, depending on levothyroxine (LT4) therapy, and then meta-analysed across. Study included 345 HT patients in total and 6 007 322 common autosomal genetic variants. Underlying hypothesis was that genetic components that are involved in regulation of thyroid volume display their effect in specific pathophysiologic conditions of thyroid gland of HT patients. We additionally performed immunohistochemical analysis using thyroid tissues and analysed differences in expression levels of identified proteins and apoptotic marker between HT patients and controls. We found genome-wide significant association of two loci, both involved in apoptosis, with thyroid volume of HT patients: rs7212416 inside apoptosis-antagonizing transcription factor  Follicle consists of epithelial follicular cells, known as thyrocytes, which surround colloid filled with thyroid hormone precursor thyroglobulin. The size of follicles, number of thyrocytes and the size of colloid are all dependent on thyroid gland biological activity and can affect thyroid gland size1 whereas its size can also be decreased, as seen in progressed autoimmune hypothyroidism5. Moreover, Hashimoto’s thyroiditis (HT), also known as autoimmune thyroiditis, can be presented as goiter on one side of the spectrum or as atrophic thyroid gland on the other6. A study demonstrated that patients with autoimmune hypothyroidism have larger dispersion of thyroid volume size in comparison to controls indicating that some patients have increased and some decreased thyroid volumes, i.e. goiter and thyroid atrophy just represent the extremes in the distribution3. Further studies linked thyroid volume size with thyroid function, as it was found that patients with smallest thyroid volumes have more pronounced hypothyroidism5, whereas patients with subclinical hypothyroidism predominantly have increased thyroid volume4. It was also suggested that progression of autoimmune thyroiditis to overt hypothyroidism is accompanied by thyroid atrophy4. In other words, as thyroid volume changes over the course of disease5, it reflects the change in biological function of thyroid gland, as well as stroma changes, including lymphocitic infiltration, local edema, and vascularity. The underlying mechanisms of progressing atrophy are not well known, but the most suspected mechanism is thyrocyte death due to apoptosis7. In summary, thyroid mass is affected by the number of thyrocytes that are maintained through the balance between their proliferation and apoptosis8. However, predominant process in HT is increased apoptosis12. Although the knowledge on mechanisms and regulation of apoptosis in thyroid gland is limited, it is suggested that the most important apoptotic pathway includes the Fas death receptor10.Thyroid volume can be enlarged in number of thyroid diseases14. In iodine sufficient areas, or in cases of iodine supplementation, thyroid volume of healthy individuals ranges within reference values16. Other factors that influence thyroid volume are age17, anthropometric features, such as body surface area (BSA)19, gender21, smoking22 and alcohol consumption23. Beside these, genetic factors are important regulators of thyroid size13. It has been estimated that genetic factors contribute to about 71% of the total variation in thyroid volume of healthy individuals13. Nevertheless, genetic determinants of healthy thyroid volume are not well understood and to date, only one genome-wide association study (GWAS) of thyroid volume in thyroid disease free individuals has been performed24. It identified four independent loci associated with thyroid volume located inside or near CAPZB, FGF7 and LOC44038924. On the other hand, genetic factors that determine thyroid volume in thyroid diseases are completely unexplored.There are several other determinants of thyroid follicular cells volume and thyroid volume, including serum TSH and iodineThis study, for the first time, analyzed genome-wide genetic variants underlying thyroid volume of HT patients. We hypothesize that in specific pathological conditions of thyroid gland, such as in HT, genetic components involved in the regulation of thyroid volume could actually be the same ones regulating the number of thyrocytes and thus, biological activity of thyroid gland. The main aim of our study was to identify genetic determinants underlying these processes and to verify findings by protein expression analysis in thyroid tissues that can further deepen the understanding of biology behind pathologic processes in HT.15. Patients were diagnosed with HT on the basis of clinical examination and diffuse thyroid autoimmune disease established by characteristic thyroid ultrasound (US) imaging (unhomogenic thyroid tissue with diffusely reduced echo levels). Diagnosis was further complemented with biochemical measurements of thyroid hormones and antibodies  according to ETA recommendations and guidelines for Management of Subclinical Hypothyroidism25. During recruitment period, from 2013 to 2016, we were sequentially recruiting newly diagnosed patients as they were referring to Outpatient clinic for thyroid disorders, mostly due to positive thyroid antibodies or abnormal TSH levels established during routine screenings, or because of suspected clinical manifestations of thyroid disorders. We additionally involved patients with previously diagnosed HT that came to physicians on follow-up. All HT patients that were recruited through our study are part of “Croatian biobank of patients with Hashimoto’s thyroiditis” (CROHT) that was established with principal aim of identifying genetic variants associated with HT26 and HT-related traits, such as thyroid antibodies27 and thyroid volume.A total of 370 HT patients were involved in this study. Patients were recruited after complete examination by nuclear medicine specialist at the Outpatient clinic for thyroid disorders at the Department for Nuclear Medicine at the University Hospital Split (Croatia). All patients were of white European ancestry, adult and living in the southern Croatia, a region considered iodine sufficient since 200328. The body surface area (BSA) in m2 was estimated as W0.425 × H0.725 × 0.007184, where W is the weight in kg and H is the height in cm29. Thyroid hormones and antibodies levels in plasma of HT patients were determined by immunoassay reaction conducted in a fully automated instrument “Liaison” Biomedica Chemiluminescence Analyzer in the Laboratory of Biochemistry, University Hospital Split. Genomic DNA of HT patients was extracted from peripheral blood leukocytes, using Nucleon Genomic DNA Extraction Kit BACC3, according to the manufacturer’s instructions . DNA concentration was determined by the Nanodrop ND-1000 Spectrophotometer . The final concentration of DNA was uniformed across all samples and brought to 100 µg/µl prior genotyping.Dimensions of thyroid gland in HT patients were determined by thyroid ultrasonography performed using Medison Accuvix V10  high frequency linear probe (8–12 MHz). Thyroid volume was calculated as a sum of volumes of both lobes of thyroid gland, where the volume of each lobe was calculated as length × width × depth × 0.479Written informed consent was obtained from all study participants, and the study was approved by two separate Ethics Committees: the University of Split, School of Medicine  and University Hospital Split . Both Ethics Committees declared that study is in accordance with the provisions of the Code of Ethics and the Helsinki Declaration.2-test was used for nominal variables.Descriptive statistics was used to summarize clinical characteristics of patients. None of continuous variables showed normal distribution (tested by Kolmogorov-Smirnov test), thus were presented as median (first quartile - third quartile), while nominal variables (gender) were presented as number of individuals (%) per category. To compare clinical characteristics between two groups of HT patients, depending on levothyroxine (LT4) therapy, Mann–Whitney-U test was used for continuous variables and χAll participants were genotyped using Illumina Infinium HumanCoreExome genotyping platform that contains 551 839 markers. We performed quality control (QC) of genotype data, using PLINK and R software, following the standard procedure.−4).In the sample QC, we excluded all samples with call rate less than 95% and all samples with heterozygosity rate deviating more than three standard deviations from the mean. We cross-checked reported sex with the sex inferred from the genotypes – no exclusions were made based on this criterion. We checked for ethnicity by performing multidimensional scaling analysis (MDS) and visually inspecting the MDS plot  as reference panel. We checked concordance tables to assess an overall imputation quality, mean genotype concordance in our dataset was 95,51%. We also compared the concordance rate and INFO (type 0) metric of directly typed SNPs to detect poorly genotyped SNPs or possible strand flips. Performed analysis showed that all directly genotyped SNPs used in imputation were well genotyped and correctly aligned to the forward strand. After imputation, we excluded all genetic variants with MAF less than 5%, variants with INFO metric less than 0.4 and variants with HWE P < 10−4. A total of 6 007 542 autosomal genetic variants passed post-imputation QC and were selected for GWAS.Following QC, we performed genotype imputation on clean dataset. All SNPSs were aligned to the forward strand prior to the imputation. Genotypes were firstly pre-phased using SHAPEIT2 software33. Residuals derived from linear regression model were quantile transformed in R and then used as a new phenotype in association analyses, instead of thyroid volume. Association analyses between 6 007 542 common autosomal genetic variants and adjusted thyroid volume were performed under the linear mixed model implemented in GEMMA software which accounts for population stratification and relatedness34.We performed GWAS of thyroid volume in two groups of HT patients separately, depending on levothyroxine (LT4) therapy. There were 130 patients that were on LT4 therapy and 215 patients without LT4 therapy. Thyroid volume was firstly adjusted for covariates under standard linear regression model in R: age, gender, TSH and BSA were used as covariates in both groups of HT patients, as they are known to influence thyroid volume. We used LT4 dosage as an additional covariate in the group of HT patients that were on LT4 therapy, as LT4 therapy reduces thyroid volume35. Manhattan plot of GWAS meta-analysis results was generated using R package qqman. Regional association plots were generated using LocusZoom software36. We checked cluster plots of all directly genotyped variants with P-value less than 0.1 located +/− 400 kb from top-associated variants, to eliminate possible spurious associations. Cluster genotyping plots were generated using Ilummina GenomeStudio software. For genetic variants associated with thyroid volume on the genome-wide level of significance (P < 5 × 10−8), we assessed the distribution of thyroid volume per genotype and generated box plots using R statistical software.Individual GWAS summary results were meta-analyzed in METAL using fixed-effects inverse variance method which weights genetic variant’s effect sizes (β-coefficients) by their standard errors (SE)37, with available genome-wide data, who underwent thyroidectomy at University Hospital of Split. Tissues were reviewed by expert pathologist for signs of HT and for confirmation of healthy (control) tissue. We analyzed expression of AATF, SMARCA2 and apoptotic marker cytochrome C (CytC) in thyroid follicles. Immunohistochemical analysis was used to test AATF/SMARCA2 and apoptotic marker expression between HT cases and controls of the same genotype i.e. if HT patients with one/two risk alleles have decreased AATF/SMARCA2 expression and increased apoptosis in comparison to controls with the same genotype. As the risk allele T of rs7212416 (AATF) has allele frequency of 85%, we were able to acquire tissues from 3 HT patients and 7 controls with TT genotypes . Due to allele frequency of 50% of the risk allele T of rs10738556 (SMARCA2), there were no thyroid specimens from individuals with homozygous TT genotypes in our Biobanks, therefore, we focused on analysis of heterozygous individuals. We were able to acquire tissues from 5 HT patients and 7 controls with heterozygous CT genotypes .Thyroid tissue paraffin blocks were obtained from HT patients who are part of our CROHT biobank and control participants from “10001 Dalmatians” biobankFormalin fixed and paraffin embedded tissue sections were deparaffinized and run through process of antigen retrieval in citrate buffer. Nonspecific binding was blocked by Protein Block . Tissue sections were incubated with primary antibodies to AATF , SMARCA2  and CytC  overnight at 4 °C. Staining was visualized by incubation with secondary antibodies labeled with red  and green  fluorochromes. Finally, samples were counterstained with DAPI .Photo-micrographs were shot by SPOT Insight digital camera , mounted on Olympus BX61 fluorescence microscope . Camera settings were set using image acquisition software CellA® at 1360 × 1024 resolution, exposition of 1/333.3 s with noise reduction filter. Ten micro-photographs of thyroid follicles, under the magnification of 200×, were shot per slide in all of 3 fluorescent channels. Furthermore, florescence intensity histograms were acquired for red and green fluorescence channels in ImageJ software . Region of positive signal was determined by using the slides stained with secondary antibodies only, thus quantifying the autofluorescence and fluorescence due to unspecific binding of secondary antibodies. Region of positive signal was defined as the one that excluded 99.99% of the signal obtained from florescence intensity histograms of slides stained with secondary antibodies only. Expression of AATF and SMARCA2 was quantified as the area under the curve (AUC) of florescence intensity histograms. On the other hand, since CytC release from mitochondria into cytoplasm is a marker of apoptosis, unlike its expression, we quantified it as a percentage of micro-photograph area covered by positive signal. AUCs and their interval estimates were calculated by using AUC analysis routine in GraphPad Prism 8.0 software . Statistical significance and effect sizes as well as respective 95%CI were calculated by t-test with Welch correction . For comparisons of percentage of area covered by positive signal t-test was used.38. Additionally, HT patients that are on LT4 therapy have significantly lower thyroid volume (P = 0.0024). There are no significant differences between two groups of HT patients in age, gender, BSA, and T3 levels. We also do not observe differences in median TgAb/TPOAb levels between two groups of patients.Clinical characteristics of HT patients divided in two sets, depending on LT4 therapy, are summarized in Table AATF  and rs10738556 located 7 kb from chromatin-remodeling SMARCA2   , rs170884 inside FAM129A , rs4747268 close to PTER , rs193145729 close to MIR8054 , rs11247367 inside IGF1R  and rs10767013 between MIR8054 and CCDC179  . This was accompanied by significant increase in apoptosis by 3.99-fold in comparison to control samples , as shown in Fig. We also analyzed expression of SMARCA2 and apoptosis in thyroid tissue of samples with rs10738556 CT genotypes. SMARCA2 levels were decreased by 1.07-fold in HT group in comparison to control group, however without reaching statistical significance , whereas we observe significant increase in apoptosis by 4.11-fold in HT group   and other near chromatin remodeling SMARCA2, associated with transcriptional regulation. Additionally, we have identified six loci suggestively associated with thyroid volume, of which two, IGF1R and FAM129A, play a role in apoptosis, thyroid morphogenesis and thyroid carcinomas, respectively. Identification of mentioned genes with clear connection to apoptosis is particularly interesting as apoptosis of thyrocytes affects the mass of thyroid gland and is considered to be the landmark of HT12. We additionally performed immunohistochemical analysis of expression levels of AATF, SMARCA2 and apoptotic marker in thyroid tissues of HT patients and controls to further explore involvement of identified proteins in pathophysiological processes of thyroid gland of HT patients.We have identified two genome-wide significant loci with thyroid volume of HT patients, one in apoptosis-antagonizing transcription factor AATF genetic variant, rs7212416, on thyroid volume. Patients with major allele on this SNP have decreased thyroid volume , have significantly decreased AATF expression and significantly increased apoptosis in comparison to controls with the same genotype  of the most associated me Table . Our hypWe also observe in GWAS analysis that the effect is stronger  in the group of HT patients that are in progressed stage of disease (overt hypothyroidism and on LT4 therapy) in comparison to newly diagnosed HT patients without LT4 therapy Table , which i44. Bruno et al. suggested interesting role of AATF as a novel effector in the regulation of p53 pathway41. In this model, DNA damage-induced phosphorylation and stabilization of AATF, set off cellular arrest by increasing transcription of p53 and p2141. An anti-apoptotic role of AATF is further suggested because activation of DNA damage checkpoints by AATF promotes DNA repair and cell survival41, whereas in the case of extensive DNA damage, a cell is destined for apoptosis and AATF is targeted for degradation39. Similarly, experimental silencing of AATF resulted with decreased cell proliferation and promotion of apoptosis45. Additionally, upon AATF silencing, pro-apoptotic genes, including Fas and FasL, were up regulated, while multiple anti-apoptotic genes were down-regulated45. Since the apoptosis via Fas death receptor is the hallmark of HT10, involvement of AATF in Fas/FasL signaling is compelling.Beside an anti-apoptotic role, AATF has several other versatile, but correlative roles in transcriptional regulation, induction of cell proliferation, cell cycle checkpoint control and response to DNA damage through activation of p5326, we were able to check allele frequencies of the most associated AATF genetic variant (rs7212416) in both groups. We do not observe difference in allele frequency distribution of this SNP between HT patients and controls implying that an effect of this SNP on thyroid volume may be triggered by pathologic conditions in thyroid of HT patients. A further indication of suggested mechanism is demonstrated by our immunohistochemical analysis where we found significantly decreased AATF levels and increased apoptosis in thyroid follicles of HT patients in comparison to controls with the same rs7212416 TT genotype. We therefore propose that SNP effect may be secondary to inflammatory events in the thyroid of HT patients where inflammatory cytokines initiate autoimmunity and play regulative roles in expression of apoptotic signaling47. Studies have shown that thyrocytes, alongside immune cells, contribute to secretion of proinflammatory cytokines of Th1 type that stimulate autocrine/paracrine apoptosis induction of thyrocytes48. However, as already marked, true association between identified genetic variant and apoptosis needs to be analyzed in additional tests.As we have previously performed GWAS study of 405 HT cases and 430 controls49, which is involved in cytokine signaling in immune system50. This chemokine has already been associated with autoimmune pathology of adrenal gland51. Furthermore, MIP-1-beta has also been linked to Fas-FasL mediated apoptosis52. As chemokines, together with Th1 cytokines, are considered the main regulators of autoimmune process in HT, a finding of association of the same genomic region with MIP-1-beta and regulation of thyroid volume is intriguing and provides multiple lines of evidence of involvement of this genomic region with apoptosis.Interestingly, a genomic region encompassing AATF has already been strongly associated with chemokine macrophage inflammatory protein 1b levels (MIP-1-beta)AATF and SMARCA2. A transcriptional regulator SMARCA2, also known as hBRM, is a component of SWI/SNF family of proteins that changes chromatin structure through altering DNA-histone contacts (www.genecards.org). hSWI/SNF chromatin remodelers have already been linked to apoptosis54. Overexpression of SMARCA2 has been associated with decrease in apoptosis of ovarian cancer cells55 and pancreatic cancer cells56. On the other hand, SMARCA2 is found to be cleaved during apoptosis57 and its truncated levels correlate with cell damage and apoptosis after virus infection58. It has been shown that SMARCA2 also interacts with HDAC159, the deacetylase known for its role in regulation of the cell cycle progression through Rb binding that is inhibited by AATF60. In brief, Bruno et al. have proven that AATF directly inactivates HDAC1/Rb/E2F1 transcriptional inhibition by competition with HDAC1 for the Rb binding site, thus inducing cellular growth and proliferation. On the other hand, a glucocorticoid receptor-induced apoptosis is found to be dependent on Rb and SMARCA261. From our results, we can speculate that both AATF and SMARCA2, through common interaction with HDAC1 and Rb, could be involved in the regulation of transcription that blocks cell proliferation and subsequently drives thyrocytes to apoptosis in thyroid gland of HT patients.This study presents yet another important link between our two major hits, We also demonstrate that HT patients (heterozygous for SMARCA2 rs10738556) have decreased SMARCA2 expression, albeit without reaching statistical significance, and significantly increased apoptosis in comparison to controls Fig. . We assuSMARCA2 is involved in the same pathway as our next suggestively associated locus, IGF1R. They both belong to hepatocellular carcinoma (HCC) pathway that has in turn been associated with abnormal thyroid hormone signaling62 further linking our genetic variants with thyroid dysfunction. IGF1R has pleiotropic roles in humans, among which two roles are relevant for our study. Firstly, it is an anti-apoptotic marker and, secondly, it has a direct role in functionality of thyroid gland. In in vitro conditions, IGF1R works together with TSH in stimulating thyrocyte growth and is actually found to be essential for regulation of thyroid function and TSH-stimulated goitrogenesis63. Important role in maintaining homeostasis and normal thyroid morphogenesis was further demonstrated in animal model64. These data suggest an important role of IGF1R with thyroid growth morphogenesis thus providing a concrete support for our finding of association of this gene with thyroid volume. We believe that this is a true candidate gene for maintaining thyroid volume albeit our study was not powerful enough to detect its association on a genome-wide level.Furthermore, FAM129A, also known as C1orf24 and NIBAN, as it was also found to act in anti-apoptotic manner, in regulation of p53-mediated apoptosis65. FAM129A is highly expressed in carcinomas including thyroid carcinomas67 and is used as one of several markers that may differentiate benign from malignant thyroid lesions, therefore, it is considered to be the best predictor for thyroid cancer71. FAM129A silencing in thyroid carcinoma cell lines directly leads to apoptosis, cell migration inhibition and cell cycle progression70. Finally, immunohistochemical staining showed that FAM129A, beside in thyroid tumors, is weekly expressed in subsets of scattered cells with oxyphilic cell metaplasia in HT and not in normal thyroid tissue71. Taken together, the current knowledge on this gene and our results, imply the role of FAM129A in the apoptosis regulation in thyroid tissue of HT patients.Finally, we bold out our second suggestively associated locus, 72. Given the fact that some studies report an increased incidence of well-differentiated thyroid cancer in chronic autoimmune thyroiditis74, it is possible that there is a genetic link between HT and thyroid cancer, i.e. that changes in expression of SMARCA2 and FAM129A in HT can predispose to development of some forms of thyroid cancer. If this holds true, then SMARCA2/FAM129A could be potential biomarkers for stratifying HT patients who maybe of higher risk of developing thyroid cancer. However, more research using prospective studies with full genetic profiling is needed to test this hypothesis, as our study was not designed to give answer on genetic correlation between HT and thyroid carcinomas.Beside the role of FAM129A in thyroid cancer, the loss of SMARCA2 was also found to be associated with thyroid cancer, particularly with anaplastic thyroid cancerDNAH5, PTER and MIR8054. Genetic variants inside these genomic regions were previously associated with number of traits, such as bacterial meningitis (DNAH5 region)75, hepatitis B (PTER region)75, cancer (MIR8054 region)76, allergic rhinitis (MIR8054 region)77.Further, we have identified another four suggestively associated variants located inside or near 78. Thyroid volume in HT patients is considered to change over the course of disease and it is smaller in patients with prolonged disease duration, representing tissue degradation5. This is exactly what we observe in our cohort, individuals with prolonged disease and in overt hypothyreosis (those that are taking LT4 therapy), have smaller thyroid volumes than newly diagnosed individuals  our GWAS study of thyroid volume in HT patients identified association of two genetic variants in AATF/SMARCA2, thus implying that regulation of thyroid volume of patients with HT has a genetic component; (b) further analysis of expression patterns showed differential expression levels of AATF and SMARCA2 between HT cases and controls of the same genotype accompanied by significantly increased apoptosis in HT patients, further suggesting their role in pathophysiological processes of thyroid gland of HT patients. Additional clarification of mechanisms through which identified genetic variants exhibit their effect is needed. Based on results of our study and function of associated proteins, we speculate that these genetic variants may display their effect under specific pathophysiologic conditions of thyroid gland of HT patients, possibly through modulation of AATF/SMARCA2 expression levels and induction of apoptosis. Our study provides suggestive evidence of involvement of six more loci in regulation of thyroid volume, of which Supplementary Material.


To investigate the correlation of plasma and local expressions of Circ CDR1as with disease severity in patients with non-traumatic osteonecrosis of femoral head (ONFH).Ninety-nine non-traumatic ONFH patients receiving surgery and 99 healthy individuals were enrolled in our study. Plasma and local Circ CDR1as were detected using real-time quantitative PCR (RT-qPCR). Radiographic progression was determined using Association Research Circulation Osseous (ARCO) classification system. Harris hip score (HHS) and visual analogue scale (VAS) were used to assess the clinical severity. Receiver operating characteristic (ROC) curve was carried out to evaluate the diagnostic value of plasma Circ CDR1as with regard to the radiographic severity.Plasma Circ CDR1as expressions were significantly higher in non-traumatic ONFH patients compared with healthy controls. In non-traumatic ONFH patients, there were no significant differences of CircCDR1as expressions between patients with alcohol-induced ONFH and steroid-induced ONFH. CircCDR1as in local necrotic tissue were significantly higher than adjacent non-affected tissue. Plasma and local Circ CDR1as expressions in patients with ARCO phase 4 were markedly upregulated compared with ARCO phase 3; plasma and local Circ CDR1as expressions in patients with ARCO phase 3 were markedly upregulated compared with ARCO phase 1/2. Plasma and local CircCDR1as expressions were positively associated with ARCO classification. In addition, plasma and local Circ CDR1as expressions were positively correlated with VAS and HHS scores. ROC curve analysis indicated that plasma Circ CDR1as may act as a decent marker for radiographic progression in non-traumatic ONFH patients.Both plasma and local expressions of CircRNA CDR1as are linked with disease severity in patients with non-traumatic ONFH. Non-traumatic osteonecrosis of the femoral head (ONFH) is a common disease characterized by death of bone cells due to insufficient blood flow , leadingCircular RNA (circRNA) is a new class of endogenously expressed non-coding RNA, which is characterized by covalently closed loop structures with neither 5' to 3' polarity nor polyadenylated tail . They arDerived from cerebellar degeneration-related protein 1 antisense transcript (CDR1AS), CDR1as is the best-known circRNA and has been uncovered as a regulator of various cellular processes . Abnormat test and correlation analysis, and a receiver operating characteristics (ROC) curve was established to estimate the value of circRNA expression as a biomarker in non-traumatic ONFH.All these previous studies indicated that Circ CDR1as may act as a potential marker to evaluate non-traumatic ONFH progression. However, there is no report about circCDR1as as biomarker to explore the correlation of diagnosis and prognosis in non-traumatic ONFH. In the current study, we examined the expression level of circCDR1as in ONFH tissues and plasmas by real-time quantitative reverse transcription-polymerase chain reactions (qRT-PCRs). Then, tests were performed between clinical information and circRNA expression level by analysis of variance (ANOVA) or A total of 99  unrelated non-traumatic ONFH patients receiving hip preserving surgery or total hip replacement (THR) were enrolled at the Panyu Hospital of Chinese Medicine  from July 2018 to July 2020. All the patients were diagnosed through symptoms, functional disability, and imaging findings. Meanwhile, 99  unrelated healthy subjects receiving regular medical check were drifted as controls. Patients were diagnosed using anteroposterior and lateral pelvic radiographs and magnetic resonance images. GC-induced ONFH was defined by a history of a mean daily dose of ≥ 16.6 mg or a highest daily dose of 80 mg of predinosolone equivalent within 1 year before the development of symptoms or radiological diagnosis in asymptomatic cases. The inclusion of alcohol-associated ONFH patients included the following: patients should have a history of alcohol intake > 400 ml/week  of pure ethanol for more than 6 months; ONFH should be diagnosed within 1 year after alcohol intake of this dose; and patients should not have other risk factors. Patients with a demonstrable history of direct trauma or possible combined causes were excluded. Healthy control subjects were matched with patients for gender and age and enrolled from subjects attending routine medical checkups. This study was approved by our ethical committee. Informed consents were obtained from both groups.For preserving surgery, marrow core decompression with bone grafting was used. Epidural anesthesia was used with the hip blocked up. A longitudinal incision was made on the external side of the femur 2 cm below the greater femur trochanter. Being monitored by the C-arm X-ray machine, the position of the guide pin was determined, and the guide pin was drilled into the center of the necrotic area beneath the femur head cartilage through the femoral neck from under the trochanter and screwed into the edge of lesion area below the head using a decompressor with tube core. The biopsy device was screwed into the lesion area, and the yellowish white wax-like loose diseased tissues (necrotic tissue) were taken out from the front end of the biopsy device and delivered for further study. For total hip arthroplasty, the femoral head was obtained according to the standard surgery procedure, and necrotic samples and adjacent non-necrotic samples were collected directly. The normal bone tissues were collected at least 3 cm away from the margin of necrotic tissue using the biopsy device.g, no later than 30 min after the collection of specimens for harvesting plasma under room temperature. All plasma samples were extracted immediately and were stored at − 80 °C for further analysis. Human bone samples (approximately 500 mg) were cryo-grinded under liquid nitrogen using a freezer-mill 6750 . 5 ml lysis/binding buffer from Dynabeads Oligo (dt)25 kit  were added to the grinded bone tissue, and it was centrifuged 9000 rpm for 15 min at room temperature. These parated median layer containing nucleic acid was added to 1 ml oligo-dt coated paramagnetic particles from Dynabeads Oligo (dt)25 kit. Total RNA was extracted from each specimen using Trizol reagent  according to the manufacturer’s instruction. RNA concentration was determined by ScanDrop Nuclear Acid Analyzer . In order to verify the integrity of RNA, 3 μl total RNA of each sample was electrophoretically separated on a 1% denatured agarose gel and subsequently detected via Chemical Mp Imaging System . If the peak area of 28S ribosome RNA (rRNA) was approximately twice than that of 18S rRNA, the integrity of total RNA was accepted and used for later experiments.Serial blood specimens were collected from patients before and after treatment. All specimens were collected in Vacutainer EDTA tubes and centrifuged for 10 min at 1000×− ΔΔCt method.The relative expression levels of CircCDR1as were quantified by quantitative reverse transcription polymerase chain reaction (qRT-PCR). All primers used in this study were designed via primer 5.0 software  and synthesized in Sangon Biotech   to ascertain the diagnosis and early diagnosis, and staging was used which includes radiographs, computed tomography (CT), bone scans, and MRI . For MRIPro-operative clinical assessments were conducted using the visual analogue scale (VAS)  score ant test or Mann–Whitney test, whereas one-way ANOVA or Kruskal–Wallis test were used for three or more groups followed by Tukey or Tamhan’s test for post hoc analysis. The Pearson rank or Spearman correlation test was employed to examine the relationship between plasma CircCDR1as expressions and ARCO classification as well as VAS score and HSS score. ROC curve analysis was employed to determine the diagnostic value of CircCDR1as with regard to ARCO classification. P values less than 0.05 were regarded as significant.All obtained data were analyzed using the Graphpad Prism 8.0 software . The Kolmogorov-Smirnov test was operated for testing the distribution  of the variables. Normal distribution was expressed as mean ± standard deviation, whereas non-normally distributed continuous variables were expressed as median (95% CI). We determined the differences of CircCDR1as levels between the two groups using student’s The non-traumatic ONFH patients included 59 males and 40 females with their ages ranged from 20 to 70 years. The disease course ranged from 3 to 250 months. The main symptoms were all unilateral or bilateral hip pain and functional disability. There were no significant differences of age, sex distribution, and BMI between non-traumatic ONFH patients and healthy controls Table .P < 0.001)  . Moreover, CircCDR1as expressions in necrotic tissues were significantly higher than adjacent non-necrotic tissue              Fig. a, but no06) Fig. b. These Our present study explored the correlation of plasma and local CircCDR1as expressions with disease severity of non-traumatic ONFH. We demonstrated for the first time that plasma CircCDR1as expressions were significantly higher compared with healthy controls. Meanwhile, in non-traumatic ONFH patients, local CircCDR1as expressions in necrotic tissue were markedly increased than in adjacent non-necrotic tissue. Moreover, increased plasma and local CircCDR1as expressions were significantly and positively correlated with ACRO stage. In addition, we also found both plasma and local CircCDR1as expression were positively related to VAS score and negatively correlated with HHS score. ROC curve analysis showed that increased plasma CircCDR1as expression may act as an early biomarker for ACRO stage. To our best of knowledge, this is the first time we identified circular RNA as a biomarker in the progression of non-traumatic ONFH.The typical pathological features of non-traumatic ONFH include decreased density of the femoral head, cystic degeneration of the bone, fracture of the trabecular structure and deformity and collapse of the femoral head . HoweverPrevious studies have shown that CircRNAs have many important biological functions. First, CircRNAs act as sponges of miRNAs to regulate the function of miRNA target genes , 27. SecRecent studies implicated CircRNAs may have an important role in ONFH, which is closely, to their functions, related to the activity of osteoblasts and osteoclasts, endothelial cell damage, weakened osteogenic differentiation, and increased adipogenic differentiation of BMSCs . TherefoIn the present study, we assessed the potential expressions of plasma and local CDR1as, a CircRNA with the disease severity of non-traumatic ONFH. First, we found plasma and local CDR1as was significantly increased in non-traumatic ONFH patients. One previous study found that CircRNA CDR1as was overexpressed in SONFH-BMSCs with decreased osteogenic differentiation . FurtherThere were several limitations that should be noted. Firstly, this study was carried out and single-centered study with relatively sample size in China, further multicenter studies with more samples are needed to identify the results. Secondly, since this is a cross-sectional study, the causal relationship between increased expression CircCDR1as and development of ONFH could not be illuminated. Therefore, the detailed mechanisms that unveiling CircCDR1as in ONFH should be explored in further study. Thirdly, we did not measure CircCDR1as expressions in healthy individual’s femoral head due to ethical reasons. Hence, we were not able to compare the rate and amount of increment between plasma CDR1 and local CDR1 to show whether plasma CDR1 or local CDR1 is a more useful determinant in that the we used different controls. Last, we did not explore the potential mechanism of CircCDR1as in the development of non-traumatic ONFH.In conclusion, we found higher levels of plasma CircCDR1as in non-traumatic ONFH patients compared with healthy controls. Also, plasma and local CircCDR1as expressions were positively correlated with radiological and symptomatic severity. Further studies investigating the intensive mechanisms of CircCDR1as in non-traumatic ONFH would provide a better insight about the potential clinical target of CircCDR1as.Therapies that targeting CircCDR1as and its related regulating signaling ways may serve as a potential interventional method in non-traumatic ONFH.