{"paper_id":"af0f0fef-a6ab-4c29-b9ee-aa9555785b75","body_text":"RESEARCH Open Access\n© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, \nsharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and \nthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included \nin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will \nneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The \nCreative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available \nin this article, unless otherwise stated in a credit line to the data.\nMarshall et al. Reproductive Biology and Endocrinology           (2023) 21:33 \nhttps://doi.org/10.1186/s12958-023-01083-9\nReproductive Biology \nand Endocrinology\n*Correspondence:\nA. Marshall\nAnne.Marshall@med.uni-heidelberg.de\n1Dept. of Gynecological Endocrinology and Fertility Disorders, University \nHospital Heidelberg, Im Neuenheimer Feld 440, 69120 Heidelberg, \nGermany\n2Dept. of Pathology, University Hospital Heidelberg, Im Neuenheimer Feld \n224, 69120 Heidelberg, Germany\nAbstract\nBackground The pathogenesis of deep infiltrating endometriosis (DIE) is poorly understood. It is considered a \nbenign disease but has histologic features of malignancy, such as local invasion or gene mutations. Moreover, it is \nnot clear whether its invasive potential is comparable to that of adenomyosis uteri (FA), or whether it has a different \nbiological background. Therefore, the aim of this study was to molecularly characterize the gene expression signatures \nof both diseases in order to gain insight into the common or different underlying pathomechanisms and to provide \nclues to pathomechanisms of tumor development based on these diseases.\nMethods In this study, we analyzed formalin-fixed and paraffin-embedded tissue samples from two independent \ncohorts. One cohort involved 7 female patients with histologically confirmed FA, the other cohort 19 female patients \nwith histologically confirmed DIE. The epithelium of both entities was microdissected in a laser-guided fashion and \nRNA was extracted. We analyzed the expression of 770 genes using the nCounter expression assay human PanCancer \n(Nanostring Technology).\nResults In total, 162 genes were identified to be significantly down-regulated (n = 46) or up-regulated (n = 116) in \nDIE (for log2-fold changes of < 0.66 or > 1.5 and an adjusted p-value of < 0.05) compared to FA. Gene ontology and \nKEGG pathway analysis of increased gene expression in DIE compared to FA revealed significant overlap with genes \nupregulated in the PI3K pathway and focal adhesion signaling pathway as well as other solid cancer pathways. In FA, \non the other hand, genes of the RAS pathway showed significant expression compared to DIE.\nConclusion DIE and FA differ significantly at the RNA expression level: in DIE the most expressed genes were those \nbelonging to the PI3K pathway, and in FA those belonging to the RAS pathway.\nKeywords Gene expression analysis, Deep infiltrating endometriosis, Adenomyosis, PI3K pathway, RAS pathway\nComparing gene expression in deep \ninfiltrating endometriosis with adenomyosis \nuteri: evidence for dysregulation of oncogene \npathways\nA. Marshall1*, K. F. Kommoss2, H. Ortmann1, M. Kirchner2, J. Jauckus1, P . Sinn2, T. Strowitzki1 and A. Germeyer1\n\nPage 2 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \nBackground\nDeep infiltrating endometriosis (DIE) and adenomyo -\nsis (FA) are very common benign gynecological condi -\ntions in women of childbearing age: 10–15% of women \nundergoing laparoscopy for benign reasons are generally \nfound to have endometriosis [ 1], and in patients with \ninfertility the incidence is even higher at 30–50% [ 2]. DIE \nis thought to occur in at least 20% of women with pelvic \nendometriosis [ 3]. FA affects 19.5% of women of child -\nbearing age [ 4] [5]. In hysterectomy specimens, the inci -\ndence of diagnosed FA is 10–35% [ 6].\nBoth conditions are defined by the presence of endo -\nmetrial glands and stroma either in the myometrium or \noutside the uterus. Originally DIE was even called “ade -\nnomyosis externa” [7].\nThere is a major discussion, if FA and especially DIE \nare related diseases or not, as in women with both enti -\nties the phenotype of FA appears to be related to the \nseverity of endometriosis, particularly as women with \nDIE had a significantly higher frequency of focal adeno -\nmyosis in the external myometrium than patients with \novarian endometriosis [ 8]. Clinically however, the two \nconditions differ markedly. FA is commonly associated \nwith dysmenorrhea, infertility, repeated implantation \nfailure and pregnancy loss [ 9– 11]. Multiparity and previ -\nous uterine surgery are discussed as risk factors for this \ncondition [12– 14]. DIE, on the other hand, is known to \ncause extensive adhesions up to complete obliteration \nof the Douglas space, as well as constriction of affected \norgans such as the bowel and bladder, and thus can cause \nnot only dysmenorrhea but also chronic abdominal pain \nand, in severe cases, bowel obstruction up to the point of \nan ileus.\nAlthough histologically benign, both DIE and FA are \ncharacterized by their propensity for local tissue invasion \nand resistance to apoptosis [ 15]. Notably, DIE has been \ndescribed as a “benign tumor” [ 16]. Recent work using \nnext-generation sequencing (NGS) has demonstrated \ndriver mutations in cancer associated genes such as \nPIK3CA, ARID1A, PPP2R1A and KRAS in both, ovarian \nendometrioma [17] and DIE [15].\nThe presence of PIK3CA- or KRAS-mutated clones in \nhistologically normal uterine endometrium in endome -\ntriosis [17] but also in patients without endometriosis has \nalso been demonstrated [ 18], so the theory of the cellu -\nlar origin of endometriosis requires further investigation. \nIn contrast, the discovery of identical mutations in the \nKRAS gene in coexisting adenomyotic and endometriotic \nlesions in several patients [ 11], supports the theory of a \ncommon pathogenesis of adenomyosis uteri and endo -\nmetriosis and a common molecular mechanism in these \ndiseases [11, 19].\nIn this study, we aimed to further characterize the \nmolecular mechanisms involved in FA and especially DIE \nto find molecular similarities and differences in both dis -\neases. To this end, we analyzed cancer-related signaling \npathways at the gene expression level using a nanostring \ngene panel encompassing the major signaling pathways of \ncarcinogenesis using epithelial cells of FA and DIE. Any \nrelevant genomic changes at the DNA level should be \nreflected in their gene expression and provide insight into \nthe common or different underlying pathomechanisms of \nthe two diseases and provide clues to the pathomecha -\nnisms of tumor development in these diseases and treat -\nment options.\nPatients and methods\nStudy population\nFor this study, we collected formalin-fixed and paraffin-\nembedded (FFPE) tissue samples for the analysis of two \nindependent cohorts of patients with DIE or FA. Patients \nunderwent surgery at the University Hospital, Heidelberg \nor cooperating clinics and the samples were histologically \nexamined and assessed at the Dept. of Pathology of the \nUniversity Hospital, Heidelberg between 2003 and 2018. \nClinical records and histology were reviewed. Exclusion \ncriteria were histological indications of cancer or dyspla -\nsia or a lesion size too small to gain sufficient material \nfor further analysis. The samples were provided by the \nTissue Bank at the National Center for Tumor Diseases \n(Heidelberg, Germany) in concordance with the Ethics \nCommittee of the University of Heidelberg (approval No. \nS-362/2017).\nStaining and laser microdissection (LMD)\nFor the RNA extraction, FFPE tissue blocks from FA and \nDIE were selected after reviewing all original tissue slides \nand were recut for hematoxylin & eosin sections, to be \nused for reference and to determine the lesion size. RNA \nwas extracted using 10–20 FFPE slides for each entity.\nFor mounting on Zeiss 1.0 PEN slides (Carl Zeiss, \nOberkochen, Germany) and for better adhesion of the \ntissue to the membrane, slides were irradiated with \nUV light/254nm for 30  min before, FFPE tissue blocks \nwere cut at 8  μm thickness and incubated overnight at \n37  °C. They were dewaxed in xylene (100%), rehydrated \nthrough decreasing concentrations of ethanol (100, 95, \n75%), stained in 1% cresyl violet acetate (Sigma-Aldrich, \nTaufkirchen, Germany) and again dehydrated in increas -\ning ethanol concentrations (75, 95, 100%). After that, tis -\nsue sections were dried and stored at 4 °C. Using a ZEISS \nPALM LMD laser capture microdissection unit, regions \nof interest (epithelium of the adenomyosis or epithelium \nof deep infiltrating endometriosis) were microdissected. \nThe isolated tissue fragments corresponded to an area \nof approximately 20.000.000  μm² for each sample. They \nwere collected in AdhesiveCap 500 opaque tubes (Carl \n\nPage 3 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \nZeiss) and stored at − 20  °C until further processing \n(Fig. 1).\nRNA isolation\nExtraction of the total RNA from microdissected tissue \nsamples was performed using the AllPrep DNA/RNA \nFFPE Kit (Qiagen, Venlo, the Netherlands) according to \nthe manufacturer’s protocol. They were quantified with \nthe Nanodrop ND-1000 spectrophotometer (NanoDrop \nTechnologies, Rockland, DE, USA).\nGene expression analysis\nWe analyzed the expression of 770 genes (Codeset: \nHuman PanCancer Pathways) and hybridisation counts \nwere measured using the nCounter technology (both by \nNanostring™ Technology, Seattle, WA). A minimum of \napproximately 50 ng of total RNA was used. Hybridiza -\ntion time per cartridge was 16  h before measurement. \nAccording to the manufacturer’s protocol, the exam -\nined genes were attached to specific tag sequences and \nhybridized for 16 h at 65 °C to a capture/reporter probe \npair equipped with a fluorescent barcode. These gene-\nspecific barcodes were then detected by the nCounter \nDigital Analyzer providing count of genes. No cases were \nexcluded.\nThe 770 genes codeset included 730 genes from 13 \ncanonical pathways (e.g., cell cycle, chromatin modeling, \napoptosis, MAPK, and PI3K) and 40 housekeeping \ngenes [ 20]. The raw data were pre-analyzed for consis -\ntency using the manufacturer’s software (nSolver version \n4.0). The geNorm pairwise variation statistics was used \nfor stepwise selection of normalization genes from the \nhousekeeping genes [ 21]. Six genes with minimal pair -\nwise variation statistics were finally selected for normal -\nization ( TLK2, VPS33B, TMUB2, C10orf76, SLC4A1AP, \nERCC3).\nStatistics\nDifferential expression analysis was carried out using a \nlinear data model in limma [22, 23], and nominal p-values \nwere corrected for multiple comparisons using Benjamini \nand Hochberg’s method [24]. All genes with an adjusted \nfalse discovery rate (FDR) of p < 0.05 and fold change of \n< 0.66 or > 1.5 were considered differentially expressed. \nDifferentially expressed genes (DEGs) were subjected to \nfunctional annotation and clusterization using DAVID \nBioinformatics Resources (version 6.8, https://david.\nncifcrf.gov/ [ 25, 26]) after conversion of gene symbols \nto Entrez IDs and uploading to DAVID using the “RDA -\nVIDWebService” BioConductor library [ 27]. Basal cyto -\nkeratin co-regulated genes were identified using DAVID \nanalysis in the “Biological Process” category and the \nKEGG pathway enrichment function with a significance \nthreshold of 0.05. The p-values of selected GO terms \nFig. 1 Light microscopy of deep infiltrating endometriosis of the rectum (A) and of adenomyosis uteri (B) stained with cresylviolet (10x). In each case the \nsecond image in the row shows the marking of the tissue for laser dissection [2]. The third image shows the tissue after laser dissection [3]\n \n\nPage 4 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \nwere corrected using Benjamini-Hochberg correction \nand described as adjusted p-values [ 24]. Otherwise, dif -\nferences between samples were tested using Wilcoxon \nsigned-rank test, and correlation was tested using Spear -\nman’s rank correlation test, and p-value of 0.05 was con -\nsidered significant. All statistical calculations were done \nusing R version 4.0 [28]. For visualization, the R packages \nComplexHeatmap and Ggplot2 were utilized.\nResults\nStudy population\nThe study cohort included 19 female patients with DIE. \nDIE lesions were defined as histologically confirmed \nendometriosis with infiltrative growth with a depth of \nmore than 5  mm into the wall of pelvic organs, e.g. in \nthe bowel or bladder (Tables  1 and 2). The control group \nincluded seven patients with FA and infiltration of less \nthan half of myometrium (superficial) or more than one \nhalf of myometrium (deep adenomyosis) (Tables 1 and 2).\nThe mean age of the patients suffering from FA was \n48.4 years, as compared to 33.4 years for patients suffer -\ning from DIE. Patients with adenomyosis had undergone \nsurgery due to bleeding disorders, while DIE was resected \nfor different reasons mainly including acute and chronic \npain or incipient intestinal obstruction. The GI-tract \n(rectum, recto-sigmoid, colon and ileum, 15 cases) was \nmostly affected by DIE, followed by the vagina (7 cases) \nand the bladder (1 case), including overlapping sites. FA \nincluded cases with superficial and deep infiltration.\nDysregulated genes in DIE vs. adenomyosis uteri\nA mathematical model was constructed for the analy -\nsis of differential gene expression in FA and DIE. When \nusing a threshold for fold changes (FC) < 0.66 or > 1.5, a \ntotal of 162 genes were identified that were up- or down -\nregulated (adj. p < 0.05). This analysis included signifi -\ncantly more genes with upregulation in DIE (116 genes), \nas compared to 46 genes with relative downregulation in \nDIE, compared to adenomyosis (FC < 0.66). When using \na stricter threshold of significance (p < 0.001 was used), \n15 genes were upregulated in DIE, and only one gene \n(FDZ2) was upregulated in adenomyosis uteri (Table  3; \nFig. 2). With regards to the functional properties, no spe -\ncific pathway could be assigned, and therefore a separate \ngene ontology analysis was performed (see below).\nIn order to relate dysregulated genes to clinical charac -\nteristics of adenomyosis and DIE, an unsupervised heat -\nmap was constructed. Here, clustering revealed a clear \nseparation of DIE and adenomyosis cases with only one \nDIE case clustering within adenomyosis (Fig.  3). But gen-\nerally, samples from DIE had generally higher pathway \nactivity scores than samples from adenomyosis (FA). In \nthis analysis, one larger gene group with upregulation in \nDIE could be separated from a smaller gene group with \nupregulation in adenomyosis (Fig.  3). However, in this \nclustering no correlation of gene expression with clinical \ncharacteristics (organ, BMI, depth of adenomyosis) was \nevident.\nPathways with activation in adenomyosis uteri and DIE\nIn order to analyze the functional properties of dysregu -\nlated genes in both diseases we performed gene ontology \nanalysis using the KEGG pathway analysis. This analysis \nrevealed upregulating of several signaling pathways in \nDIE, and interestingly, the PIK3CA pathway was most \nsignificantly upregulated. Other gene ontology groups \nincluded pathways involved in virus infection, focal adhe-\nsion, endocrine resistance and malignancy (Fig. 4a).\nThe identification of virus infection pathways was an \nunexpected finding, but further analysis revealed that \nthese virus-pathway associated genes had 12 genes in \ncommon, and 11 of these genes were also common to \nthe PIK3CA pathway. Therefore, it is believed that the \nPIK3CA pathway upregulation is the root cause for \nshowing virus-pathway associated pathways in this analy-\nsis, and that this analysis does not point to a virus related \ncause of DIE. Upregulated pathways in adenomyosis \nincluded RAS, PI3K-AKT, RAP1 and calcium signaling \npathways (Fig. 4b).\nDiscussion\nTo our knowledge, this is the first study comparing \nisolated epithelium cells of deep infiltrating endome -\ntriosis with epithelium of adenomyosis uteri using the \nnanostring technology. This is of particular interest, as \nadenomyosis and endometriosis lesions are often sur -\nrounded by many stromal and inflammatory cells, which \ncause some blurring of studies at the molecular level [29]. \nAnglesio et al., Inoue et al. and Moore et al. have recently \nshown that the somatic mutation occurs in the epithelial \ncomponent of DIE [ 15], as well as of FA [ 11] and in the \nhistologically normal endometrium of healthy patients \n[18]. In the latter studies, analysis of the laser microdis -\nsected epithelium has been shown to yield promising \nresults [ 11, 15, 17, 18, 30]. Therefore, this method was \nalso used in the current work.\nIn our studies, we show for the first time that the epi -\nthelium of DIE and FA differ significantly at the RNA \nexpression level. Interestingly, these differences in RNA \nexpression between both entities are independent of the \nsite of DIE manifestation and the body mass index.\nLooking first at the KEGG analysis of genes whose \nexpression is elevated in DIE compared to FA, it appears \nthat the PI3K pathway is significantly activated. This is \nconsistent with the results of whole-exome studies by \nAnglesio 2017 and Suda 2018, which detected somatic \ndriver mutations at the DNA level in the PIK3CA gene in \nthe epithelium of DIE lesions [ 15] and in the epithelium \n\nPage 5 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \nTable 1 Cohort description including age (years), symptoms/presentation in clinic, site of manifestation, and severity according to \nrASRM/Enzian classification for each of the 26 patients enrolled in the study\nCase Age/\nyears\nSymptoms/\npresentation in clinic\nmanifestation site rASRM/Enzian \nclassification\n1/FA 47  Tumor left ovary (12cm) Endometriosis of the left adnexa or parametria, Adenomyosis uteri \ninterna\nASRM III FA\n2/FA 52 Bleeding disorder Adenomyosis uteri interna 0/FA\n3/FA 38 Symptomatic uterus myomatosus Adenomyosis uteri interna 0/FA\n4/FA 36 Dysmenorrhea Adenomyosis uteri interna 0/FA\n5/FA 65 Recurrent postmenopausal \nbleeding\nAdenomyosis uteri interna 0/FA\n6/FA 45 Bleeding disorder Adenomyosis uteri interna 0/FA\n7/FA 56 Bleeding disorder Adenomyosis uteri interna 0/FA\n1/DIE 33 Endoscopic findings suspicious \nfor endometriosis in the proximal \nrectum\nPartial sigmoid resectate with a florid, deeply infiltrating endometriosis ASRM IV ENZIAN \nC3\n2/DIE 46 Large endometrioma in the recto-\nvaginal septum with vaginal pole \nand intestinal infiltration\n(1) Endometriosis lesions in the area of the PE from the sigmoid wall \n(2) Extensive endometrioma in the rectal area with transition into the \nvaginal wall\nENZIAN A3C3\n3/DIE 43 Dysmenorrhea Partial resection of the urinary bladder with extensive tumor-like \nendometriosis\nENZIAN FB\n4/DIE 28 Dysmenorrhea Endometriosis lesions of the urinary bladder wall, colon wall and in the \nsmooth muscular soft tissue\nASRM III\nENZIAN A3 C3 FB\n5/DIE 45 Subileus Terminal ileum resectate with endometriosis FI\n6/DIE 44 Size-progressive mass in the area of \nthe sigmoid colon\nPartial sigmoid resection with central endometriosis ASRM II ENZIAN \nC3 FO\n7/DIE 30 Symptomatic endometriosis with \nvaginal and rectal involvement\nRectum resectate including vagina part with extensive endometriosis \nlesions\nASRM IV ENZIAN \nA3C3\n8/DIE 41 Symptomatic endometriosis Rectosigmoid with numerous, deeply infiltrating endometriosis lesions \nintramurally.\nASRM II ENZIAN \nA2C2\n9/DIE 25 Deeply infiltrated endometriosis in \nthe area of the posterior vaginal wall \nas well as Douglas’ space\nPartial colon resection with infiltrating endometriosis. ASRM II ENZIAN \nA2 C2 FO\n10/ DIE 32 Deep infiltrating endometriosis of \nDouglas, pelvic peritoneum, blad-\nder wall, vagina, and subphrenic on \nright side\n(1) Vaginal wall with multiple and partially hemorrhaged, deeply infil-\ntrating endometriosis lesions (2) Bladder wall with deeply infiltrating \nendometriosis\nASRM II ENZIAN \nA2 FB Fo\n11/DIE 30 Recurrent perianal bleeding during \nmenstruation.\nRectal resectate with multifocal manifestations of endometriosis lo-\ncated in the muscular wall as well as in the mesorectal adipose tissue.\nASRM II ENZIAN \nA1-2C2\n12/DIE 36 Extensive intraperitoneal and pelvic \nendometriosis\n1.Partial colon resectate (C. sigmoideum) with numerous endometriosis \nlesions 2. Ileocecal resectate with additional endometriosis lesions in \nthe wall.\nASRM IV ENZIAN \nC2-3 FB FI\n13/DIE 19 Dysmenorrhea Partial colon resection with an invasive endometriosis with infiltration \nof the muscularis propria\nASRM II ENZIAN \nA2 C2 FB\n14/DIE 23 Deep infiltrating Douglas endo-\nmetriosis with involvement of the \nvagina\nDouglas PE with marked, apparently deep infiltrating endometriosis. ASRM (negative)\nENZIAN A2-3 \nC2-3\n15/DIE 34 Dysmenorrhea Tumor-like endometriosis in the PE from the septum rectumvaginale \nwith vaginal pole\nASRM IV ENZIAN \nB2-3 C2 FB\n16/DIE 31 Monthly hematochezia Resectate of the sigmoid colon with multiple endometriosis lesions of \nthe entire intestinal wall with continuity to the intestinal lumen and \nserosa.\nASRM IV ENZIAN \nA3C3 FI\n17/DIE 34 Deeply infiltrated endometriosis Rectosigmoid resectate with extended endometriosis lesions ASRM IV ENZIAN \nA3 C3\n18/DIE 29 Dysmenorrhea Recto-vaginally manifested, spreading to the rectum to the submucosa \nand to the vaginal wall\nASRM IV ENZIAN \nA3 C3\n19/DIE 31 J Known deeply infiltrated \nendometriosis\nInfiltrating endometriosis (vaginal). ASRM III ENZIAN \nA3\n\nPage 6 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \nof ovarian endometriosis, as well as in the eutopic endo -\nmetrium of healthy patients [ 17], among others. Since \nmost PIK3CA  mutations in cancers show gain of func -\ntion and growth advantages [ 31], it was concluded that \nthe presence of the same mutations in endometriotic epi-\nthelial cells has functional significance in the pathogen -\nesis of the disease [ 17, 32]. We show that this may also \nbe reflected in the activation of the pathway at the RNA \nlevel. Previous work has also indicated dysregulation of \nthe PI3K pathway in endometriosis: Yin et al. 2012 dem -\nonstrated an increase in pAKT, albeit in stromal cells \nfrom endometriomas, compared to cells from the eutopic \nendometrium of healthy women, and Guo et al. 2015 \nalso showed that phosphorylated mTor is increased in \nectopic endometrial lesions compared to eutopic endo -\nmetrium from endometriosis patients [ 33, 34]. Also, in a \nrecent work by Madanes, the authors are able to demon -\nstrate increased expression of PI3K, reduced expression \nof PTEN, and increased levels of pAkt in the ectopic and \neutopic endometrium of patients with peritoneal endo -\nmetriosis [35].\nThe PI3K-AKT-mTOR pathway is one of the most fre -\nquently dysregulated signaling pathways in carcinoma \ndiseases [ 36]. Its significant activation in DIE compared \nto FA may explain the different behavior of the two enti -\nties under study.\nWe believe, that the fact, that the KEGG analysis \nrevealed activation of virus associated gene groups (HPV, \nKSAHV and CMV) is due to the large intersection of \ngenes activated in these gene groups with genes that are \nsignificantly activated in the PI3K pathway, rather than a \nvirus-related cause of DIE, as discussed in detail above.\nThe focal adhesion pathway plays an essential role in \ncell motility, cell proliferation and cell differentiation. Its \nincreased activation in DIE compared to FA could also \nexplain the more progressive behavior of DIE. Interest -\ningly, in a recent analysis of the proteome of the eutopic \nendometrium of endometriosis patients, Méar et al. also \ndemonstrated an increased activation of the PI3K path -\nway and the focal adhesion pathway compared to healthy \ncontrols [37].\nThe KEGG analysis of genes that are downregulated in \nDIE and upregulated in adenomyosis compared to DIE \nshows that the RAS pathway in particular is upregulated \nin adenomyosis. This fits well with the findings of Inoue et \nal., who demonstrated a mutation in the KRAS gene with \nconsecutive activation of KRAS in 37.1% of adenomyosis \nTable 2 Overview of clinical and pathological patient data\nParameter Adenomyosis DIE\nPatients (n) 7 19\nAge (years) 48.43(sd 9.42) 33.37(sd 7.58)\nBMI 25.36(sd 5.28) 23.16(sd 3.61 unk. 3)\nSmoking history 3 pos (4) (unk.3) 2 pos(15) (unk. 4)\nManifestation side\nuterus 7 0\nterminal Ileum 0 1\ncolon 0 7\ncolon/rectum 0 2\nrectum 0 1\nrectum/vagina 0 4\nvagina 0 3\nbladder 0 1\nPathology\nFA 7 0\nsuperficial 3\ndeep 4\nDIE 0 19\nTable 3 Genes with significant relative upregulation (n = 15) or downregulation (n = 1) in DIE vs. adenomyosis (> 1.5 fold, Benjamini–\nHochberg adjusted P ≤ 0.001)\nLog fold change AveExpr t P value adjusted P value B\nAKT1 0.99 9.95 5.65 5.17E-06 4.19E-04 4.12\nBAD 0.91 7.00 5.82 3.29E-06 3.43E-04 4.55\nCAPN2 1.35 9.67 5.85 3.03E-06 3.43E-04 4.62\nCTNNB1 1.27 10.53 6.11 1.54E-06 3.43E-04 5.26\nFZD2 -0.80 6.72 -5.13 2.12E-05 9.75E-04 2.78\nGNAS 1.08 10.65 5.28 1.40E-05 7.86E-04 3.17\nGRB2 0.89 8.76 5.47 8.40E-06 5.57E-04 3.66\nHSPB1 1.95 11.10 5.87 2.88E-06 3.43E-04 4.67\nJAK1 0.82 9.24 5.24 1.57E-05 8.20E-04 3.06\nKMT2D 0.74 8.19 5.77 3.76E-06 3.43E-04 4.42\nMAP2K2 0.80 8.91 5.41 9.91E-06 6.03E-04 3.50\nNCOR1 0.81 9.32 5.59 6.17E-06 4.50E-04 3.95\nPPP2R1A 1.05 10.10 6.13 1.45E-06 3.43E-04 5.32\nPRKACA 1.20 8.65 6.48 5.73E-07 3.43E-04 6.20\nTRAF7 0.95 8.99 5.84 3.11E-06 3.43E-04 4.60\nU2AF1 0.85 10.13 5.12 2.14E-05 9.75E-04 2.77\n\nPage 7 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \ncases [ 11]. They detected a mutation in PI3KCA in only \ntwo of 70 patients, which may explain the increased \nexpression of PI3K pathway-associated genes in DIE in \nour study compared with FA. In contrast, in patients with \nboth adenomyosis and endometriosis lesions, Inoue et \nal. were able to detect the same KRAS mutation in both \nlesions [11]. By comparing both tissues in our study and \nlooking at the relative gene expressions of both entities, \nthe absolute activation of the RAS pathway is shown to \nbe lower in endometriosis patients, which would explain \nwhy it is not detected in the KEGG analysis. In direct \ncomparison of the two entities at the level of gene expres-\nsion however, the PI3K pathway appears to be the domi -\nnant pathway in DIE and the RAS pathway in FA.\nThis could explain, for example, the different sensitiv -\nity of the two entities to certain therapeutic approaches. \nWhile DIE responds well to therapy with the progestin \ndienogest, progesterone resistance is often described in \nadenomyosis patients, which according to Inoue et al. is \ndue to the KRAS mutations [11, 32].\nIf we look at the changes of single genes, it is striking \nthat Frizzled class receptor 2 (FDZ2) is the only gene that \nis significantly upregulated in adenomyosis compared to \nDIE. FZD2 is discussed as an important trigger of TGF-ß \ninduced epithelial–mesenchymal transition (EMT) [ 38] \nand cell migration [ 39]. Accordingly, the induction of \nEMT and ultimate fibrosis by TGF-β1 appears to play a \ncritical role in the pathogenesis of adenomyosis [ 40]. In \naddition, EMT promoted by FZD2 also plays an impor -\ntant role in the metastasis of endometrial cancer [ 41], \ntherefore suggesting that the invasive behavior of epithe -\nlial cells in FA has a cancerogenic aspect.\nThe two genes upregulated most in DIE compared \nto FA, Heat Shock Protein Family B (Small) Member \n1 ( HSPB1) and Calpain 2 ( CAPN2), have not yet been \ndescribed in endometriosis, despite the fact, that they \nplay a role in proliferation and invasion in various solid \ntumors [42– 45] and may serve this same function in DIE.\nCatenin Beta 1 ( CTNNB1), on the other hand, which is \nlikewise upregulated in DIE epithelial cells compared to \ncells from adenomyosis, is discussed as a key factor in the \nFig. 2 Volcano plot showing genes with most significant dysregulation in DIE vs. adenomyosis (adj. p-adjusted < 0.001 and log2 FC > 1.5). 15 genes were \nhighly significantly upregulated in DIE, and one gene (FZD2) was downregulated (log2 FC < 0.66).\n \n\nPage 8 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \nregulation of proliferation and invasion of endometriosis \n[46, 47]. Since both papers demonstrate an upregulation \nof CTNNB1 in endometrial stromal cells in endome -\ntriosis lesions, we can raise the question of an additional \nimportant role for CTNNB1 action in the epithelium of \nendometriosis lesions.\nLimitations of this study may include the following: \n[1] The group of patients suffering from deep infiltrating \nendometriosis is younger than the group of patients with \nadenomyosis who underwent hysterectomy. However, \nsome of the younger patients were also on therapy with \na GnRH analogue, which hormonally corresponds to \na menopausal status and therefore attenuates any age-\nrelated differences. [ 2] By comparing adenomyosis and \nDIE without comparison to normal endometrium, we \ncan only show relative expression differences, but not \nabsolute differences compared to healthy tissue. Never -\ntheless, especially in view of the frequent co-occurrence \nof the two entities and the presumed common molecu -\nlar origin, we consider our results at the gene expression \nFig. 3 Heatmap of supervised hierarchical clustering of differential genes (adj. p < 0.05, log2 fold change > 1.5 or < 0.66 and adjusted p-value < 0.05, \nn = 162) for the FA versus DIE group. This includes 116 upregulated genes in DIE and 46 genes with upregulation in adenomyosis. Gene expression with \ncases of adenomyosis (FA) is clearly distinct from cases with deep infiltration endometriosis (DIE).\n \n\nPage 9 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \nlevel to be of further value, particularly with regard to \npossible different therapeutic approaches. [3] The sample \nsize is relatively small with 7 patients with adenomyosis \nand 19 patients with DIE. Further studies with a larger \nnumber of patients to evaluate the possible influence \nof age, therapy concept or site of manifestation of DIE \nwould be useful.\nConclusions\nDeep infiltrating endometriosis and adenomyosis uteri \ndiffer significantly at the RNA expression level: for deep \ninfiltrating endometriosis, the genes most expressed were \nthose belonging to the PI3K pathway, and for adenomyo -\nsis, those belonging to the RAS pathway.\nFig. 4 ( a) KEGG enrichment analyses for differentially upregulated genes (p < 0.05, n = 116), 12 most significantly upregulated pathways are shown. This \nanalysis revealed upregulating of signaling pathways in DIE, most significantly the PI3K pathway, but also pathways involved in virus infection, focal adhe-\nsion, endocrine resistance and malignancy. (b) Same KEGG analysis, for differentially downregulated genes (p < 0.05, n = 46), 4 most significant pathways \n(p < 0.0001) are shown. Here, RAS, PI3K-AKT, RAP1 and Calcium signaling pathways are significant\n \n\nPage 10 of 11\nMarshall et al. Reproductive Biology and Endocrinology            (2023) 21:33 \nAbbreviations\nARID1A  AT-rich interaction domain1A\nBMI  Body mass index\nC10orf76  chromosome 10, open-reading frame 76\nCAPN2  Calpain 2\nCMV  cytomegalovirus\nCTNNB1  Catenin Beta 1\nDEG  differentially expressed genes\nDIE  deep infiltrating endometriosis\nDNA  deoxyribonucleic acid\nEMT  epithelial mesenchymal transition\nERCC3  ERCC excision repair 3\nFA  Adenomyosis uteri\nFC  Fold change\nFDR  False discorvery rate\nFFPE  formalin-fixed and paraffin-embedded\nFZD2  Frizzled class receptor 2\nGI  Gastrointestinal tract\nGO  Gene ontology\nHPV  Human papillomavirus\nHSPB1  Heat Shock Protein Family B (Small) Member 1\nKRAS  KRAS Proto-oncogene, GTPase\nKSAHV  kaposi sarcoma associated herpesvirus infection\nLMD  laser microdissection\nNGS  next-generation sequencing\npAKT  phosphorylated AKT Serine/Threonine Kinase \nPI3K  Phosphatidylinositol 3-kinase\nPI3KCA  Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic \nSubunit Alpha\nPPP2R1A  Protein phosphatase 2, regulatory subunit A\nPTEN  Phosphatase and Tensin Homolog\nRAP1  Ras-related protein 1\nRAS  Rat sarcoma\nrASRM  Revised American Society for Reproductive Medicine \nclassification\nRNA  Ribonucleic acid\nSLC4A1AP  Solute Carrier Family 4 Member 1 Adaptor Protein\nTGF-ß  Transforming growth factor ß\nTLK2  Tousled Like Kinase 2\nTMUB2  Transmembrane and Ubiquitin Like Domain Containing 2\nVPS33B  Vacuolar Protein Sorting-Associated Protein 33B\nAcknowledgements\nWe would like to thank the working group of Prof. Dr. rer. nat. Stefanie Rössler \nfor the many fruitful discussions and support during the evaluation as well as \nMTA Jutta Scheurer for the great support during the LMD. We would also like \nto thank Professor Stenzinger’s research group for continuous support of the \nproject.\nAuthor contribution\nThe study conception and design was conducted by A.M., P .S. and A.G. \nMaterial preparation, data collection and analysis were performed by A.M, \nF.K., H.O., M.K., J.J. and P .S. Supervision of the project was conducted by A.G. \nand T.S. Financial support and equipment were made available by T.S. The \nfirst draft of the manuscript was written by A.M., P .S. and A.G. and all authors \ncommented on the following versions of the manuscript. All authors read and \napproved the final manuscript.\nFunding\nOpen Access funding enabled and organized by Projekt DEAL. A.M. has \nreceived financial support for the project from the Kußmaul and Walter-Erb \nFoundation. The other authors received no financial support for the research \nand authorship of this article. For the publication fee we acknowledge \nfinancial support by Deutsche Forschungsgemeinschaft within the funding \nprogramme „Open Access Publikationskosten“ as well as by Heidelberg \nUniversity\nData Availability\nData or material are available on reasonable request.\nDeclarations\nEthics approval and consent to participate\nPatient consent was obtained at the time of hospitalization for the use of \ndiagnostic materials for research. The tissue samples were provided by the \nTissue Bank at the National Center for Tumor Diseases (Heidelberg, Germany) \nin concordance with the Ethics Committee of the University of Heidelberg \n(approval No. S-362/2017).\nConsent for publication\nAll authors read and approved the manuscript in the present form.\nCompeting interests\nThe author(s) declared no potential conflicts of interest with respect to the \nresearch, authorship, and/or publication of this article.\nReceived: 22 February 2023 / Accepted: 21 March 2023\nReferences\n1. Giudice LC. Clinical practice. Endometriosis. N Engl J Med. \n2010;362(25):2389–98.\n2. Meuleman C, Vandenabeele B, Fieuws S, Spiessens C, Timmerman D, \nD’Hooghe T. High prevalence of endometriosis in infertile women with \nnormal ovulation and normospermic partners. Fertil Steril. 2009;92(1):68–74.\n3. Chapron C, Fauconnier A, Vieira M, Barakat H, Dousset B, Pansini V et al. 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