{"paper_id":"28378e31-ddf8-4046-95f3-2caddb520945","body_text":"Title: Endometriosis lesions are 1 \noligoclonal structures derived from the 2 \nnormal endometrium 3 \n 4 \nAuthors: Sigurgeir Ólafsson1,*, Ásgeir Ö. Arnþórsson, Kirsten Kübler2-5, Ásgeir Sigurðsson, Helga 5 \nSigrún Gunnarsdóttir6, Hákon Jónsson1, Bergrún Ásbjörnsdóttir7, Louise le Roux1, Jóna 6 \nSæmundsdóttir1, Valgerdur Steinthorsdottir1, Guðmundur Norddahl1, Ingileif Jónsdóttir1, Jón 7 \nGunnlaugur Jónasson6,7, Droplaug Magnúsdóttir1, Ólafur Magnússon1, Anna M. Jónsdóttir6, 8 \nRagnheiður O. Árnadóttir8, Kári Stefánsson7 9 \nAffiliations: 10 \n1. Amgen deCODE genetics, Reykjavik, Iceland 11 \n2. Berlin Institute of Health at Charité – Universitätsmedizin Berlin, Berlin, Germany 12 \n3. Department of Hematology, Oncology and Cancer Immunology, Charité – 13 \nUniversitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-14 \nUniversität zu Berlin; Berlin, Germany 15 \n4. German Cancer Consortium (DKTK), Partner Site Berlin, and German Cancer Research 16 \nCenter (DKFZ), Heidelberg, Germany 17 \n5. Broad Institute of MIT and Harvard, Cambridge, Massachusetts, US 18 \n6. Dept. of Pathology, Landspitali University Hospital, Reykjavik, Iceland 19 \n7. Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland 20 \n8. Dept. of Obstetrics and gynaecology, Landspitali University Hospital, Reykjavik, Iceland 21 \n*Please address correspondence to sigurgeo@decode.is.  22 \n 23 \nAbstract 24 \nEndometriosis is characterized by the presence of endometrium-like tissue outside the uterus. The 25 \norigin of this ectopic tissue is debated, with leading theories including retrograde menstruation and 26 \nembryonic remnants. Using somatic mutations as markers, we show that endometriosis lesions consist 27 \nof unrelated clones of epithelial cells, with stromal cells being distinct, and that lesions at different 28 \nbody sites have different sets of clones. We observed that mutation burdens, signatures and driver 29 \nlandscapes are similar in cells from lesions and normal endometrium and the distribution of somatic 30 \nmutations along the length of chromosomes is consistent with uterine origin. Furthermore a large-31 \nscale screen of the normal endometrium of endometriosis patients identified clones that are ancestral 32 \nto endometriosis, indicating that the ectopic tissue in endometriosis originates from the normal 33 \nendometrium. 34 \n  35 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n \nMain text: 36 \nEndometriosis is a chronic condition characterized by the presence of endometrial tissue, epithelium 37 \nand stroma, outside the uterus. It is associated with pelvic pain and infertility and is thought to affect 38 \nas many as 10% of women of reproductive age (1, 2). Lesions present as any of three anatomical 39 \nsubtypes: (i) Superficial endometriosis, where lesions are confined to the pelvic area; (ii) deep 40 \ninfiltrating endometriosis, where lesions are found deep within pelvic structures and/or invading 41 \nvisceral organs; or (iii) as endometriomas, which are large cysts on the ovaries filled with dark brown 42 \nendometrial fluid and lined on the inside with a thin sheet of endometrial cells. Patients often present 43 \nwith multiple lesions distributed throughout the pelvis and beyond (1, 2).  44 \nThe origins of the ectopic tissue continue to be debated (3, 4). In the 1920s, John A. Sampson 45 \nput forth his theory of retrograde menstruation, which maintains that endometrial cells, epithelium and 46 \nstroma, refluxes through the fallopian tubes into the peritoneal cavity to implant on the pelvic surfaces 47 \n(5). While intellectually compelling, this theory is supported by limited functional evidence in 48 \nhumans, and several competing theories have been proposed. These include the theory of coelomic 49 \nmetaplasia (6, 7), which states that endometrial cells arise through transformation of mesothelial cells 50 \nin the peritoneum, and the Mullerian remnants theory, which states that lesions arise from cells which 51 \nfailed to properly differentiate or were displaced during fetal development (8, 9). Finally, the stem cell 52 \nrecruitment theory states that endometriosis lesions originate from stem cells travelling through the 53 \nangiolymphatic circulation (10), but opinions are divided as to whether these stem cells would be of 54 \nuterine origin or derived from other stem-cell niches, in particular the bone marrow (11, 12). 55 \nIn addition to the unclear etiology of the lesions, the mechanism of endometriotic lesion 56 \ndissemination within the body is incompletely understood (4). Specifically, do lesions spread through 57 \nshedding of cells from an initial founder lesion, reminiscent of cancer metastases, or are lesions 58 \nindependently seeded by distinct progenitor cells?  59 \nAll cells of the body continuously accrue somatic mutations throughout life (13, 14). The 60 \nglands of the normal endometrium, which are structures of clonal cells, accumulate ~30 mutations per 61 \ncell per year (15). These mutations are naturally occurring lineage tracing markers, with mutations 62 \nshared by different cells implying a common progenitor. While the majority of somatic mutations are 63 \nthought to be functionally neutral, cancer driver mutations are also detected within normal tissues. 64 \nThe normal endometrium in particular, is rich in mutations in genes commonly mutated in 65 \nendometrial adenocarcinomas, including PIK3CA, KRAS, ARHGAP35 and others (15–17). Driver 66 \nmutations have also been reported at a high prevalence in endometriotic lesions (17–19), although 67 \ntheir role in the etiology of the disease remains unclear.  68 \nHere we used laser capture microdissection to isolate individual endometrial glands from 69 \nendometriotic lesions and patient-matched normal endometrium (15, 20). We performed whole-70 \ngenome sequencing (WGS) of individual endometrial glands to identify the somatic mutations present 71 \nand build phylogenetic trees to reveal the developmental histories and relationships between lesions 72 \nand to compare the somatic mutation landscape between normal tissue and endometriotic lesions.  73 \n 74 \nResults 75 \nSomatic mutations in endometriosis mirror those found in normal endometrium 76 \nWe obtained tissue biopsies of endometriotic lesions and matched normal endometrium from 25 77 \nwomen undergoing laparoscopic surgery as part of their treatment for endometriosis at Landspitali 78 \nUniversity Hospital in Iceland between 2022 and 2024 (Methods, Table S1). We used laser capture 79 \nmicrodissection to isolate samples of a few hundred endometrial or stromal cells for WGS (15, 20) 80 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n \n(Figure 1A, Methods). The dataset comprises 403 microdissections in total; 315 microdissections of 81 \nepithelium from endometriosis biopsies, 3 samples of adjacent stroma, 3 from adenomyosis and 85 82 \nglands from matched normal endometrium sequenced to a median depth of 19X (range 5-106X, 83 \nFigure S1, Table S2). After filtering and quality control (Methods), 213,280 substitutions and 11,071 84 \nindels were used in the analyses described below. 85 \nWe fit a linear mixed-effects model to jointly estimate the effects of the age of the donor and lesional 86 \nstatus after correcting for sequencing coverage, median variant allele fraction (VAF) and non-random 87 \nsampling (21) (Methods). We estimate a yearly increase of 23.3 mutations per cell (7.1-39.5 95% CI, 88 \nP=0.0078, Likelihood ratio test (LRT), Figure 1B) and found that lesional samples do not have a 89 \nmutation burden different from normal (P=0.62, LRT). We used a Bayesian hierarchical Dirichlet 90 \nprocess to extract COSMIC mutational signatures for all samples. In line with previous work (13, 15), 91 \nwe found the mutation spectra of all samples to be dominated by SBS1, SBS5 and SBS18. These 92 \nsignatures contributed an equal number of mutations in cells from lesional and normal tissues, 93 \nsuggesting that epithelial cells in endometriotic lesions are subject to the same mutational processes as 94 \nthe cells of the normal endometrium and to a similar magnitude (Figure 1C). 95 \nWe used the dNdScv software (22) (v. 0.0.1.0) to identify genes where the ratio of the mutation rate at 96 \nnon-synonymous sites (dN) and synonymous sites (dS) is greater than 1, indicating positive selection 97 \nof non-synonymous mutations. Combining mutations from lesional and normal biopsies, six genes 98 \nshowed evidence of positive selection after Benjamini-Hochberg correction for multiple testing 99 \n(Figure 1D, Table S3). No additional genes could be identified in analyses stratified by lesional status 100 \n(Table S3). Taking into account differences in the number of samples and clonal structure between 101 \nlesional and normal biopsies (Methods), no gene was differentially mutated in lesional samples 102 \ncompared with normal endometrium after correction for multiple testing. KRAS mutations have been 103 \npreviously implicated in endometriosis (17–19) and are specially highlighted in Figures S4-S27. 104 \nKRAS hotspot mutations were found in 50/315 (16%) of microdissections from endometriosis 105 \ncompared with 3/85 (4%) normal glands. However, the KRAS mutations in endometriosis often 106 \npreceded clonal expansions and so only represent 12 independent mutation events. Thus, although we 107 \nobserved 4-times as many hotspot mutations in KRAS in lesional compared with normal samples, this 108 \ndifference is not statistically significant (Figure 1E, P=0.1, likelihood ratio test of missense mutations, 109 \nCoselens (23)) after accounting for differences in clonal structure between samples.  110 \nTaken together, these results show that somatic mutations in epithelial cells in endometriosis mirror 111 \nthose found in normal endometrium.  112 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n 113 \nFigure 1: Somatic mutations in endometriosis and normal endometrium. A) An overview of the 114 \ncohort and sampling. B) Mutation burden in endometrial glands isolated from lesional and normal 115 \ntissue in 5-year year-of-birth intervals. C) Substitution mutational signatures identified in 116 \nmicrodissected samples. Each bar represents one microdissected sample. D) An overview of the 117 \nmutations identified in each of the genes showing evidence of positive selection in our cohort. E) The 118 \nchange in the number of driver mutations observed per 1000 mutations between lesional and normal 119 \nendometrial cells. Error bars represent 95% confidence intervals.  120 \n  121 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n \nIndividual lesions are derived from multiple distinct cells 122 \nTo gain insights into the cellular histories of endometriotic lesions, we reconstructed phylogenetic 123 \ntrees (MPBoot software (24) (v. 1.1.0)) for cells isolated from each patient (Figure 2, Figures S4-124 \nS27). Inspecting the trees from all 25 patients, three broad insights emerge from our data: 125 \nFirst, individual endometriosis lesions typically comprise multiple unrelated clones of epithelial cells. 126 \nThis shows that epithelial cells within a lesion are generally not seeded by a single cell but rather by 127 \nmultiple cells which have diverged from one another during embryonic development and thus share 128 \nfew mutations in common. An example is shown in Figure 2A. Multiple microdissections from the 129 \nsame 2-3 mm strip of endometrioma (Figure 2A-right) were sequenced and found to share few-to-no 130 \nmutations in common. These cells are also unrelated to cells isolated from other biopsies of the same 131 \nendometrioma, revealing the endometrioma to be a polyclonal structure.   132 \nFigure 2B shows a second example of this. An endometrioma was sectioned, with samples taken 133 \nevery 3-4 mm, yielding biopsies A through F. While in this case a large clone (carrying a KRAS G12D 134 \nmutation) was found to span all the biopsies, other independent clones were also found within the 135 \nendometrioma. The endometrioma clones are also unrelated to a superficial lesion on the fallopian 136 \ntube (gray) displaying a dichotomous tree structure. This superficial lesion exemplifies how samples 137 \nfrom the same clade of the tree are sometimes separated by dozens to hundreds of mutations. The 138 \ncoalescent events defining these clades are rarely polytomies but rather display a bifurcation pattern 139 \nconsistent with continued local growth of the clone. This branching structure suggests that epithelial 140 \ncells continue to expand locally within the host tissue post seeding. 141 \nThe second insight emerging, as suggested by previous work (25), is that stroma cells from 142 \nendometriosis lesions are unrelated to the epithelial cells. We sequenced 3 samples of stroma directly 143 \nadjacent to glands of epithelial cells of different patients (Figure 2C). We observed no sharing of 144 \nmutations and in fact, very few mutations were found in the stroma samples. This suggests that the 145 \nstroma in endometriosis lesions is a polyclonal mix of cells and that endometriosis lesions, which 146 \ncomprise both stroma and epithelium, are not seeded by a single ancestor cell giving rise to both cell 147 \ntypes. Stroma cells are either recruited to the site by epithelial cells or else stroma cells originating in 148 \nthe normal endometrium travel together with the epithelial cells during retrograde menstruation.  149 \nThe third observation we make, which holds true for all 14 patients in our cohort with lesional 150 \nbiopsies from multiple body sites, is that epithelial cells isolated from distant body sites have always 151 \ndiverged early in molecular time. An example is shown in Figure 2D. Lesions of superficial 152 \nendometriosis from the bladder, left pelvis, right adnexa and the right ovary all consists of unrelated 153 \ncell clones, with no sharing of mutations between cells from different sites. Oligoclonality of 154 \nindividual lesions is further exemplified by the lesion located on the peritoneum of the bladder 155 \npresenting as rows of cyst-like structures growing in close proximity but with each cyst being clonally 156 \nderived from an independent cell (Figure 2D-right).  157 \nThe lack of sharing of mutations between lesions from different body sites suggests that 158 \nendometriosis does not spread through the pelvis or beyond from an initial founder lesion, similar to 159 \nmetastatic spread of cancer. Rather, each individual lesion is independently seeded and while local 160 \nclone growth is possible, we find no evidence of clones leaping between sites of the body. That the set 161 \nof clones found in any two endometriosis lesions from the same patient are always distinct, indicates 162 \nthat endometriosis seeding likely occurs repeatedly throughout life in susceptible individuals. 163 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n 164 \n 165 \nFigure 2: Examples of phylogenetic trees from endometriosis patients. Tips of trees circled in red 166 \nare microdissections marked with circles in black in the H&E tissue images to the right. The x-axis is 167 \nthe same across all panels. A) Patient 3, presenting with endometrioma and adenomyosis. Multiple 168 \nindependent cell clones are highlighted within a 3-4mm wide section of the endometrioma. B) Patient 169 \n30, presenting with endometrioma, which was sectioned into slices separated by 3-4 mm of tissue, 170 \ncreating biopsies A-F. A large clone spanning all biopsies and carrying a KRAS G12D mutation was 171 \nidentified but other independent clones are also found within the endometrioma, showing oligoclonal 172 \norigin. C) Patient 37, presenting with endometrioma as well as superficial and deep-infiltrating 173 \nlesions. Cells isolated from different lesions do not share mutations. The histopathological image 174 \nshows an example of a gland and adjacent stroma. No mutations could be detected in the stroma 175 \nsample, most likely due to polyclonal composition. D) Patient 18, presenting with superficial 176 \nendometriosis lesions on multiple body sites. No sharing of mutations is observed between sites of the 177 \nbody. The highlighted lesion manifests as rows of cyst-like structures on the peritoneum of the 178 \nbladder. Each cyst is derived from an independent cell, sharing no mutations with its neighbor. 179 \n  180 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n \nEndometriosis cells retain an endometrial identity throughout their lives 181 \nCompeting theories of endometriosis origin have proposed that lesional cells arise from a non-182 \nendometrial origin such as metaplasia from local lineages (6, 7) or recruited circulating stem cells (11, 183 \n12). We tested these cell-of-origin hypotheses using our somatic mutation data from endometriosis 184 \nlesions. The genomic distribution of somatic mutations along the length of each chromosome has been 185 \nshown to vary with cell-type specific chromatin organization and epigenetic state (26). Thus, somatic 186 \nmutations preserve a record of the epigenetic landscape experienced by a cell lineage. This principle 187 \nhas been leveraged to infer the cell-of-origin in cancer by correlating distribution of mutations along 188 \nthe genome with chromatin modifications from candidate normal tissue types (27). 189 \nWe applied this framework to endometriosis lesions and eutopic endometrium by constructing 190 \nmutation profiles and comparing them to epigenomes from 65 normal cell/tissue types. For cells 191 \nisolated from endometriosis lesions, normal endometrium was the best match (Figure 3; P=6×10−7, t-192 \ntest; Table S4). As expected, cells isolated from the normal endometrium also had a mutational profile 193 \nmost concordant with endometrium epigenomes (P=2×10−5), suggesting that eutopic endometrial and 194 \nectopic lesional cells share a closely related cell lineage consistent with a common endometrial origin. 195 \nWe also explored the origin of lesion types (superficial, deep infiltrating and endometrioma) 196 \nseparately and again found that normal endometrium was the top-ranked match for each type of 197 \nendometriosis lesion (P<0.01, Fig. S28).  198 \nWe next asked whether a metaplastic transformation could explain lesion development. Under a 199 \nmetaplasia model, mutations acquired after a lineage adopts a new cell identity should reflect the 200 \nepigenetic state of the transformed cell type, while earlier mutations should retain the ancestral cell 201 \ntype signature. To test this, we classified mutations as early or late depending on their placement on 202 \nbranches of the phylogenetic trees (Methods). When we restricted the analysis to early mutations, 203 \nnormal endometrium remained the best match (P=0.002, Fig. S29), even though we included ovarian 204 \nsurface epithelium epigenome as a proxy for coelomic mesothelium. We also tested the circulating 205 \nstem cell model by comparing early lesional mutations to hematopoietic and mesenchymal stem cell 206 \nepigenomes (Fig. S28 and S29), but they showed weaker concordance than endometrium epigenomes, 207 \nproviding no support for a stem cell origin of the epithelial lesions.  208 \nTogether, these findings consistently support an endometrial lineage origin for endometriosis lesions 209 \nand argue against a metaplastic origin or a circulating stem cell founder model.  210 \n 211 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n 212 \nFigure 3: Cell-of-origin analysis for endometriosis (left) and normal endometrium (right) . 213 \nPerformance of predicting mutational profiles from histone marks of 65 epigenomes representing 214 \ndistinct cell/tissue types; bars are colored according to tissue category; black points depict values 215 \nobtained from 10-fold cross-validation; P-values were obtained by comparing the 10-fold cross 216 \nvalidation values using a t-test (Abbreviations: HSCs, hematopoietic stem cells; MSCs, mesenchymal 217 \nstem cells). 218 \n 219 \nLarge-scale screening of the entire endometrium 220 \nThe retrograde menstruation theory and theories proposing angiolymphatic spread of endometrial 221 \nstem cells assume that the cells seeding endometriotic lesions are of uterine origin. We hypothesized 222 \nthat if this is the case, it might sometimes be possible to detect residues of the seeding clones within 223 \nthe normal endometrium. We obtained the entire uteri from three patients undergoing hysterectomies 224 \nas part of their endometriosis treatments from which lesional biopsies were also available. We used a 225 \ncapture-recapture design to test the hypothesis of uterine origin as follows: 226 \nWe first used LCM to isolate glands from lesional biopsies for WGS as described above. We built 227 \nphylogenetic trees from this material and identified independent clones of endometriosis epithelial 228 \ncells. We defined sets of 10 mutation-barcodes which uniquely identify each clone from the most 229 \nancestral branch possible (Figure 4A). We next isolated the entire endometrium from the uteri and 230 \ndivided these up into hundreds of small (approximately 2x2mm) segments, while noting the relative 231 \nlocation of each segment (Figure 4B). Lacking positive controls for this assay, we also targeted the 232 \nKRAS G12 and G13 codons and PIK3CA codons E542, E545 Q546. Mutations in these oncogenic 233 \nhotspots are common in endometrial tissue (15, 16, 28) but rare in contaminating cell types like blood 234 \nand stroma. We reasoned that the presence of these mutations in some of the endometrial segments 235 \nwould indicate that our assay was sufficiently sensitive to detect individual mutant endometrial clones 236 \ngiven the sequencing depth and the size of the tissue segments.   237 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\nFor each segment of normal endometrium, we used PCR to selectively amplify the regions 238 \nsurrounding the mutations identified in the endometriosis lesions before sequencing, thus achieving a 239 \nmedian targeted coverage in the thousands (Methods). As expected, we identified multiple segments 240 \ncarrying missense mutations in the “positive control” KRAS and PIK3CA hotspot codons in each of 241 \nthe three patients (Figure 4A, Supplementary Text). Furthermore, endometriosis clones could be 242 \nconfidently pinpointed to precise locations of the normal endometrium for two of the patients (Figure 243 \n4C and Supplementary Text), revealing those endometriosis lesions to be of uterine origin. Notably, 244 \none of the clones traced back to the normal endometrium is from a deep-infiltrating endometriosis 245 \nlesion dissected from the sacrouterine ligament (Figure 4), showing that seeding of clones from the 246 \nnormal endometrium is not limited to superficial lesions. For the patient highlighted in Figure 4, if 247 \n80% of the mutations in the ancestral branch (highlighted in red) occurred before the endometriosis 248 \ndiverged from the normal endometrium, and mutations accumulate linearly with age, then the seeding 249 \ncan be roughly estimated to have occurred when the patient was 17.4-19.2 years old. The results for 250 \neach patient are further described in the Supplementary Text.  251 \n 252 \n253 \nFigure 4: Screening for endometriosis clones in the normal endometrium of patient 42 . A) a 254 \nphylogenetic tree for endometriosis lesions removed during surgery. Branches highlighted in blue and 255 \nred were selected for the screen. The branch highlighted in red is ancestral to Clone #6, found in a 256 \ndeep-infiltrating endometriosis lesion. B) The normal endometrium of the patient was isolated and 257 \ndissected yielding 286 segments for the screen. Missense mutations affecting PIK3CA E542, E545 or 258 \nQ546 and KRAS G12 and G13 were detected in multiple segments highlighted in blue and purple 259 \nrespectively. Gray denotes parts of endometrium that were retained for clinical work or segments that 260 \ndid not yield sequencing data. Red highlights segments sharing mutations with Clone #6. C) 261 \nHistograms showing the variant allele frequencies (VAF) of each of the mutations comprising the 262 \nbarcode for Clone #6 in all segments of normal endometrium sequenced. Eight of the ten mutations 263 \ncould be identified in segments PB13, PB12 and PA9.  264 \n 265 \n  266 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n \nDiscussion 267 \nOur data reveal the cellular histories of endometriotic lesions at unprecedented resolution. They 268 \nconfirm that epithelial cells in endometriosis are of uterine origin. Mutational burden, mutational 269 \nsignatures and driver landscapes are highly similar in the normal endometrium as in all types of 270 \nendometriosis lesions and the distribution of mutations along the chromosomes is reflective of an 271 \nepigenetic landscape matching the normal endometrium. Furthermore, we isolated cell clones from 272 \nendometriosis lesions and were able to locate the origin of both superficial and deep-infiltrating 273 \nendometriosis clones within the normal endometrium. For both clones identified, the high fraction of 274 \nmutations shared between the lesion and the normal tissue is consistant with lesions diverging from 275 \nthe normal endometrium close to- or after age at menarche.  276 \nWhile our data suggest that cells from the normal endometrium pass into the pelvis via retrograde 277 \nmenstruation, we are unable to determine if this is the only mechanism of spread or whether 278 \ndissemination is also possible via the angiolymphatic system. However, the presence of multiple 279 \nunrelated clones within as little as 1 mm of tissue suggests polyclonal seeding of macroscopic 280 \nsegments of endometrium where cells adhere to one another as they travel through the fallopian tube. 281 \nThis is consistent with Sampson’s observation of “bits” of endometrium traveling through the 282 \nfallopian tubes (5) but we cannot exclude a second mechanism involving precise homing of 283 \nsuspended cells to the same location in the body via the angiolymphatic system.  284 \nIn our screening of entire uteri, we were able to confidently locate 2/14 clones tested within the 285 \nnormal endometrium. In addition to extra-uterine origin, several factors may explain why the 286 \nremaining clones were not identified. The clones may have migrated entirely to the lesional site 287 \nwithout leaving detectable residual cells in the normal endometrium. Alternatively, residual cells in 288 \nthe endometrium may have been outcompeted and replaced by other endometrial clones over time 289 \nfollowing seeding. It is also possible that these clones resided in tissue segments that were not 290 \nsequenced, were substantially contaminated with non-epithelial cell types, or were sequenced at 291 \ninsufficient depth to enable reliable detection. The detected clones were isolated from a superficial 292 \nand a deep-infiltrating endometriosis lesion. Unfortunately, no clones from endometriomas were 293 \navailable for the screen. However, given the similarities of endometriomas to other lesion types at all 294 \nother levels of analysis, a single causal mechanism is both plausible and parsimonious.  295 \nOur results have implications for treatment of endometriosis, in particular because they show that it is 296 \nlikely that the seeding of endometriosis by retrograde menstruation is a repeated event. They support 297 \nmanagement strategies focused on limiting the number of menstrual cycles to prevent the seeding of 298 \nnew lesions and justify delaying surgery as there is no evidence of existing lesions seeding additional 299 \nlesions. Oncogenic KRAS mutations have been implicated in the pathogenesis of endometriosis (17) 300 \nand several agents targeting KRAS are either on the market or under development for treatment of 301 \ncancers, raising questions about the utility of repurposing such agents for the treatment of 302 \nendometriosis. We note that although we found KRAS mutations in 16% of microdissections, and 303 \nthese often precede clonal expansions within lesions, the mutations are not ancestral to entire lesions, 304 \nindicating that treatment with KRAS inhibitors would unlikely be curative.  305 \nIn this study we have provided functional evidence supporting eutopic origin of endometriosis cells as 306 \nproposed by Sampson in the 1920s. 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No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n \nAcknowledgments 489 \nWe thank the patients who selflessly donated tissue samples for this study. We further thank the 490 \nlaboratory and informatics teams at Amgen deCODE Genetics for their contribution. 491 \nFigure 1A was created in BioRender. Olafsson, S. (2026) https://BioRender.com/d9xryv5. 492 \nFunding 493 \nThis study was funded by Amgen deCODE Genetics. K.K. received support from the Private 494 \nExcellence Initiative Johanna Quandt of the Stiftung Charité. 495 \nAuthor contributions  496 \nSO conceived the project with contributions from KK, AS, VS, IJ, JGJ, ROA and KS. SO and AOA 497 \nperformed microdissections and processed whole uteri with contributions from HSG, BA and GN. 498 \nHSG performed fixation, sectioning, staining and imaging of tissue samples. SO analyzed the 499 \nsequencing data and carried out all statistical analyses except for cell-of-origin analyses with 500 \ncontributions from HJ and BA. KK performed cell-of-origin analyses. AS designed PCR primers and 501 \noversaw PCR reactions used to amplify regions around mutational barcodes in whole-uteri screening. 502 \nDM and OM prepared sequencing libraries and oversaw sequencing of all samples. AMJ performed 503 \nhistopathological assessments of tissues and provided guidance for laser capture. ROÁ recruited and 504 \nconsented patients for the study and performed surgeries from which samples were obtained. KS 505 \nsupervised the project. SO wrote the manuscript with contributions from all authors.  506 \n 507 \nCompeting interests 508 \nSO, HJ, LR, JS, VS, GN, IJ, DM and OM are current employees of Amgen deCODE Genetics. AOA, 509 \nAS and KS were employees of Amgen deCODE Genetics at the time of patient recruitment, data 510 \ngeneration and initial drafting of the manuscript. K.K declares no competing interests. 511 \n 512 \nData and materials availability 513 \nThe data supporting the findings of this study are available in the supplementary material of this 514 \narticle. Table S1 contains patient-level meta-data. Table S2 contains meta-data and statistics 515 \ncalculated at the level of individual microdissections. Table S3 contains the dN/dScv statistics for 516 \neach coding gene. Table S4 contains results from the cell-of-origin analysis. Table S5 contains all 517 \nmutations in KRAS and PIK3CA identified in the large-scale screen of entire endometrium. Table S6 518 \ncontains sequences of the PCR primers used in the screen. Read counts for each mutation call, VAF 519 \nhistograms for each sample and tree R-objects will be made available on 520 \nwww.decode.com/summarydata upon article acceptance. 521 \nThe dNdScv software (RRID:SCR_023123) is freely available at 522 \nhttps://github.com/im3sanger/dndscv 523 \nThe Coselens software (RRID:SCR_022578) is freely available at 524 \nhttps://github.com/ggruenhagen3/coselens  525 \nThe hdp software is freely available at https://github.com/nicolaroberts/hdp  526 \n 527 \nList of Supplementary Materials. 528 \nMaterials and Methods 529 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint \n\n \nSupplementary Text 530 \nFigs. S1 to S29 531 \nCaptions for Tables S1 to S6 532 \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint","source_license":"CC0","license_restricted":false}