Endometriosis lesions are oligoclonal structures derived from the normal endometrium

24 Endometriosis is characterized by the presence of endometrium-like tissue outside the uterus. The 25 origin of this ectopic tissue is debated, with leading theories including retrograde menstruation and 26 embryonic remnants. Using somatic mutations as markers, we show that endometriosis lesions consist 27 of unrelated clones of epithelial cells, with stromal cells being distinct, and that lesions at different 28 body sites have different sets of clones. We observed that mutation burdens, signatures and driver 29 landscapes are similar in cells from lesions and normal endometrium and the distribution of somatic 30 mutations along the length of chromosomes is consistent with uterine origin. Furthermore a large-31 scale screen of the normal endometrium of endometriosis patients identified clones that are ancestral 32 to endometriosis, indicating that the ectopic tissue in endometriosis originates from the normal 33 endometrium. 34 35 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint Main text: 36 Endometriosis is a chronic condition characterized by the presence of endometrial tissue, epithelium 37 and stroma, outside the uterus. It is associated with pelvic pain and infertility and is thought to affect 38 as many as 10% of women of reproductive age (1, 2). Lesions present as any of three anatomical 39 subtypes: (i) Superficial endometriosis, where lesions are confined to the pelvic area; (ii) deep 40 infiltrating endometriosis, where lesions are found deep within pelvic structures and/or invading 41 visceral organs; or (iii) as endometriomas, which are large cysts on the ovaries filled with dark brown 42 endometrial fluid and lined on the inside with a thin sheet of endometrial cells. Patients often present 43 with multiple lesions distributed throughout the pelvis and beyond (1, 2). 44 The origins of the ectopic tissue continue to be debated (3, 4). In the 1920s, John A. Sampson 45 put forth his theory of retrograde menstruation, which maintains that endometrial cells, epithelium and 46 stroma, refluxes through the fallopian tubes into the peritoneal cavity to implant on the pelvic surfaces 47 (5). While intellectually compelling, this theory is supported by limited functional evidence in 48 humans, and several competing theories have been proposed. These include the theory of coelomic 49 metaplasia (6, 7), which states that endometrial cells arise through transformation of mesothelial cells 50 in the peritoneum, and the Mullerian remnants theory, which states that lesions arise from cells which 51 failed to properly differentiate or were displaced during fetal development (8, 9). Finally, the stem cell 52 recruitment theory states that endometriosis lesions originate from stem cells travelling through the 53 angiolymphatic circulation (10), but opinions are divided as to whether these stem cells would be of 54 uterine origin or derived from other stem-cell niches, in particular the bone marrow (11, 12). 55 In addition to the unclear etiology of the lesions, the mechanism of endometriotic lesion 56 dissemination within the body is incompletely understood (4). Specifically, do lesions spread through 57 shedding of cells from an initial founder lesion, reminiscent of cancer metastases, or are lesions 58 independently seeded by distinct progenitor cells? 59 All cells of the body continuously accrue somatic mutations throughout life (13, 14). The 60 glands of the normal endometrium, which are structures of clonal cells, accumulate ~30 mutations per 61 cell per year (15). These mutations are naturally occurring lineage tracing markers, with mutations 62 shared by different cells implying a common progenitor. While the majority of somatic mutations are 63 thought to be functionally neutral, cancer driver mutations are also detected within normal tissues. 64 The normal endometrium in particular, is rich in mutations in genes commonly mutated in 65 endometrial adenocarcinomas, including PIK3CA, KRAS, ARHGAP35 and others (15–17). Driver 66 mutations have also been reported at a high prevalence in endometriotic lesions (17–19), although 67 their role in the etiology of the disease remains unclear. 68 Here we used laser capture microdissection to isolate individual endometrial glands from 69 endometriotic lesions and patient-matched normal endometrium (15, 20). We performed whole-70 genome sequencing (WGS) of individual endometrial glands to identify the somatic mutations present 71 and build phylogenetic trees to reveal the developmental histories and relationships between lesions 72 and to compare the somatic mutation landscape between normal tissue and endometriotic lesions. 73 74

75 Somatic mutations in endometriosis mirror those found in normal endometrium 76 We obtained tissue biopsies of endometriotic lesions and matched normal endometrium from 25 77 women undergoing laparoscopic surgery as part of their treatment for endometriosis at Landspitali 78 University Hospital in Iceland between 2022 and 2024 (Methods, Table S1). We used laser capture 79 microdissection to isolate samples of a few hundred endometrial or stromal cells for WGS (15, 20) 80 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint (Figure 1A, Methods). The dataset comprises 403 microdissections in total; 315 microdissections of 81 epithelium from endometriosis biopsies, 3 samples of adjacent stroma, 3 from adenomyosis and 85 82 glands from matched normal endometrium sequenced to a median depth of 19X (range 5-106X, 83 Figure S1, Table S2). After filtering and quality control (Methods), 213,280 substitutions and 11,071 84 indels were used in the analyses described below. 85 We fit a linear mixed-effects model to jointly estimate the effects of the age of the donor and lesional 86 status after correcting for sequencing coverage, median variant allele fraction (VAF) and non-random 87 sampling (21) (Methods). We estimate a yearly increase of 23.3 mutations per cell (7.1-39.5 95% CI, 88 P=0.0078, Likelihood ratio test (LRT), Figure 1B) and found that lesional samples do not have a 89 mutation burden different from normal (P=0.62, LRT). We used a Bayesian hierarchical Dirichlet 90 process to extract COSMIC mutational signatures for all samples. In line with previous work (13, 15), 91 we found the mutation spectra of all samples to be dominated by SBS1, SBS5 and SBS18. These 92 signatures contributed an equal number of mutations in cells from lesional and normal tissues, 93 suggesting that epithelial cells in endometriotic lesions are subject to the same mutational processes as 94 the cells of the normal endometrium and to a similar magnitude (Figure 1C). 95 We used the dNdScv software (22) (v. 0.0.1.0) to identify genes where the ratio of the mutation rate at 96 non-synonymous sites (dN) and synonymous sites (dS) is greater than 1, indicating positive selection 97 of non-synonymous mutations. Combining mutations from lesional and normal biopsies, six genes 98 showed evidence of positive selection after Benjamini-Hochberg correction for multiple testing 99 (Figure 1D, Table S3). No additional genes could be identified in analyses stratified by lesional status 100 (Table S3). Taking into account differences in the number of samples and clonal structure between 101 lesional and normal biopsies (Methods), no gene was differentially mutated in lesional samples 102 compared with normal endometrium after correction for multiple testing. KRAS mutations have been 103 previously implicated in endometriosis (17–19) and are specially highlighted in Figures S4-S27. 104 KRAS hotspot mutations were found in 50/315 (16%) of microdissections from endometriosis 105 compared with 3/85 (4%) normal glands. However, the KRAS mutations in endometriosis often 106 preceded clonal expansions and so only represent 12 independent mutation events. Thus, although we 107 observed 4-times as many hotspot mutations in KRAS in lesional compared with normal samples, this 108 difference is not statistically significant (Figure 1E, P=0.1, likelihood ratio test of missense mutations, 109 Coselens (23)) after accounting for differences in clonal structure between samples. 110 Taken together, these results show that somatic mutations in epithelial cells in endometriosis mirror 111 those found in normal endometrium. 112 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint 113 Figure 1: Somatic mutations in endometriosis and normal endometrium. A) An overview of the 114 cohort and sampling. B) Mutation burden in endometrial glands isolated from lesional and normal 115 tissue in 5-year year-of-birth intervals. C) Substitution mutational signatures identified in 116 microdissected samples. Each bar represents one microdissected sample. D) An overview of the 117 mutations identified in each of the genes showing evidence of positive selection in our cohort. E) The 118 change in the number of driver mutations observed per 1000 mutations between lesional and normal 119 endometrial cells. Error bars represent 95% confidence intervals. 120 121 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint Individual lesions are derived from multiple distinct cells 122 To gain insights into the cellular histories of endometriotic lesions, we reconstructed phylogenetic 123 trees (MPBoot software (24) (v. 1.1.0)) for cells isolated from each patient (Figure 2, Figures S4-124 S27). Inspecting the trees from all 25 patients, three broad insights emerge from our data: 125 First, individual endometriosis lesions typically comprise multiple unrelated clones of epithelial cells. 126 This shows that epithelial cells within a lesion are generally not seeded by a single cell but rather by 127 multiple cells which have diverged from one another during embryonic development and thus share 128 few mutations in common. An example is shown in Figure 2A. Multiple microdissections from the 129 same 2-3 mm strip of endometrioma (Figure 2A-right) were sequenced and found to share few-to-no 130 mutations in common. These cells are also unrelated to cells isolated from other biopsies of the same 131 endometrioma, revealing the endometrioma to be a polyclonal structure. 132 Figure 2B shows a second example of this. An endometrioma was sectioned, with samples taken 133 every 3-4 mm, yielding biopsies A through F. While in this case a large clone (carrying a KRAS G12D 134 mutation) was found to span all the biopsies, other independent clones were also found within the 135 endometrioma. The endometrioma clones are also unrelated to a superficial lesion on the fallopian 136 tube (gray) displaying a dichotomous tree structure. This superficial lesion exemplifies how samples 137 from the same clade of the tree are sometimes separated by dozens to hundreds of mutations. The 138 coalescent events defining these clades are rarely polytomies but rather display a bifurcation pattern 139 consistent with continued local growth of the clone. This branching structure suggests that epithelial 140 cells continue to expand locally within the host tissue post seeding. 141 The second insight emerging, as suggested by previous work (25), is that stroma cells from 142 endometriosis lesions are unrelated to the epithelial cells. We sequenced 3 samples of stroma directly 143 adjacent to glands of epithelial cells of different patients (Figure 2C). We observed no sharing of 144 mutations and in fact, very few mutations were found in the stroma samples. This suggests that the 145 stroma in endometriosis lesions is a polyclonal mix of cells and that endometriosis lesions, which 146 comprise both stroma and epithelium, are not seeded by a single ancestor cell giving rise to both cell 147 types. Stroma cells are either recruited to the site by epithelial cells or else stroma cells originating in 148 the normal endometrium travel together with the epithelial cells during retrograde menstruation. 149 The third observation we make, which holds true for all 14 patients in our cohort with lesional 150 biopsies from multiple body sites, is that epithelial cells isolated from distant body sites have always 151 diverged early in molecular time. An example is shown in Figure 2D. Lesions of superficial 152 endometriosis from the bladder, left pelvis, right adnexa and the right ovary all consists of unrelated 153 cell clones, with no sharing of mutations between cells from different sites. Oligoclonality of 154 individual lesions is further exemplified by the lesion located on the peritoneum of the bladder 155 presenting as rows of cyst-like structures growing in close proximity but with each cyst being clonally 156 derived from an independent cell (Figure 2D-right). 157 The lack of sharing of mutations between lesions from different body sites suggests that 158 endometriosis does not spread through the pelvis or beyond from an initial founder lesion, similar to 159 metastatic spread of cancer. Rather, each individual lesion is independently seeded and while local 160 clone growth is possible, we find no evidence of clones leaping between sites of the body. That the set 161 of clones found in any two endometriosis lesions from the same patient are always distinct, indicates 162 that endometriosis seeding likely occurs repeatedly throughout life in susceptible individuals. 163 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint 164 165 Figure 2: Examples of phylogenetic trees from endometriosis patients. Tips of trees circled in red 166 are microdissections marked with circles in black in the H&E tissue images to the right. The x-axis is 167 the same across all panels. A) Patient 3, presenting with endometrioma and adenomyosis. Multiple 168 independent cell clones are highlighted within a 3-4mm wide section of the endometrioma. B) Patient 169 30, presenting with endometrioma, which was sectioned into slices separated by 3-4 mm of tissue, 170 creating biopsies A-F. A large clone spanning all biopsies and carrying a KRAS G12D mutation was 171 identified but other independent clones are also found within the endometrioma, showing oligoclonal 172 origin. C) Patient 37, presenting with endometrioma as well as superficial and deep-infiltrating 173 lesions. Cells isolated from different lesions do not share mutations. The histopathological image 174 shows an example of a gland and adjacent stroma. No mutations could be detected in the stroma 175 sample, most likely due to polyclonal composition. D) Patient 18, presenting with superficial 176 endometriosis lesions on multiple body sites. No sharing of mutations is observed between sites of the 177 body. The highlighted lesion manifests as rows of cyst-like structures on the peritoneum of the 178 bladder. Each cyst is derived from an independent cell, sharing no mutations with its neighbor. 179 180 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint Endometriosis cells retain an endometrial identity throughout their lives 181 Competing theories of endometriosis origin have proposed that lesional cells arise from a non-182 endometrial origin such as metaplasia from local lineages (6, 7) or recruited circulating stem cells (11, 183 12). We tested these cell-of-origin hypotheses using our somatic mutation data from endometriosis 184 lesions. The genomic distribution of somatic mutations along the length of each chromosome has been 185 shown to vary with cell-type specific chromatin organization and epigenetic state (26). Thus, somatic 186 mutations preserve a record of the epigenetic landscape experienced by a cell lineage. This principle 187 has been leveraged to infer the cell-of-origin in cancer by correlating distribution of mutations along 188 the genome with chromatin modifications from candidate normal tissue types (27). 189 We applied this framework to endometriosis lesions and eutopic endometrium by constructing 190 mutation profiles and comparing them to epigenomes from 65 normal cell/tissue types. For cells 191 isolated from endometriosis lesions, normal endometrium was the best match (Figure 3; P=6×10−7, t-192 test; Table S4). As expected, cells isolated from the normal endometrium also had a mutational profile 193 most concordant with endometrium epigenomes (P=2×10−5), suggesting that eutopic endometrial and 194 ectopic lesional cells share a closely related cell lineage consistent with a common endometrial origin. 195 We also explored the origin of lesion types (superficial, deep infiltrating and endometrioma) 196 separately and again found that normal endometrium was the top-ranked match for each type of 197 endometriosis lesion (P<0.01, Fig. S28). 198 We next asked whether a metaplastic transformation could explain lesion development. Under a 199 metaplasia model, mutations acquired after a lineage adopts a new cell identity should reflect the 200 epigenetic state of the transformed cell type, while earlier mutations should retain the ancestral cell 201 type signature. To test this, we classified mutations as early or late depending on their placement on 202 branches of the phylogenetic trees (Methods). When we restricted the analysis to early mutations, 203 normal endometrium remained the best match (P=0.002, Fig. S29), even though we included ovarian 204 surface epithelium epigenome as a proxy for coelomic mesothelium. We also tested the circulating 205 stem cell model by comparing early lesional mutations to hematopoietic and mesenchymal stem cell 206 epigenomes (Fig. S28 and S29), but they showed weaker concordance than endometrium epigenomes, 207 providing no support for a stem cell origin of the epithelial lesions. 208 Together, these findings consistently support an endometrial lineage origin for endometriosis lesions 209 and argue against a metaplastic origin or a circulating stem cell founder model. 210 211 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint 212 Figure 3: Cell-of-origin analysis for endometriosis (left) and normal endometrium (right) . 213 Performance of predicting mutational profiles from histone marks of 65 epigenomes representing 214 distinct cell/tissue types; bars are colored according to tissue category; black points depict values 215 obtained from 10-fold cross-validation; P-values were obtained by comparing the 10-fold cross 216 validation values using a t-test (Abbreviations: HSCs, hematopoietic stem cells; MSCs, mesenchymal 217 stem cells). 218 219 Large-scale screening of the entire endometrium 220 The retrograde menstruation theory and theories proposing angiolymphatic spread of endometrial 221 stem cells assume that the cells seeding endometriotic lesions are of uterine origin. We hypothesized 222 that if this is the case, it might sometimes be possible to detect residues of the seeding clones within 223 the normal endometrium. We obtained the entire uteri from three patients undergoing hysterectomies 224 as part of their endometriosis treatments from which lesional biopsies were also available. We used a 225 capture-recapture design to test the hypothesis of uterine origin as follows: 226 We first used LCM to isolate glands from lesional biopsies for WGS as described above. We built 227 phylogenetic trees from this material and identified independent clones of endometriosis epithelial 228 cells. We defined sets of 10 mutation-barcodes which uniquely identify each clone from the most 229 ancestral branch possible (Figure 4A). We next isolated the entire endometrium from the uteri and 230 divided these up into hundreds of small (approximately 2x2mm) segments, while noting the relative 231 location of each segment (Figure 4B). Lacking positive controls for this assay, we also targeted the 232 KRAS G12 and G13 codons and PIK3CA codons E542, E545 Q546. Mutations in these oncogenic 233 hotspots are common in endometrial tissue (15, 16, 28) but rare in contaminating cell types like blood 234 and stroma. We reasoned that the presence of these mutations in some of the endometrial segments 235 would indicate that our assay was sufficiently sensitive to detect individual mutant endometrial clones 236 given the sequencing depth and the size of the tissue segments. 237 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint For each segment of normal endometrium, we used PCR to selectively amplify the regions 238 surrounding the mutations identified in the endometriosis lesions before sequencing, thus achieving a 239 median targeted coverage in the thousands (Methods). As expected, we identified multiple segments 240 carrying missense mutations in the “positive control” KRAS and PIK3CA hotspot codons in each of 241 the three patients (Figure 4A, Supplementary Text). Furthermore, endometriosis clones could be 242 confidently pinpointed to precise locations of the normal endometrium for two of the patients (Figure 243 4C and Supplementary Text), revealing those endometriosis lesions to be of uterine origin. Notably, 244 one of the clones traced back to the normal endometrium is from a deep-infiltrating endometriosis 245 lesion dissected from the sacrouterine ligament (Figure 4), showing that seeding of clones from the 246 normal endometrium is not limited to superficial lesions. For the patient highlighted in Figure 4, if 247 80% of the mutations in the ancestral branch (highlighted in red) occurred before the endometriosis 248 diverged from the normal endometrium, and mutations accumulate linearly with age, then the seeding 249 can be roughly estimated to have occurred when the patient was 17.4-19.2 years old. The results for 250 each patient are further described in the Supplementary Text. 251 252 253 Figure 4: Screening for endometriosis clones in the normal endometrium of patient 42 . A) a 254 phylogenetic tree for endometriosis lesions removed during surgery. Branches highlighted in blue and 255 red were selected for the screen. The branch highlighted in red is ancestral to Clone #6, found in a 256 deep-infiltrating endometriosis lesion. B) The normal endometrium of the patient was isolated and 257 dissected yielding 286 segments for the screen. Missense mutations affecting PIK3CA E542, E545 or 258 Q546 and KRAS G12 and G13 were detected in multiple segments highlighted in blue and purple 259 respectively. Gray denotes parts of endometrium that were retained for clinical work or segments that 260 did not yield sequencing data. Red highlights segments sharing mutations with Clone #6. C) 261 Histograms showing the variant allele frequencies (VAF) of each of the mutations comprising the 262 barcode for Clone #6 in all segments of normal endometrium sequenced. Eight of the ten mutations 263 could be identified in segments PB13, PB12 and PA9. 264 265 266 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint

267 Our data reveal the cellular histories of endometriotic lesions at unprecedented resolution. They 268 confirm that epithelial cells in endometriosis are of uterine origin. Mutational burden, mutational 269 signatures and driver landscapes are highly similar in the normal endometrium as in all types of 270 endometriosis lesions and the distribution of mutations along the chromosomes is reflective of an 271 epigenetic landscape matching the normal endometrium. Furthermore, we isolated cell clones from 272 endometriosis lesions and were able to locate the origin of both superficial and deep-infiltrating 273 endometriosis clones within the normal endometrium. For both clones identified, the high fraction of 274 mutations shared between the lesion and the normal tissue is consistant with lesions diverging from 275 the normal endometrium close to- or after age at menarche. 276 While our data suggest that cells from the normal endometrium pass into the pelvis via retrograde 277 menstruation, we are unable to determine if this is the only mechanism of spread or whether 278 dissemination is also possible via the angiolymphatic system. However, the presence of multiple 279 unrelated clones within as little as 1 mm of tissue suggests polyclonal seeding of macroscopic 280 segments of endometrium where cells adhere to one another as they travel through the fallopian tube. 281 This is consistent with Sampson’s observation of “bits” of endometrium traveling through the 282 fallopian tubes (5) but we cannot exclude a second mechanism involving precise homing of 283 suspended cells to the same location in the body via the angiolymphatic system. 284 In our screening of entire uteri, we were able to confidently locate 2/14 clones tested within the 285 normal endometrium. In addition to extra-uterine origin, several factors may explain why the 286 remaining clones were not identified. The clones may have migrated entirely to the lesional site 287 without leaving detectable residual cells in the normal endometrium. Alternatively, residual cells in 288 the endometrium may have been outcompeted and replaced by other endometrial clones over time 289 following seeding. It is also possible that these clones resided in tissue segments that were not 290 sequenced, were substantially contaminated with non-epithelial cell types, or were sequenced at 291 insufficient depth to enable reliable detection. The detected clones were isolated from a superficial 292 and a deep-infiltrating endometriosis lesion. Unfortunately, no clones from endometriomas were 293 available for the screen. However, given the similarities of endometriomas to other lesion types at all 294 other levels of analysis, a single causal mechanism is both plausible and parsimonious. 295 Our results have implications for treatment of endometriosis, in particular because they show that it is 296 likely that the seeding of endometriosis by retrograde menstruation is a repeated event. They support 297 management strategies focused on limiting the number of menstrual cycles to prevent the seeding of 298 new lesions and justify delaying surgery as there is no evidence of existing lesions seeding additional 299 lesions. Oncogenic KRAS mutations have been implicated in the pathogenesis of endometriosis (17) 300 and several agents targeting KRAS are either on the market or under development for treatment of 301 cancers, raising questions about the utility of repurposing such agents for the treatment of 302 endometriosis. We note that although we found KRAS mutations in 16% of microdissections, and 303 these often precede clonal expansions within lesions, the mutations are not ancestral to entire lesions, 304 indicating that treatment with KRAS inhibitors would unlikely be curative. 305 In this study we have provided functional evidence supporting eutopic origin of endometriosis cells as 306 proposed by Sampson in the 1920s. We have further shown that lesions are independently seeded and 307 generally consist of several independent cell clones. It is our hope that understanding of endometriosis 308 etiology will, in time, contribute to the development of treatments and preventions against this often 309 debilitating and understudied disease. 310 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint

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Van L oo , S. McLa r e n , D . C. We dge , A. 483 F ullam, L. B. Al exan dr ov , J . M. Tu bio, L . S tebbing s , A. Menz i e s , S . Wi daa , M . R. S tr atto n, P . H. 484 Jone s , P. J . C ampbell , High b ur d e n and p e r v asive p o sitive s e lection o f s oma t i c mu tatio ns in 485 no r ma l human s ki n. Sc ienc e (80- . ) . 34 8, 880– 886 (201 5). 486 487 488 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint Acknowledgments 489 We thank the patients who selflessly donated tissue samples for this study. We further thank the 490 laboratory and informatics teams at Amgen deCODE Genetics for their contribution. 491 Figure 1A was created in BioRender. Olafsson, S. (2026) https://BioRender.com/d9xryv5. 492 Funding 493 This study was funded by Amgen deCODE Genetics. K.K. received support from the Private 494 Excellence Initiative Johanna Quandt of the Stiftung Charité. 495 Author contributions 496 SO conceived the project with contributions from KK, AS, VS, IJ, JGJ, ROA and KS. SO and AOA 497 performed microdissections and processed whole uteri with contributions from HSG, BA and GN. 498 HSG performed fixation, sectioning, staining and imaging of tissue samples. SO analyzed the 499 sequencing data and carried out all statistical analyses except for cell-of-origin analyses with 500 contributions from HJ and BA. KK performed cell-of-origin analyses. AS designed PCR primers and 501 oversaw PCR reactions used to amplify regions around mutational barcodes in whole-uteri screening. 502 DM and OM prepared sequencing libraries and oversaw sequencing of all samples. AMJ performed 503 histopathological assessments of tissues and provided guidance for laser capture. ROÁ recruited and 504 consented patients for the study and performed surgeries from which samples were obtained. KS 505 supervised the project. SO wrote the manuscript with contributions from all authors. 506 507 Competing interests 508 SO, HJ, LR, JS, VS, GN, IJ, DM and OM are current employees of Amgen deCODE Genetics. AOA, 509 AS and KS were employees of Amgen deCODE Genetics at the time of patient recruitment, data 510 generation and initial drafting of the manuscript. K.K declares no competing interests. 511 512 Data and materials availability 513 The data supporting the findings of this study are available in the supplementary material of this 514 article. Table S1 contains patient-level meta-data. Table S2 contains meta-data and statistics 515 calculated at the level of individual microdissections. Table S3 contains the dN/dScv statistics for 516 each coding gene. Table S4 contains results from the cell-of-origin analysis. Table S5 contains all 517 mutations in KRAS and PIK3CA identified in the large-scale screen of entire endometrium. Table S6 518 contains sequences of the PCR primers used in the screen. Read counts for each mutation call, VAF 519 histograms for each sample and tree R-objects will be made available on 520 www.decode.com/summarydata upon article acceptance. 521 The dNdScv software (RRID:SCR_023123) is freely available at 522 https://github.com/im3sanger/dndscv 523 The Coselens software (RRID:SCR_022578) is freely available at 524 https://github.com/ggruenhagen3/coselens 525 The hdp software is freely available at https://github.com/nicolaroberts/hdp 526 527 List of Supplementary Materials. 528

529 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint Supplementary Text 530 Figs. S1 to S29 531 Captions for Tables S1 to S6 532 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 26, 2026. ; https://doi.org/10.64898/2026.02.25.708037doi: bioRxiv preprint