{"paper_id":"2bebe5c0-0990-4c6f-a1e5-ea93bb470ac0","body_text":"1\nVol.:(0123456789)Scientific Reports |         (2022) 12:5775  | https://doi.org/10.1038/s41598-022-09386-4\nwww.nature.com/scientificreports\nTranscriptomic analysis of cumulus \ncells shows altered pathways \nin patients with minimal and mild \nendometriosis\nCaroline Mantovani Da Luz 1,2*, Michele Gomes Da Broi 1,2, Larissa de Oliveira Koopman 1,2,  \nJessica Rodrigues Plaça 3, Wilson Araújo da Silva‑Jr 3,4, Rui Alberto Ferriani 1,2, \nJuliana Meola 1,2 & Paula Andrea Navarro 1,2\nEndometriosis is a chronic inflammatory disorder that is highly associated with infertility. This \nassociation seems to be related to oocyte impairment, mainly in the initial stages of endometriosis \n(minimal and mild), where no distortions or adhesions are present. Nonetheless, invasive oocyte \nanalyses are not routinely feasible; thus, indirect assessment of oocyte quality is highly desirable, \nand, in this context, cumulus cells (CCs) may be more suitable targets of analysis. CCs are crucial in \noocyte development and could be used as an index of oocyte quality. Therefore, this prospective case–\ncontrol study aimed to shed light on the infertility mechanisms of endometriosis I/II by analyzing the \nCCs’ mRNA transcription profile (women with endometriosis I/II, n = 9) compared to controls (women \nwith tubal abnormalities or male factor, n = 9). The transcriptomic analyses of CCs from patients with \nminimal and mild endometriosis revealed 26 differentially expressed genes compared to the controls. \nThe enrichment analysis evidenced some altered molecular processes: Cytokine‑cytokine receptor \ninteractions, Chemokine signaling, TNF signaling, NOD‑like receptor signaling, NF‑kappa B signaling, \nand inflammatory response. With the exception of CXCL12, all enriched genes were downregulated \nin CCs from patients with endometriosis. These findings provide a significant achievement in the \nfield of reproductive biology, directing future studies to discover biomarkers of oocyte quality in \nendometriosis.\nEndometriosis is a chronic inflammatory and estrogen-dependent disorder characterized by functional endome-\ntrial-like tissue outside the  uterus1. According to the American Society for Reproductive Medicine, endometriosis \ncan be classified into four stages: I-minimal, II-mild, III-moderate, and IV-severe2. It affects 6–10% of women of \nreproductive  age3, and approximately 30–50% of them are estimated to be  infertile4. The mechanisms underlying \nthe etiopathogenesis of infertility in patients with endometriosis remain unclear, especially in the initial stages \n(minimal and mild), when no distortions and adhesions in the reproductive tract are  present5.\nSeveral studies suggest that the infertility presented by these patients may be due to compromised oocyte \nquality and, consequently, embryonic  quality5–15, or the interaction between the endometrium and the  embryo1,16. \nIn assisted reproduction techniques, women with endometriosis have shown decreased oocyte quality and fer -\ntilization  rates15,17.\nNonetheless, invasive oocyte analyses are not routinely feasible since human oocytes are rarely donated to \nresearch centers, and their application in invasive techniques precludes subsequent use in assisted reproduction \nprocedures. Thus, the indirect evaluation of oocyte quality may contribute to understanding endometriosis-\nrelated  infertility18–20.\nIndirect oocyte quality assessments are highly desirable, and, in this context, cumulus cells (CCs) may be \nmore feasible targets of analysis. CCs are a specialized type of granulosa cells (GCs) 21 that surround the oocyte \nOPEN\n1Division of Human Reproduction, Department of Gynecology and Obstetrics, Ribeirão Preto Medical School, \nUniversity of São Paulo, 3900 Bandeirantes Avenue, Ribeirão Preto, São Paulo 14049-900, Brazil. 2National \nInstitute of Hormones and Women’s Health, CNPq, Porto Alegre, Rio Grande Do Sul 90035-003, Brazil. 3Center \nfor Integrative Systems Biology - CISBi, NAP/USP , Ribeirão Preto, São Paulo 14049-900, Brazil. 4Department \nof Genetics, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, São Paulo 14049-900, \nBrazil. *email: carollinemantovani@gmail.com\n\n2\nVol:.(1234567890)Scientific Reports |         (2022) 12:5775  | https://doi.org/10.1038/s41598-022-09386-4\nwww.nature.com/scientificreports/\nsince antrum  formation22 and contribute to oocyte development and  maturation22,23. CCs are closely linked to \nthe oocyte through a network of gap junctions and paracrine  signals24,25 and are metabolically codependent; \ntherefore, any change in the CCs can affect oocyte  quality15,26,27. Some studies have shown that genetic alterations \nin the CCs of infertile women with endometriosis may be related to lower oocyte  quality10,12,28. However, no \nstudy has evaluated the transcriptome of CCs from infertile patients with minimal and mild endometriosis. In a \nprevious study conducted by our group, CCs from patients with advanced (moderate and severe) endometriosis, \nwith and without endometrioma, presented changes in the transcription profile related to acetylation, mitochon-\ndrial function (oxidative phosphorylation), and steroid  biosynthesis29. Poli-Neto et al. (2020) demonstrated that \nthe eutopic endometrium of women in the initial stages of endometriosis had a predominant proinflammatory \nprofile, and the cellular microenvironments and immune cell profiles were different in the initial and advanced \nstages of the  disorder30. Since the initial stages display particular morphological and physiological findings, is the \ntranscriptome also altered as in advanced stages of endometriosis? The present study aimed to analyze the mRNA \ntranscription profile of CCs from infertile women with minimal and mild endometriosis compared to infertile \nwomen without endometriosis to gather knowledge on the mechanisms of endometriosis I/II-related infertility.\nMaterials and methods\nEthical approval. The Research Ethics Committee of the Clinical Hospital of Ribeirão Preto Medical School \napproved this prospective case–control study (Protocol No. 15113/2012, approval date December 5, 2012). All \nmethods were carried out following the Code of Ethics of the World Medical Association and the Declaration of \nHelsinki. All participants provided written informed consent.\nEligibility criteria. The endometriosis group consisted of patients in the initial stages of endometriosis \n(minimal and mild), diagnosed by video laparoscopy, in the absence of male infertility. The control group con-\nsisted of patients who underwent diagnostic video laparoscopy to rule out the presence of endometriosis and \nwho were diagnosed with male and/or tubal factor-related infertility.\nOther eligibility criteria for both groups were age ≤  39 years, body mass index (BMI) ≤  34.9 kg/m2, and non-\nsmoker. Participants presenting the following were excluded from the study: polycystic ovary syndrome or \nchronic anovulation; untreated endocrinopathies (diabetes or hypothyroidism); cardiovascular disease; dyslipi-\ndemia; rheumatologic and auto-immune diseases, and active infection.\nControlled ovarian stimulation protocol. All patients included in this study (endometriosis I/II and \ncontrol patients) underwent controlled ovarian stimulation. After synchronizing the cycles using oral contra-\nceptive pretreatment, controlled ovarian stimulation was carried out according to the needs of each patient and \nfollowing the clinical protocols adopted in our Assisted Reproduction Program, as previously  described29.\nOocytes were retrieved from 34 to 36 h after the administration of hCG, and the luteal phase was maintained \nby vaginal administration of micronized progesterone (600 mg/day, Utrogestan®, Besins, Brazil).\nSample collection. Samples were collected from all eligible patients who agreed to participate in the study \nfrom August 2014 to February 2016. The cumulus-oocyte complex was collected on oocyte retrieval, as previ-\nously described by Da Luz et al. (2021)29. CCs were mechanically removed by microdissection, transferred to a \ncryotube containing 100 μL of cryopreserves (RNAlater®, Life Technologies, USA), and stored in liquid nitrogen \nat -196 °C until total RNA extraction.\nTotal RNA. Total RNA was extracted from the CCs using the AllPrep DNA/RNA/miRNA Kit (Qiagen®, Ger-\nmany). The total RNA samples were diluted in 20 μL of the recommended solution, and the concentrations were \nquantified with the Qubit RNA BR Assay Kit (Invitrogen, USA) in a Qubit 2.0 Fluorometer (Invitrogen, USA). \nAccording to the manufacturer’s instructions, RNA integrity was evaluated using the Agilent RNA 6000 Nano \nKit (Agilent Technologies, USA) in an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Samples with an \nRNA Integrity Number (RIN) ≥ 7.0 were considered appropriate.\nPool strategy. As explained in our previous  study29, CCs have a considerably low concentration of total \nRNA; thus, we chose to cluster the samples in pools, a strategy described in several reports with  CCs27,31–37 and \nrecommended as  effective38. Pools of 3 patients each yielded sufficient total RNA (≥ 120 ng) and enabled to keep \nthe pool size small, as  recommended38. All pools had RINs ≥ 7.0.\nThe following clinical characteristics were considered for clustering the pools (in order of importance): age, \nnumber of oocytes collected; BMI; controlled ovarian stimulation protocol, and time after video laparoscopy. \nThe samples in the pools were clustered heterogeneously, and the pools were homogeneous with each other. This \nstrategy was adopted because we compared the groups, not individual samples.\nLibrary and RNA‑Seq. Library construction was carried out following the TruSeq® RNA Sample Prepara-\ntion v2 (Illumina Inc, USA) protocol. RNA-Seq was performed using the TruSeq Cluster Generation Kit v5 \n(Illumina Inc, USA), following the manufacturer’s instructions. The six libraries were distributed into 2 lanes \nand underwent paired-end sequencing (PE 2 × 101 bp) using the HISEQ2500 Illumina Platform through High \nOutput run.\nBioinformatics. The quality control of the nucleotide sequences was conducted using the FastQC v0.11.2 \nprogram. The PRINSEQ v0.20.4 program was used to remove nucleotides that did not meet the quality. The \n\n3\nVol.:(0123456789)Scientific Reports |         (2022) 12:5775  | https://doi.org/10.1038/s41598-022-09386-4\nwww.nature.com/scientificreports/\nmapping and quantification of the reads were performed using the STAR program (v. 020,201)39, with GRCh37.\np7 as the reference genome, and Ensembl Release 85 for gene annotation. Subsequently, the normalization and \ndifferential expression between the groups were carried out using the Bioconductor DESeq2 (version 1.15.40)40 \nin the R statistical  environment41. We considered differentially expressed genes (DEGs) the genes with adjusted \np value (FDR) < 0.05. Heatmap plots were unsupervised and generated with the aheatmap function.\nEnrichment analysis. DEGs were analyzed using the DAVID-Bioinformatics Resources 6.8. database for \nrelevant molecular processes and pathways. The proteins encoded by the DEGs were analyzed with the STRING \n11.5 database for relevant interactions and pathways. The platforms Kyoto Encyclopedia of Genes and Genomes \n(KEGG) and Gene Ontology (GO) were also consulted when appropriate.\nStatistics. Exploratory data analysis was performed using measures of central tendency, dispersion, and \nbox-plot plots. Clinical characteristics (age, BMI, infertility time, and the number of oocytes collected) were \ncompared between groups using the Mann–Whitney test. All analyses were conducted using the SAS program, \nversion 9.4. Data were presented as median, minimum, and maximum, and significance was defined as p < 0.05.\nEthics approval. The Research Ethics Committee of the University Hospital approved this prospective \ncase–control study (Protocol No. 15113/2012, approval date 12/05/2012), which was carried out following The \nCode of Ethics of the World Medical Association.\nConsent to participate. All participants provided written informed consent (Protocol No. 15113/2012, \napproval date 12/05/2012).\nConsent for publication. All authors have read the manuscript and approved its publication.\nResults\nFlowchart. During the recruitment period, a total of 54 patients were deemed eligible. However, seven \npatients did not agree to participate in the study. Thus, 47 patients provided written informed consent and \nbegan controlled ovarian stimulation for intracytoplasmic sperm injection. Ten patients did not undergo oocyte \nretrieval, whereas 37 did. There was no oocyte in 4 of them, and five patients exhibited few CCs, impeding dona-\ntion for the study. Therefore, the obtained CCs were donated by 28 patients. Total RNA was isolated from the \nCCs, and RNA integrity was assessed, although it was unacceptable in 10 samples. We obtained 18 samples, nine \ncontrols, and nine patients with endometriosis I/II; the samples were clustered in pools of three patients each. \nThe flowchart is depicted in Fig. 1.\nClinical variables. No significant differences between groups were observed regarding age, BMI, infertility \ntime, and the number of oocytes (Table 1). The majority of patients (83.3%; N:15) were submitted to the flexible \nantagonist protocol plus rFSH or menotropin. Only 3 (16.7%; 1 control and two endometriosis I/II patients) \nunderwent the minimal stimulation protocol.\nRNA next‑generation sequencing. Approximately 50 million reads per sample were obtained with \nRNA-Seq, and the average mapping quality was 92.8%. RNA-Seq provides the differential gene expression pro-\nfiles in CCs of patients with endometriosis I/II compared to the controls. Such analysis enabled us to obtain a list \nof 26 DEGs (21 down-regulated and five up-regulated, in endometriosis I/II) with adjusted p < 0.05 as significant \n(Fig. 2).\nFigure 1.  Flowchart of the study.\n\n4\nVol:.(1234567890)Scientific Reports |         (2022) 12:5775  | https://doi.org/10.1038/s41598-022-09386-4\nwww.nature.com/scientificreports/\nFunctional enrichment analysis. The 26 DEGs of endometriosis I/II were used to perform the enrich-\nment analysis. The main enriched pathways potentially associated with infertility-related endometriosis in the \nCCs were: Cytokine-cytokine receptor interactions, Chemokine signaling pathway, Tumor-Necrosis Factor \n(TNF) signaling pathway, Nucleotide-binding Oligomerization Domain (NOD)-like receptor signaling path-\nway, and Nuclear Factor (NF)-kappa B signaling pathway (Table 2). Some genes were present in more than one \npathway, all of which share multiple genes. Interestingly, all enriched genes underwent a negative regulation of \nTable 1.  Clinical variables of women with endometriosis I/II and controls. Data presented as median \n(minimum and maximum). E I/II, minimal and mild endometriosis; BMI, Body Mass Index. p value < 0.05.\nClinical variables Control (N = 9) E I/II (N = 9) P value\nAge (years) 34 (30; 39) 36 (33; 39) 0.34\nBMI (kg/m2) 24.23 (19.88; 30.22) 25.52 (22.60; 32.05) 0.72\nInfertility time (months) 84 (24; 139) 30 (11; 192) 0.18\nNumber of oocytes retrieved 9 (2; 13) 8 (2; 15) 0.89\nFigure 2.  Unsupervised heatmap of the 26 DEGs of CCs from patients with endometriosis I/II compared to the \ncontrols.\n\n5\nVol.:(0123456789)Scientific Reports |         (2022) 12:5775  | https://doi.org/10.1038/s41598-022-09386-4\nwww.nature.com/scientificreports/\nexpression in the CCs of the patients with endometriosis I/II compared to the controls, except for the CXCL12, \nwhich was up-regulated. The enrichment analysis also highlighted an essential biological process, the inflam-\nmatory response with 12 altered genes (CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL12, TNFAIP3, \nTNFRSF9, IL1A, IRAK2, and NFKB1) (p < 0.001).\nOf the 26 DEGs, 25 were protein-coding genes. These genes encode 25 proteins, which were used to enrich \nprotein–protein interaction networks (Fig. 3). The protein–protein interaction enrichment p value was < 1.0e−16.\nThe same pathways enriched using DEGs were found in the protein analysis (Fig. 3). The interaction enrich-\nment showed that these proteins have significantly more interactions among themselves than expected for a \nrandom set of proteins of the same size and degree distribution drawn from the genome. Such enrichment \nindicates that the proteins are at least partially biologically connected as a group.\nDiscussion\nEndometriosis related to infertility seems to be associated with oocyte impairment, mainly in the initial stages of \nendometriosis, when no distortions or adhesions in the reproductive tract are  present6,42,43. Exposure to hostile \nenvironments with macrophages, cytokines, and reactive oxygen species in the peritoneal and follicular fluid \ncould lead to dysfunctional folliculogenesis and worsen oocyte  quality5,13,44,45. Within this environment, the \ncross-talk between oocyte and CCs is crucial for oocyte  development24,25. Therefore, CCs reflect oocyte status \nand could be used as an index of oocyte  quality10,12,27,28. A large-scale analysis is essential to comprehend the \nbiological changes in CCs. This study was the first to evaluate the transcriptome of CCs from infertile patients \nwith endometriosis I/II compared to women without the disease.\nThe differential gene expression profile in the CCs of patients with endometriosis I/II showed 26 DEGs \ncompared to the controls, demonstrating that endometriosis I/II is related to the deregulation of the CCs’ tran-\nscriptome. Subsequently, enrichment analysis showed altered molecular mechanisms in the CCs of patients with \nendometriosis I/II; Cytokine-cytokine receptor interactions, Chemokine signaling, TNF signaling, NOD-like \nreceptor signaling, and NF-kappa B signaling. These pathways are related to immunity, and, except for CXCL12, \nall enriched genes are downregulated in endometriosis CCs. Allegra et al. (2014) also found deregulated genes \nin all these pathways from CCs of women with severe endometriosis by microarray  analysis27.\nIt is known that endometriosis is a chronic inflammatory disease that can cause excessive reactive oxygen \nspecies (ROS) accumulation and, consequently, intra-follicular oxidative stress, even in infertile women with \nendometriosis I/II 46. Lin et al. (2020) found an increase in ROS in the granulosa cells of patients with endo-\nmetriosis and suggested that this process induces cell senescence, contributing to endometriosis-associated \n infertility47. This proinflammatory and ROS-filled microenvironment can trigger immune system pathways like \nthose found in our study.\nNOD-like receptors are known as recognition receptors, responsible for recognizing pathogen-associated \nmolecular patterns released by damaged  cells48. The activation of these receptors leads to the transcription \nof several genes, including NFKB, which induces inflammatory cytokines and  chemokines49,50. In the ovary, \ncytokines and chemokines promote leukocyte recruitment and activation, steroidogenesis, follicular growth, and \n ovulation51,52. In the literature, several cytokines are found down-regulated in endometriosis CCs when compared \nto the controls. Moreover, follicular fluid cytokines appear to be related to successful pregnancy following IVF \n treatments52. The TNF signaling pathway acts in several processes, including cell proliferation, differentiation, \nand apoptosis, in addition to the modulation of immune and inflammatory  responses53. In part of this pathway, \nthe gene TNFAIP6 plays an essential role in forming the extracellular matrix of the cumulus-oocyte  complex54,55. \nAllegra et al. (2014) showed the down-regulation of the TNFAIP6 gene in CCs of patients with  endometriosis27. \nAll of these pathways are essential for ovulation, as well as fertilization. The expansion of the cumulus-oocyte \ncomplex can be improved by activating Toll-like receptors, followed by genes such as NFKB, besides cytokines and \n chemokines50. An inflammatory process marks the rupture of the follicle. Moreover, sperm induces the release \nof cytokines and chemokines from CCs, enhancing the fertilization  process56,57. Therefore, alterations in these \nintricate molecular mechanisms may compromise oocyte quality and decrease fertilization rates.\nThe main limitation of this study was its small sample size, resulting from the strict eligibility criteria adopted \nand the low RNA integrity and concentrations in CCs. Also, pooling samples might not be beneficial when the \ngene expression levels display low variability and reduce samples. However, small RNA sample pools effectively \nreduce the variability and compensate for the loss of  replicates38. Furthermore, the data obtained from studies \nTable 2.  In silico enrichment analysis of 26 DEGs of CCs from patients with endometriosis I/II. The in silico \nenrichment analysis was performed using the DAVID-Bioinformatics Resources 6.8 tool and KEGG database. \n↑ = Positively regulated. All other genes were negatively regulated. P value < 0.05.\nPathways Genes P value\nCytokine-cytokine receptor interaction CXCL12↑, CCL20, CXCL1, CXCL3, CXCL5, CXCL8, TNFRSF9, CSF3, and IL1A 2.3e−4\nChemokine signaling CXCL12↑, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, and NFKB1 2.3e−7\nTNF signaling CCL20, CXCL1, CXCL2, CXCL3, TNFAIP3, BIRC3, ICAM1 and NFKB1 4.5e−9\nNOD-like receptor signaling CXCL1, CXCL2, CXCL8, TNFAIP3, BIRC3, and NFKB1 3.6e−4\nNF-kappa B signaling CXCL12↑, CXCL8, TNFAIP3, BIRC3, ICAM1, and NFKB1 2.1e−6\n\n6\nVol:.(1234567890)Scientific Reports |         (2022) 12:5775  | https://doi.org/10.1038/s41598-022-09386-4\nwww.nature.com/scientificreports/\nusing samples collected after different controlled ovarian stimulation protocols may not necessarily be extrapo-\nlated to natural cycles.\nIn conclusion, the present study shows, for the first time, that endometriosis I/II could promote alterations in \nthe transcriptome of CCs. These results provide a better understanding of the mechanisms (Cytokine-cytokine \nreceptor interactions, Chemokine, TNF , NF-kappa B, NOD-like receptor signaling, and inflammatory response) \nthat may affect oocyte competence acquisition in patients with endometriosis I/II. These pathways share a vari-\nety of genes and cannot be considered an individualized process. This differential transcription profile provides \na significant achievement in the field of reproductive biology, directing future studies with a larger cohort to \ndiscover biomarkers of oocyte quality in endometriosis, be they pathways or genes.\nFigure 3.  In silico enrichment analysis of 25 proteins encoded by DEGs of the CCs from patients with \nendometriosis I/II. Note Protein-coding gene = identified outside the nodes. Nodes = proteins (inside the nodes \nis the protein 3D structure). The colors of the nodes represent the pathways in which they were enriched. White \nnodes are the second shell of interactions. Edges = protein–protein associations. There are 86 edges. The white \nnodes on the left side, without edges, have no protein–protein associations. FDR, False Discovery Rate. Created \nin STRING. Permanent link: https:// versi on- 11-5. strin gdb. org/ cgi/ netwo rk? netwo rkId= bAvx7 jSmic 41.\n\n7\nVol.:(0123456789)Scientific Reports |         (2022) 12:5775  | https://doi.org/10.1038/s41598-022-09386-4\nwww.nature.com/scientificreports/\nData availability\nThe RNA-sequencing data underlying this article is available in the repository Sequence Read Archive (SRA) \nof the National Center for Biotechnology Information (NCBI) (Permanent link: https:// www. ncbi. nlm. nih. gov/ \nsra/? term= PRJNA 808988), study number: PRJNA808988. Other datasets generated during the current study \nare available from the corresponding author on reasonable request.\nReceived: 30 September 2021; Accepted: 11 March 2022\nReferences\n 1. Giudice, L. C. & Kao, L. C. Endometriosis. 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J. \nEndocrinol. 180, 203–212. https:// doi. org/ 10. 1677/ joe.0. 18002 03 (2004).\nAcknowledgements\nThe authors would like to thank the staff of the Human Reproduction Division of the RPMS of the University \nof São Paulo, particularly Maria Cristina Picinato, Roberta Giorgenon, Camila Kokudai, and Thalita Berteli for \ncollecting the CCs, and Lilian Eslaine da Silva and Cristiana Carolina Padovan for their technical assistance.\nAuthor contributions\nC.M.L. was responsible for the study design, sample collection and preparation, acquisition of data, and inter -\npretation of the results, as well as manuscript writing. M.G.B. provided substantial contributions to the study \ndesign, data interpretation, and manuscript review. L.O.K. contributed with sample analysis, acquisition of data, \nand manuscript review. J.R.P . was responsible for the bioinformatics analysis and contributed to data interpreta-\ntion. W .A.S. contributed to data interpretation and manuscript review. R.A.F . provided important intellectual \ncontent, sample collection, and manuscript review. J.M. contributed to data interpretation and critically revising \nthe manuscript for important intellectual content. P .A.N. contributed to the study design, data interpretation, \ncritical review of the manuscript, and was the project coordinator. All authors have approved the final version \nand submission of the manuscript.\nFunding\nThis work was supported by the Research Support Foundation of the State of São Paulo (grant No. 2014/05878-7 \nand fellowship No. 2014/026830, C.M.L.), and National Council for Scientific and Technological Development \nthrough the Brazilian National Institute of Hormones and Women’s Health (fellowship No. 88887.141452/2017-\n00, M.G.B.).\nCompeting interests \nThe authors declare no competing interests.\nAdditional information\nCorrespondence and requests for materials should be addressed to C.M.D.L.\n\n9\nVol.:(0123456789)Scientific Reports |         (2022) 12:5775  | https://doi.org/10.1038/s41598-022-09386-4\nwww.nature.com/scientificreports/\nReprints and permissions information is available at www.nature.com/reprints.\nPublisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access  This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the \nCreative Commons licence, and indicate if changes were made. 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