{"paper_id":"475c1bac-0890-456d-ac41-8f12cb63c060","body_text":"1 \n \nSingle cell analysis of menstrual endometrial tissues defines phenotypes associated with \nendometriosis \n \nAndrew J. Shih1, Robert P. Adelson1, Himanshu Vashistha1, Houman Khalili1, Ashima Nayyar1, \nRadha Puran1, Rixsi Herrera1, Prodyot K. Chatterjee1, Annette T. Lee1,3, Alexander M. \nTruskinovsky2,3 Kristine Elmaliki1, Margaret DeFranco1, Christine N. Metz1,3+ and Peter K. \nGregersen1,3+ \n \n1Feinstein Institutes for Medical Research, Northwell Health, 350 Community Drive, Manhasset, \nNY, USA \n2Department of Pathology, North Shore University Hospital, Northwell Health, 300 Community \nDrive, Manhasset, NY, USA \n3Donald and Barbara Zucker School of Medicine, 500 Hofstra Blvd, Hempstead, NY, USA \n \nCorresponding authors: \n \n+Peter K. Gregersen MD \nProfessor and Director, Robert S. Boas Center for Genomics and Human Genetics \nFeinstein Institutes for Medical Research-Northwell Health \n350 Community Drive \nManhasset NY 11030 \nTelephone 516-562-1542 \nMobile: 516-297-1164 \nEmail: pgregers@northwell.edu \n \n+Christine N Metz, PhD \nProfessor \nFeinstein Institutes for Medical Research \nNorthwell Health \n350 Community Drive \nManhasset, NY 11030 \nTel: (516) 562-3403 \nCell: (516) 721-2702 \nEmail: cmetz@northwell.edu \n \n \n \n \n \n \n  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\n2 \n \nAbstract \nBackground. Endometriosis is a common, complex disorder which is underrecognized and \nsubject to prolonged delays in diagnosis. It is accompanied by significant changes in the eutopic \nendometrial lining. \nMethods. We have undertaken the first single cell RNA-sequencing (scRNA-Seq) comparison \nof endometrial tissues in freshly collected menstrual effluent (ME) from 33 subjects, including \nconfirmed endometriosis patients (cases) and controls as well as symptomatic subjects (who \nhave chronic symptoms suggestive of endometriosis but have not been diagnosed). \nResults. We identify a unique subcluster of proliferating uterine natural killer (uNK) cells in ME-\ntissues from controls that is almost absent from endometriosis cases, along with a striking \nreduction of total uNK cells in the ME of cases (p<10-16). In addition, an IGFBP1+ decidualized \nsubset of endometrial stromal cells are abundant in the shed endometrium of controls when \ncompared to cases (p<10-16) confirming findings of compromised decidualization of cultured \nstromal cells from cases. By contrast, endometrial stromal cells from cases are enriched in cells \nexpressing pro-inflammatory and senescent phenotypes. An enrichment of B cells in the cases \n(p=5.8 x 10\n-6) raises the possibility that some may have chronic endometritis, a disorder which \npredisposes to endometriosis. \nConclusions. We propose that characterization of endometrial tissues in ME will provide an \neffective screening tool for identifying endometriosis in patients with chronic symptoms \nsuggestive of this disorder. This constitutes a major advance, since delayed diagnosis for many \nyears is a major clinical problem in the evaluation of these patients. Comprehensive analysis of \nME is expected to lead to new diagnostic and therapeutic approaches to endometriosis and \nother associated reproductive disorders such as female infertility. \n \nKeywords: menstrual blood, menstrual effluent, inflammation, senescence, fibrosis, \nbiomarkers, decidualization \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n3 \n \nBackground \nEndometriosis is a common and heterogeneous disorder that is characterized by the growth of \nendometrial-like tissues outside of the uterus, most commonly in the peritoneal cavity and \nassociated with inflammation (1). While the pathogenesis of endometriosis is not understood, \nretrograde menstruation of endometrial cells and tissues via the fallopian tubes is one accepted \ntheory for the development of endometriosis lesions in the peritoneal cavity (2, 3). However, \nretrograde menstruation occurs in nearly all women (4), yet endometriosis occurs in \napproximately one in ten females in their reproductive years (3). Thus, other factors must \ncontribute to the development of endometriosis. While there is a significant genetic component \nto endometriosis (5), very little is known about how these putative risk alleles function. On the \nother hand, the eutopic endometrium of patients with endometriosis is significantly different \nwhen compared to the endometrium of those without endometriosis, with inflammatory changes \nnoted in the setting of endometriosis(6-9).  We have undertaken a detailed analysis of \nendometrial tissues and cells present in menstrual effluent (ME), since ME is the critical \nbiological sample transferred to the pelvic cavity, where most endometriosis lesions grow.  \n \nMost previous investigations of ME have involved the phenotypic analysis by \nimmunofluorescence, flow cytometry, and/or in vitro culture of single cell suspensions collected \nusing menstrual cups (10-14). Our previous flow cytometry studies showed that uterine natural \nkiller (uNK) cells were relatively depleted in ME from endometriosis cases vs. controls (11). \nHowever, this study was limited by the analysis of relatively few cell types in ME, with no \nassessment of specific subsets of cells. In addition, we demonstrated a defect in decidualization \ncapacity of endometrial stromal cells grown from the ME of patients with endometriosis when \ncompared to ME-stromal cells grown from healthy controls (11, 15). While these earlier results \npotentially provided a basis for a screening test for endometriosis, these analyses relied on \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n4 \n \nlaborious and expensive cell culture and in vitro assays, making them impractical for clinical \napplication. \n \nHerein we investigated fresh ME as an unexplored and important biological specimen for the \ndevelopment of non-invasive diagnostics based on the direct analysis of endometrial tissue \nfragments. We show that ME contains large numbers of shed fragments from endometrial \ntissues. Using enzymatic digestion of ME and associated tissues followed by single cell RNA-\nsequencing (scRNA-Seq) analysis, we compared the major cellular differences and gene \nexpression profiles found in ME collected from healthy controls (without symptoms of \nendometriosis) and patients diagnosed with endometriosis (confirmed by laparoscopic surgery \nwith positive confirmation by pathology), as well as patients with symptoms of endometriosis \n(e.g., recurrent dysmenorrhea, persistent abdominal bloating, dyspareunia, dysuria, and/or \ndyschezia) who are not yet diagnosed. In order to gain insight into the pathogenesis of \nendometriosis, we particularly focused on the phenotypes of stromal and uNK cells in ME \nthrough scRNA-Seq because these are abundant and have been previously shown to be \nabnormal in eutopic endometrium of patients. \n \nMethods \nHuman subjects and menstrual effluent collections \nMenstrual effluent (ME) was collected as previously described (11, 15). Briefly, women of \nreproductive age (N=33, age 20-45 years, average age 33.6 years) living in North America who \nwere not pregnant or breastfeeding, who were menstruating, and who were willing to provide \nME samples were recruited mainly via social media and consented to the ROSE study (IRB#13-\n376A)  \n (https://feinstein.northwell.edu/institutes-researchers/institute-molecular-medicine/robert-s-\nboas-center-for-genomics-and-human-genetics/rose-research-outsmarts-endometriosis). \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n5 \n \nWomen with histologically confirmed endometriosis (determined following excision laparoscopic \nsurgery and documented in a pathology report without revised American Society for \nReproductive Medicine (rASRM) staging/classification) were enrolled as ‘endometriosis’ \nsubjects (N=11). Women who reported chronic symptoms consistent with endometriosis (e.g., \nrecurrent dysmenorrhea, persistent abdominal bloating, dyspareunia, dysuria, and/or \ndyschezia), but not yet diagnosed with endometriosis (or not) were enrolled as ‘symptomatic’ \nsubjects (N=13). Control subjects living in North America who self-reported no gynecologic \nhistory suggestive of a diagnosis of endometriosis (and the absence of polycystic ovarian \nsyndrome, and pelvic inflammatory disease) were recruited mainly via social media and enrolled \nas ‘controls’ (N=9). \n \nEndometriosis, symptomatic, and control subjects collected their ME using an ‘at home’ ME \ncollection kit for 4-8 hours on the day of their heaviest menstrual flow (typically day 1 or 2 of the \ncycle) with a menstrual cup (provided by DIVA International), except for one subject who \ncollected ME using a novel menstrual collection sponge (as previously described (15)). After \ncollection, ME was shipped priority overnight at 4◦ C to the laboratory for processing. ME \ncollected from menstrual cups was mixed 1:1 with DMEM for processing. For the saturated \nmenstrual collection sponge, ME tissue was collected after rinsing the sponges with PBS to \ncollect cells and tissue. Demographic and gynecologic/health data (including hormone usage, \nmenstrual cycle information, and pain/pain medications) for controls, endometriosis subjects, \nand symptomatic subjects (and the total cohort) are shown in Table 1. \n \nImmunostaining of ME-derived tissue fragments \nME-derived tissue fragments were obtained from controls, symptomatic subjects, and \nendometriosis patients (n=2 each); tissue fragments were collected by pouring ME over a 70µ \nfilter, fixed, and transferred to the clinical pathology lab for paraffin embedding and hematoxylin \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n6 \n \nand eosin (H&E) staining. CD10 was chosen for immunohistochemical analysis because it is a \nsensitive marker of eutopic endometrial stroma (16) and because adjunctive use of CD10 \nimmunostaining with H&E staining enhances the histologic detection of endometriosis (17). \nCD56 was chosen because uNK cells stain brightly with CD56. H&E slides and immunostained \nslides were examined microscopically and imaged by a pathologist. Representative images are \nshown in Fig. 1. \n \nProcessing menstrual effluent for scRNA-Seq analyses \nWhole (unfractionated) ME (2.5-10ml) was digested with Collagenase I (1mg/ml, Worthington \nBiochemical Corporation, Lakewood, NJ) and DNase I (0.25mg/ml, Worthington Biochemical \nCorporation) at 37°C for 10-30 min using the gentleMACS\nTM Tissue Octo Dissociator (Miltenyi \nBiotec, Cambridge, MA) using C tubes and Program 37CMulti_E_01 (31 min). After digestion, \nthe sample was sieved over a 70µ filter and washed with DMEM 10% fetal bovine serum (FBS) \nto neutralize digestion enzymes; the flow through was sieved over a 40µ filter and washed with \nDMEM 10%FBS. After collecting the single cells (from the flow through) following centrifugation \n(350xg for 5 min), Neutrophils were removed using the EasySepTM HLA Chimerism Whole \nBlood CD66b Positive Selection Kit (STEMCELL, Cambridge, MA), according to the \nmanufacturer’s protocol. The neutrophil pellet was frozen at -80°C and used as a source of \nsubject DNA for genotyping (see below). The resultant cells were depleted of red blood cells \nusing the EasySep™ RBC Depletion Reagent (STEMCELL), according to the manufacturer’s \nprotocol, and then washed and subjected to density gradient centrifugation using Ficoll-Paque \nPLUS (Sigma-Aldrich, St. Louis, MO) to collect mononuclear cells, according to manufacturer’s \ndirections. To collect ME-tissue, whole ME (2.5-10ml) was sieved over a 70µ filter and washed \nwith DMEM; the ME-tissues trapped on the filter was collected and digested with Collagenase I \n(1mg/ml, Worthington Biochemical Corporation, Lakewood, NJ) and DNase I (0.25mg/ml, \nWorthington Biochemical Corporation) at 37°C for 10 min and processed as described above for \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n7 \n \nwhole ME, except without a density gradient centrifugation step. The resultant whole ME cells \nwere enumerated, and viability was assessed using ViaStain™ AOPI Staining Solution and the \nNexcelom Cellometer (Lawrence, MA). Preparations with >80% viability were processed for \nscRNA-Seq. Cells were immediately fixed in methanol for scRNA-Seq, as described by Chen for \nperipheral blood mononuclear cells (18)). Briefly, cells were washed and resuspended in a \n200µl Ca++ and Mg++-free PBS, followed by dropwise addition of chilled 100% methanol (800µl, \nfinal 80% w/v). Fixed cells were stored at -20°C for 20min and then stored at -80°C until used \nfor scRNA-Seq.   A pilot experiment was performed with a single ME sample, which was \nprocessed and either prepared immediately for scRNA-Seq (without methanol fixation and \nfreezing) or was fixed in methanol and frozen, as described above to optimize our scRNA-Seq \nmethods. The data showed almost identical scRNA-Seq results using both methods (see \nAdditional File 1: Fig. S1). Thus, all ME samples were methanol fixed and frozen, allowing for \ncost-effective ‘batching’ in scRNA-SEQ.  \n \nProcessing of samples for single cell sequencing \nMethanol-fixed cells were removed from -80°C and placed on ice for 5 min before centrifugation \n(1000xg for 5 min). Methanol-PBS supernatant was completely removed and cells were \nrehydrated in 0.04% bovine serum albumin (BSA) + 1mM dithiothreitol (DTT) + 0.2 U/ul RNase \nInhibitor in 3X SSC (saline sodium citrate buffer solution) Buffer (Sigma). An aliquot of fixed \ncells was stained with Trypan Blue and visualized under the microscope. The cells were \ncounted and pooled from different donors at equal ratios, filtered using 35µ strainer (Falcon), \nrecounted and brought up to a final conc. of 2,000 cells/µl and proceeded immediately for GEM \ngeneration and barcoding on a 10X Chromium using Next GEM 3’ v3.1 reagents (10X \nGenomics). Libraries were constructed following 10X Genomics’ recommendations and quality \nwas assessed on a High Sensitivity DNA chip on a BioAnalyzer 2100 (Agilent) before loading \n(1.8 pM) and sequencing on an Illumina Nextseq 500 using a High Output kit v2.5 (150 cycles).  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n8 \n \n \nFive subjects were pooled together into a single 10x lane with at least one of each phenotype \nper run with a total of 8 runs for ME-tissue and 3 runs of whole ME. The ME-tissue runs had \n44,135 total cells, of which 5,147 had ambiguous calls in Demuxlet, 2,632 were doublets and \n36,356 were singlets; only the singlets were analyzed. The whole-ME runs had 30,090 total \ncells, of which 5,556 had ambiguous calls, 2,776 were doublets, and 21,758 singlets (and hence \nanalyzed). A total of 43,054 cells were analyzed in this study  following filtering and QC  \n(thresholds of > 10% mitochondrial reads < 500 nUMI (number of unique molecular identifiers) \nor > 50000 nUMI or > 6000 unique features per cell). \n \nSingle cell RNA-Sequencing and analyses and statistics \nSamples were converted from raw bcl files to gene by cell matrices using CellRanger 6.0 \naligned to 10x Genomics' GRCh38-3.0.0 reference. Individuals were demultiplexed via \nDemuxlet (19) using genotypes taken from SNPs on the Illumina GSAv3 genotyping array, run \non DNA prepared from neutrophils isolated from ME. The thresholds in Demuxlet were adjusted \nto the expected doublet rate and those marked as doublets were removed. Downstream \nanalysis and visualization were done using Seurat 4.0 (20). Briefly, there were at least 25,000 \nreads per cell on average per 10x run and the mean number of genes captured was 1,388 (± \n896 (mean ± standard deviation [SD]). There was no significant difference between the various \nclinical groups (controls, cases, symptomatic) in these values. Genes were filtered out if they \nwere expressed in less than 3 cells while cells were filtered out if they had > 10% mitochondrial \nreads, 500 < nUMI < 50000 and > 6000 unique features. For the analysis of ME-tissue samples, \nonly subjects with information on at least 500 cells per subject were retained. After filtering the \ncell yields were comparable in each group (mean ± SD: 1,256 ±732 and 1,319 ±767 in ME-\ntissue and whole ME, respectively). Gene expression normalization and cell clustering was \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n9 \n \ndone using the SCTransform pipeline (21) with percent mitochondrial reads regressed out and \nperson specific batch effects corrected using Harmony (22). Identification of cell clusters was \ndone using known marker genes (Additional File 2: Table S1) (23-30) with differential gene \nexpression calculated using a Wilcoxon rank sum test. Enrichment of cell clusters of specific \nphenotypes was done using MASC (mixed-effects modeling of associations of single cells) \n( https://github.com/immunogenomics/masc ), which essentially uses a percentage of cells per \ncluster while also taking into account technical covariates; 10x library batch, preparation (whole \nME or ME-tissue),  nUMI per cell, percent mitochondrial reads and phase are accounted for. All \ndatasets are deposited in the National Center for Biotechnology Information/Gene Expression \nOmnibus (GEO) accession number \nGSE203191.  \n \n \nResults \nEndometrial tissue fragments are present in fresh menstrual effluent. \nWe carried out histological assessment of fresh menstrual effluent (ME)-associated tissues \nisolated from ME. Representative H&E sections of ME-derived tissue fragments from four \nsubjects (1 control, 2 laparoscopically/histologically confirmed endometriosis subjects, and 1 \nsymptomatic subject) show the presence of endometrial tissues with mucosal and glandular \nepithelium and areas of stroma. The endometrium had typical late secretory/menstrual \nmorphology with expanded stroma containing scattered inflammatory cells, and secretory and \ninactive-type glands (Fig. 1A-D, upper panels). Immunostaining of ME-derived tissue sections \nreveals a range of stromal cells stained with antibodies to CD10, a clinically used marker of \nendometrial stroma (16, 17), and an abundance of uNK cells (stained with antibodies to CD56 \n(NCAM), an archetypical marker of NK cells [Fig. 1A-D, lower panels]). \n \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n10 \n \nSingle cell RNA sequencing (scRNA-Seq) of digested freshly processed ME reveals the \npresence of a heterogenous mixture of immune and non-immune cells. \nWe have analyzed ME samples from 33 subjects, including age-matched healthy controls \n(N=9), endometriosis cases (N=11), and subjects with chronic symptoms suggestive of \nendometriosis but not yet diagnosed (N=13) (see Table 1). ME samples from either whole ME \n(unfractionated) or ME samples enriched for tissues (“ME-tissue”) were digested with \ncollagenase I and DNase I, depleted of neutrophils, and processed for scRNA-Seq, as \ndescribed in the methods. As shown in Fig. 2 a graph-based clustering approach using \nSeurat distinguishes multiple cell clusters shown on the UMAP (uniform manifold approximation \nand projection) plot. There is striking diversity of the cell types defined by the cluster analysis. A \nmajor group of uterine NK cells is designated cluster uNK1, with a small associated cluster \ndesignated uNK2. Sets of clusters related to CD8+and CD4+ T cells are shown in the central \nportion of the plot. Endometrial stromal cells and epithelial cells are identified in major clusters in \nthe right side of the UMAP plot. Subclusters of endometrial stromal cells are described below in \ndetail. Based on (31), Epithelial1 appears to be a mix of lumenal and glandular epithelial cells, \nEpithelial2 is comprised of ciliated epithelial cells, and Epithelial3 is a separate set of CD326-\nexpressing cells that do not overlap with Epithelial 1 or Epithelial2. Distinct clusters of B cells \nand myeloid cells can also be delineated, along with a small cluster of plasmacytoid dendritic \ncells (pDC). The positive gene markers used to generate the cell clusters shown in Fig. 2 are \nincluded in Additional File 2: Table S1. Overall, the various cell clusters are well represented \nwhether unfractionated whole ME or tissue-enriched ME is processed for scRNA-Seq. Some \ndifferences in cell subset frequencies can be observed; in particular, epithelial cells  were \nenhanced when tissue-enriched ME was utilized for sample processing (see Additional File 3: \nFig. S2). \n \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n11 \n \nCell clusters from ME containing endometrial tissue differ between endometriosis cases and \nhealthy controls; relative depletion of uterine NK cells and enrichment of B cells in \nendometriosis cases. \nWe compared the relative frequency of the various cell clusters in the freshly processed ME \nobtained from the diagnosed endometriosis cases (N=11) compared with controls (N=9), as \nshown in Fig. 3. By inspection of Fig. 3, it is apparent that both clusters of uNK cells (uNK1 and \nuNK2) are markedly depleted in the cases vs. controls (average percentage of uNK \napproximately 8% in cases, 28% in controls), as well as an increase in the proportion of B cells \nin cases (~9%) vs. controls (~3%). The odds ratios and confidence intervals for these two cell \nenrichment patterns are shown in Fig. 4, along with the patterns of enrichment of all the other \nmajor cell clusters. While there is some variation among many of the different cell clusters, a \nformal analysis shows the most striking differences are observed for uNK cells, which are \nenriched in controls (and depleted in cases; uNK1, P <10E-16; uNK2, P <10E-16), along with a \nrelative enrichment in the proportion of B cells in the cases diagnosed with endometriosis (and \nrelatively depleted in controls; P <10E-16). Note that the stromal cell cluster is not significantly \ndifferent between cases and controls (P > 0.05). \n \nWe also explored whether the various proportions of cell clusters of the ME preparations from \nthe “symptomatic” but undiagnosed group of subjects (N=13) are different from ME preparations \nfrom controls. This is clearly the case, as shown in Additional File 4: Fig. S3. Here, we show the \nrelative enrichment of uNK cells is maintained in controls in comparison to the symptomatic \ngroup (uNK1, P <10E-16; uNK2, P = 0.0025), similar to that observed with ME from cases. B \ncells also show a significant relative enrichment in symptomatic as well as diagnosed cases, \ncompared with controls (Additional File 4: Fig. S3) (symptomatic vs. control, P = 5.8 x10\n-6), \nsimilar to that observed with ME from cases. Perhaps not surprisingly, these significant \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n12 \n \ndifferences in symptomatic cases vs. controls are less striking than the differences in \nendometriosis cases vs. controls, given the likely heterogeneity of the symptomatic group. \n \nDecidualized stromal cell subclusters are reduced in endometriosis. \nPrevious studies have reported reduced decidualization capacity in endometrial stromal cells \ngrown from biopsies of patients with endometriosis (32). We have also observed impaired \ndecidualization using stromal cells grown directly from ME (11, 15). Therefore, we examined \nwhether this trend could be observed in fresh stromal cells analyzed by scRNA-Seq. The \nstromal cell numbers or percentages did not significantly differ between the control and \nendometriosis groups, as shown in Fig. 3 and Fig. 4. However, subclustering of the stromal cell \ncluster clearly identified 5 subclusters of interest within the stromal cell population (Fig. 5A). We \nhave designated these subclusters based on the dominant transcripts expressed in each of \nthese subclusters, as shown in the violin plots in Fig. 5B. Two of the five subclusters (2 and 4) \nare not different between cases and controls (the top genes of subclusters 2 and 4 are \ndescribed in Additional File 5: Table S2). The subclusters showing significant enrichment in \neither cases or controls (subclusters 1, 3, and 5) are indicated by the Log\n2 (odds ratios, [OR]) \nbelow the UMAP plot (Fig. 5C). \n \nIt is striking that an apparently decidualized stromal cell subcluster (expressing IGFBP1 mRNA) \nis significantly enriched in controls compared with endometriosis cases (Fig. 5A-B). In addition \nto IGFBP1, the top differentially expressed genes in this subcluster (compared to other stromal \ncell subclusters) include LEFTY2, DCN, LUM, MDK, C1QTNF6, APOE/D, DCN, and other \nprogesterone sensitive and decidualization/fertility gene markers (see left panel (subcluster 3) in \nFig. 6 and Additional File 6: Table S3). (33-41) (42-46, 47 , 48-103) (104-108) (109-113).\n This \nsuggests that a phenotype of “decidualization” can be measured directly in stromal cells derived \nfrom fresh ME and is associated with control vs. disease phenotype. A modest enrichment of a \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n13 \n \nsubcluster expressing IL11 was observed in cases, as indicated in Fig. 5A-C. In addition to IL11, \nthis subcluster is associated with transcripts for MMP3, MMP1, MMP9, SERPINB2, S100A6, \nand CXCL8, among other genes associated with inflammation, fibrosis and senescence, as well \nas endometriosis, as shown in the middle panel (subcluster 1) of Fig. 6 (and Additional File 6: \nTable S3). A third subcluster, designated by high expression of the gene encoding matrix Gla \nprotein (MGP), is also enriched in the stromal cells of cases (Fig. 5A-C). This subset expresses \nnumerous extracellular matrix genes that have been associated with presence of perivascular \nstromal cells, senescence, and cell adhesion/cell spreading, including FN1 (which encodes \nfibronectin-1), a known risk locus for endometriosis (114). Fig. 6 (right panel (subcluster 5) and \nAdditional File 6: Table S3) also shows the list of top genes expressed in this subset. Additional \nFile 7: Fig. S4 demonstrates that the IGFBP1+ and MGP+ subclusters map to stromal cells \nsubsets defined in the decidua found in the first trimester of pregnancy by Vento-Tormo et al \n(26).\n \n \nFinally, we examined the differences between cases and controls in the two uNK subclusters \npresent in digested endometrial tissues in ME (uNK1 and uNK2, see Fig. 2). We noted a distinct \nsubcluster of uNK cells (uNK2) that is characterized by the expression of genes associated with \ncell proliferation such as MKI67 (which encodes Ki67) and TOP2A (which encodes \ntopoisomerase 2A) (see Additional File 8: Fig. S5 for a full uNK subcluster analysis). As \ndiscussed below, this cluster also mapped nearly exactly (97%) with a proliferative subset of \nuNK cells that has been defined by scRNA-Seq in decidua obtained during the first trimester of \npregnancy (26). This is consistent with the proliferation of uNK cells and overall accumulation of \nuNK cells in the course of decidualization in control subjects vs. cases, as shown in Fig. 3 and \nFig. 4. \n  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n14 \n \nDiscussion \nThese studies show for the first time that the phenotype of eutopic endometrial tissue shed into \nthe menstrual effluent is distinct in patients with endometriosis compared to control subjects. \nThere are three major observations. First, the endometrial stromal cells show a relative \ndeficiency of progesterone-sensitive gene markers associated with endometrial stromal cell \ndecidualization in patients with endometriosis (e.g., IGFBP1, LEFTY2, LUM, DCN, etc). This is \nconsistent with previous studies showing impaired decidualization of cultured endometrial \nstromal cells obtained from endometrial and ectopic endometriosis biopsies (32, 115), as well as \nfrom menstrual effluent (11, 15). Secondly, there is a striking reduction in the proportion of uNK \ncells in the ME-derived endometrial tissue of patients with endometriosis compared with \ncontrols. This was suggested by our previous studies of free cells present in ME using flow \ncytometry methods [11], but it is clearly a major distinguishing feature of the eutopic \nendometrium of endometriosis patients. Thirdly, our data suggest an enrichment of B cells in the \neutopic endometrium of patients with endometriosis, a finding that is consistent with the \nhypothesis that chronic inflammation and/or chronic endometritis is a predisposing factor in the \ndevelopment of endometriosis (116). \n \nA deficiency in the decidualization capacity of stromal cells cultured from biopsies of the eutopic \nendometrium has been reported previously (32), and is also found in ME-derived stromal cells \ncollected at the time of menstruation (11, 15). Our scRNA-Seq data clearly shows the reduction \nof the IGFBP1+-expressing decidualized stromal cell subclusters in endometriosis cases vs. \ncontrols (Fig. 5C). The relationship of this finding to the pathogenesis of endometriosis is not \nestablished. One possibility is that this differentiation deficiency leaves behind non-decidualized \nendometrial stromal cells that exhibit proinflammatory, pro-fibrotic, and/or senescent \nphenotypes. These ‘pathogenic’ cells may then initiate or promote lesions following retrograde \ntransfer into the peritoneal cavity. The enrichment of an IL11-expressing stromal cell subcluster \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n15 \n \nin the endometriosis ME samples that express many estrogen-responsive, pro-inflammatory, \npro-fibrotic and senescence gene markers (shown in Fig. 6 and Additional File 6: Table S3) \nprovides some support for this possibility, but this needs confirmation in larger datasets. The \nsignificant increase in the MGP+ stromal subcluster in endometriosis (Fig. 5C) is also of \npotential interest. As shown in Fig. 6 (right panel), the MGP+ stromal cell subcluster expresses \nmany genes that are associated with the extracellular matrix, including FN1 (encoding \nfibronectin-1) which has been associated with an increased risk for endometriosis in GWAS \nstudies (117). Interestingly, most of the top markers found in the IL11+ and the MGP+ \nsubclusters are either associated with senescence or induce senescence (e.g., IL11 and \nSERPINB2 [Fig. 6 and Additional File 6: Table S3]). Inflammation and senescence are key \nfeatures of endometriosis and reduced uterine receptivity and infertility (118-120). \n \nAnother possibility is that the overall environment of the eutopic endometrium predisposes to \nreduced stromal cell decidualization, independent of any direct role or effect on stromal cell \nsubsets in the disease. A chronic inflammatory endometrial environment might lead to, or be \nassociated with, other changes that put individuals at risk for endometriosis. For example, the \npresence of chronic endometritis has been reported to be a significant risk factor for \nendometriosis (116, 121); chronic endometritis is also associated with reduced stromal cell \ndecidualization (122). Interestingly, the presence of B cells in endometrial tissue, particularly \nplasma cells, is a requirement for the clinical diagnosis of chronic endometritis (116). We note \nthe significant increase in B cells in shed endometrium of endometriosis patients (Fig. 3 and 4) \nand symptomatic subjects (Additional File 4: Fig. S3) when compared to controls. This may \nreflect an inflammatory state, as B cells play an important role in mediating or regulating \ninflammatory and autoimmune diseases (123). The numbers of B cells available for detailed \nanalysis have not allowed us to fully understand the phenotype of these cells; this is an area for \nfuture study. \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n16 \n \nWe have  demonstrated that uNK cells are remarkably depleted in the ME-derived endometrial \ntissues of patients with endometriosis (Fig. 3 and Fig. 4 and Additional File: Fig. S3). This may \nreflect compromised decidualization in these subjects. uNK cells are a characteristic feature of \ndecidualizing tissues (124) and are also prominent in the decidua of early pregnancy (26). To \nour knowledge, this is the first report of proliferating uNK cells found in ME. Crosstalk between \nstromal cells and uNK cells is a feature that promotes decidualization and uterine \nreceptivity/placental vascular remodeling (125). uNK cells do not appear to play a major role in \ndecidualization in uNK deficient IL15 knockout mice (126). It remains unclear whether uNK cells \nor stromal cells are the primary driver of the decidualization impairment in endometriosis. \nHowever, uNK cells do play a role in the maintenance of decidual integrity as reported by \nAshkar et al (127). Brighton and co-workers emphasized the important role of uNK cells in \nclearing senescent decidual cells in the cycling human endometrium and their clearance is \nproposed to be important for optimal fertility (128). A lack of uNK cells in the endometrium may \ncontribute to increased numbers of senescent cells observed in the stromal subclusters among \nendometriosis subjects and may contribute to endometriosis-associated infertility. However, it is \nplausible that a lack of decidualizing endometrial stromal cells (with concomitant reduced \nproduction of IL-15 and uNK chemoattractants) reduces the infiltration and proliferation of uNK \nin decidualizing zones. Defective uNK cell function has recently been proposed in the setting of \nendometriosis with infertility (129). We did not observe IL15 expression by ME-stromal cells; this \nis not surprising as IL-15 expression by stromal cells peaks before the mid-secretory phase. \nInterestingly, we did observe enhanced expression of IL2RB (which encodes a component of \nthe IL-15 receptor) in the uNK cells of controls compared with the endometriosis group. Since \nuNK cells are reported to play a role in infertility (124, 130), and infertility is a common feature of \nendometriosis, further analysis of the uNK subset will clearly be of interest. \n \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n17 \n \nTaken together, these observations suggest a set of interactions that may drive the \ndevelopment and/or progression of endometriosis at several levels, as summarized in Fig. 7. \nStromal cell decidualization may be inhibited by a number of factors, including chronic \ninflammation, stress and/or progesterone resistance. This may divert stromal cells into a more \nproinflammatory/senescent state. Furthermore, deficient decidualization may also compromise \nthe infiltration of uNK cells into the decidua, and therefore reduce the clearance of senescent \ncells. Clearly, host genetic variation may influence these processes at every level of these \ninteractions. \n  \nIt is encouraging that many of our findings in patients with pathologically confirmed \nendometriosis are also present in a proportion of subjects with chronic symptoms that are \nsuggestive of endometriosis, even in the absence of a confirmed tissue diagnosis. The delay in \ndiagnosis of endometriosis is widely recognized as a major barrier in the management of this \ndisease, with delays of up to a decade in some subjects before the disease is recognized (131). \nWe recognize the limitation that the symptomatic group lacks a diagnosis and therefore, we \ncannot assess the predictive ability of our results. To address this limitation, a clinical trial is \nunderway to enroll symptomatic subjects who are being evaluated by diagnostic laparoscopy as \npart of their standard care by collaborating surgeons; scRNA-Seq profiles of their ME collected \nprior to surgery will be validated based on the results of their laparoscopic diagnosis.\n Such a \nstudy design will be required to establish the positive and negative predictive value of menstrual \ntissue analysis in a real-world clinical setting where an endometriosis screening test might be \napplied. \n \nDue to the cost and complexity of the analysis, an scRNA-Seq approach is unlikely to become a \ndiagnostic test for endometriosis. However, we propose that the data obtained from scRNA-Seq \ncan be leveraged to develop future diagnostic and/or screening tests.  What should such  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n18 \n \nscreening tests involve? It will likely include an assessment of gene expression patterns among \nME-derived stromal cells or uNK cells (or specific stromal cell and uNK subsets). An initial \nanalysis of stromal cell clusters suggests several potentially useful gene expression differences \namong cases vs. controls (Additional File 9: Fig. S6). Differences are also observed in uNK cells \n(Additional File 10: Fig. S7) or indeed may be found in other cell types as well. . On the other \nhand, if it can be adapted to a clinical diagnostic test, scRNA-Seq of these tissues is likely to be \nthe most informative approach, perhaps having more global utility to establish complex and \nheterogenous disease subtypes, as well as predicting or following response to therapy. \nAdditional phenotypes that can be uncovered using scRNA-Seq analysis on larger populations \nmay yet yield additional biomarkers that can be incorporated into a more targeted multivariate \nbiomarker analysis for diagnostic purposes. \n \nIn any case, the integration of our findings into a unified picture of the pathogenesis of \nendometriosis will require additional scRNA-Seq studies of larger heterogenous populations, at \ndifferent stages of disease development and include deeper analysis of T cells, B cells, myeloid \ncells, and epithelial cells. Abnormalities of the eutopic endometrium are widely recognized \nfeatures of endometriosis [6-9, 18, 34] and this can provide diagnostic value, regardless of \nwhether retrograde menstruation plays a causative role. In addition, it is likely that scRNA-Seq \napproaches of the endometrium via ME may allow for improved classification of clinically \nmeaningful disease subsets and as a means for assessing patients’ responses to therapies, as \nwell as uterine-associated fertility status. For example, many of the genes that exhibit changes \nin the stromal cell subclusters are associated with either estrogen or progesterone \nresponsiveness (Fig. 6), and these differences could be used to guide or assess responses to \nhormonal therapies and for assessing aspects of uterine receptivity/fertility. \n \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n19 \n \nOn the other hand, if disease causation is due to retrograde menstruation of abnormal \nendometrial tissues, ME analysis provides an opportunity to explore new therapies. For \nexample, based on the enrichment of pro-senescent genes in endometriosis endometrial stomal \ncells (vs. control cells) and the deficit of uNK cells in endometriosis subjects, we propose \ninvestigating senescence as a central feature of endometriosis. Once demonstrated, this may \nhave important potential therapeutic implications, since various senotherapeutics (senolytic and \nsenomorphic agents) have now been shown to improve chronic inflammatory diseases in pre-\nclinical models and human clinical trials (132, 133). This is significant since none of the current \nmedical therapies for endometriosis have been shown to alter disease progression. \n \nConclusions \nIn summary, these scRNA-Seq data of ME collect from endometriosis cases and healthy \ncontrols represent a first attempt to globally characterize the cellular diversity of endometrium \nthat is shed at the time of menstruation. More detailed studies in larger datasets are clearly \nrequired, particularly regarding diversity in T cells, B cells and myeloid cells, as well as epithelial \ncells. We propose that a comprehensive assessment of cellular phenotypes in ME tissues will \nopen a new window on both diagnosis as well as preventive treatment for patients at risk for \nendometriosis as well as other uterine and reproductive disorders. \n \nAbbreviations \nBSA: Bovine serum albumin; DTT: Dithiothreitol; FBS: Fetal bovine serum; GEO: Gene \nExpression Omnibus; H&E: Hematoxylin and eosin; MASC: Mixed-effects modeling of \nassociations of single cells; ME: Menstrual effluent; nUMI: Number of unique molecular \nidentifiers; OR: Odds ratios; pDC: Plasmacytoid dendritic cells; SASP: Senescence associated \nsecretory phenotype; scRNA-Seq: Single cell RNA-sequencing; SD: Standard deviation; SSC: \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n20 \n \nSaline sodium citrate; UMAP: Uniform manifold approximation and projection; uNK: Uterine \nnatural killer \n \nDeclarations \nAcknowledgements \nWe are grateful to the Endometriosis Foundation of America and to Dr. Tamer Seckin for \nproviding early support for this work, and the for the ongoing support from the Northwell Health \nInnovation Award. We also thank Anthony Liew, Cassie Pond and Maruf Chowdhury who \nprovided valuable technical support for this project. We are especially grateful to the many \nextraordinary patients and volunteers without whose participation this project could not have \nbeen accomplished.  \n \nEthics Approval and Consent to Participate \nAll procedures for the collection of samples from research subjects were performed with the \napproval of the institutional review board (IRB) of the Feinstein Institutes/Northwell Health. All \nparticipants signed informed consent prior to enrollment and study participation. \n \nConsent for Publication \nAll authors give their consent for publication of this manuscript. \n \nAvailability of Data and Materials  \nThe original data and materials presented in the study are available from the corresponding \nauthors upon reasonable request. scRNA-Seq data is available at National Center for \nBiotechnology Information/Gene Expression Omnibus (GEO) (accession number \nGSE203191).  \n \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n21 \n \nCompeting Interests \nThe authors declare that they have no competing interests. \n \nFunding \nThis work was supported by the Northwell Health Innovations Award and the Endometriosis \nFoundation of America \n \nAuthors’ Contributions \nConceptualization: CNM, PKG. Recruitment and enrollment: KE, MDF. Data collection: AJS, \nRPA, KE, HK, MDF.  Formal analysis: PKG, CNM, AJS, RPA.  Sample processing: RP, PKC, \nHV, RH, AN. Pathology slide review: AMT. Library construction: HK. Funding acquisition: CNM, \nPKG. Supervision of scRNA-Seq: ATL. Writing—original draft: PKG, CNM, AJS, RPA. Writing—\nreview & editing: HK, PKG, CNM, RPA, AJS. All authors read and approved the final \nmanuscript. \n \n  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n22 \n \nFigures \n \nFig. 1. ME contains endometrial tissues. Histological analysis of endometrial tissues isolated \nfrom the menstrual effluent (ME) from 4 separate subjects: A) control subject, B-C) two subjects \nwith pathologically confirmed endometriosis, and D) subject chronic symptoms of endometriosis \n(not yet diagnosed). Upper panels for A-D: H&E staining is shown in two panels at two \nmagnifications for each individual: A) 40X (left) and 200X (right); B) 100X and 200X; C) 100X \nand 200X; and D) 40X and 200X; arrowheads point to glandular epithelium. Sections show \ntypical late secretory/menstrual endometrium with expanded stroma containing scattered \ninflammatory cells and secretory and inactive type glands. Lower panels for A-D: \nimmunostaining with anti-CD10 and anti-CD56 antibodies to detect stromal cells (left) and \nuterine NK (uNK) cells (right), respectively, at 100X. Scale bars are shown in each image.  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n23 \n \nFig. 2. Cellular composition of digested ME based on scRNA-Seq. UMAP plot for all 33 digested \nmenstrual effluent (ME) samples (controls=9; endometriosis cases=11; symptomatic cases=13). \nSeveral well-delineated cell clusters include a large cluster of uterine NK cells (uNK1), as well \nas clearly separated stromal cells, epithelial cells, and B cells. Several clusters each of T cells \nand myeloid cells are also defined, as well as a small cluster of plasmacytoid dendritic cells \n(pDC). A small cluster of approximately 60 unknown cells is in the lower right corner. The \npositive gene markers used to generate the cell clusters shown are included in Additional File 2 :\nTable S1. \n \nd \n). \n: \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n24 \n \nFig. 3. Distinct cellular composition differences in digested ME from endometriosis cases vs. \ncontrols are revealed by scRNA-Seq. The data taken from the UMAP plot in Fig. 2 is separated \ninto two groups: controls (n=9, providing 14,327 cells) and endometriosis cases (n=11, providing\n11,924 cells). The most striking difference is the increased fractions of uterine NK cells (uNK1 \nand uNK2) in the endometrial tissues of controls as compared to cases. In contrast, B cells are \nsignificantly enriched in cases. A formal analysis of enrichment is given in Fig. 4 and confirms \nthe significant enrichment of uNK cells and B cells in controls and cases, respectively. The \npositive gene markers used to generate the cell clusters shown are included in Additional File 2 : \nTable S1. \n \nng \n: \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n25 \n \nFig. 4. Analysis of enrichment of cell subsets in ME comparing endometriosis cases and \ncontrols. These data are taken from data shown in Fig. 3. The Log2 odd ratios (OR) with cell \nsubsets enriched in controls on the left and cell subsets enriched in cases on the right. It is \napparent that uterine NK (uNK) cells, both uNK1 and uNK2, are significantly enriched in \ncontrols, while B cells show the greatest enrichment in cases. Note: Epithelial cells are excluded \nfrom this analysis because their enrichment was affected by the tissue preparation method \nused.  \n \n \ned \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n26 \n \nFig. 5. Analysis of the stromal cell subclusters. A) UMAP plot of the five stromal cell subclusters \nare shown. B) Violin plots showing the defining gene expression per subcluster for subclusters \n1-5. C) Log2 (Odds Ratio) shows that subcluster 3 (IGFBP1+) is significantly enriched in controls \n(Log2 OR = -1.3, case vs. control). In contrast, subcluster 1 (IL11+) and subcluster 5 (MGP+) \nare enriched in diagnosed subjects. The top transcripts characterizing these three distinct \nstromal cell subclusters are summarized in Fig. 6 and emphasize the enrichment of the \ndecidualized stromal cells – subcluster 3 (IGFBP1+) – in controls. \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n27 \n \n \nFig. 6. Distinct subclusters of decidualized stromal cells and pro-inflammatory stromal cells \ndistinguish ME from controls and endometriosis cases. Upper panel: A summary of genes \nenriched in the stromal cell subclusters which are significantly enriched in cases (subclusters 1 \n[IL11+] and 5 [MGP+]) or controls (subcluster 3 [IGFBP1+]). Note: Subclusters 2 and 4 were not \nsignificantly different in cases vs. controls; see Additional File 6; Table S3 for the listing of genes \ndifferentially expressed in these clusters. Lower panel: Characteristic features of stromal cell \nsubcluster gene markers. The decidualized stromal cell subcluster (IGFBP1+, subcluster 3) is \nprominently enriched in genes that are associated with decidualization and uterine receptivity \nand are progesterone responsive. In contrast, the non-decidualized stromal cell subsets that are \nenriched in cases (MGP+ [subcluster 5] and IL11+ [subcluster 1]) are variably enriched in \nestrogen responsive genes, and remarkably enriched in genes associated with inflammation, \nfibrosis, and cellular senescence. Note: MGP+ (subcluster 5) is also enriched in cell adhesion \nand cell spreading gene markers. \n \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n28 \n \nFig. 7. A disease model for endometriosis. Defective endometrial stromal cell decidualization \nmay be driven by multiple factors including inflammation, chronic endometritis, stress, and/or \nprogesterone resistance. This, in turn, may direct stromal cell differentiation in the direction of \nchronic inflammation and senescence, with accompanying senescence associated secretory \nphenotypes (SASPs), which include pro-inflammatory mediators and proteases. The \nsenescence phenotype may also impair decidualization. Reduced decidualization may also \ncompromise the infiltration and proliferation of uNK cells, which are likely to be important for \nsenescent cell removal. Further analysis of other cells in menstrual effluent will be important to \nprovide further support for this model. \n \n \n \n \n \n \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n29 \n \n \nTable 1. Subject Group Characteristics – Control (CTRL), Dx (Diagnosed), Sx (Symptomatic) \nCTRL  Dx  Sx  TOTAL         P value (CTRL vs Dx)  \nAge (years)   33.4±5.4 35.2±4.4 32.3±8.2 33.6±6.3  0.42  \n(mean±SD \n \nBMI (kg/m²)   24.2±7.1 28.4±5.4 26.8±7.6 26.5±6.8  0.15 \n(mean±SD) \n \nAge at menarche (years) 12.0±0.9 11.6±1.6 12.6±1.7 12.0±1.5  0.51  \n(mean±SD) \n \nRace/ethnicity           0.34 \n Caucasian  7/9 (78%)  0/11 (91%) 13/13 (100%) 30/33 (91%) \n Black   1/9 (11%) 0/11 (0%) 0/13 (0%) 1/33 (3%)  \n Mixed   1/9 (11%) 0/11 (0%) 0/13 (0%) 1/33 (3%)  \n Other   0/9 (0%) 1/11 (9%) 0/13 (0%) 1/33 (3%)  \n Hispanic  0/9 (0%) 0/11 (0%)  1/13 (8%) 1/33 (3%) \n \nT y p i c a l  c y c l e  l e n g t h  ( d a y s )           0.52 \n 21-25 days  0/9 (0%) 1/11 (9%) 3/13 (23%) 4/33 (12%) \n 26-31 days  8/9 (89%) 7/11 (64%) 8/13 (62%) 23/33 (70%) \n 32-39 days  1/9 (11%) 2/11 (18%) 2/13 (15%) 5/33 (15%) \n >40 days  0/9 (0%) 1/11 (9%) 0/13 (0%) 1/33 (3%) \n \nTypical bleed time (days)          0 . 3 6  \n <3 d   0/9 (0%) 2/11 (18%) 0/13 (0%) 2/33 (6%) \n 3-5 d   5/9 (56%) 6/11 (55%) 8/13 (62%) 19/33 (58%) \n 6-8 d   4/9 (44%) 3/11 (23%) 5/13 (38%) 12/33 (36%) \n \nTypical flow            0 . 9 6  \n Light   1/9 (11%) 1/11 (9%) 0/13 (0%) 2/33 (6%) \n Moderate  2/9 (22%) 2/11 (18%) 3/13 (23%) 7/33 (21%) \n Moderately Heavy 3/9 (33%) 5/11 (45%) 9/13 (69%) 17/33 (52%) \n Heavy   3/9 (33%) 3/11 (23%) 1/13 (8%) 7/33 (21%) \n \nH o r m o n e  u s e             0.35 \n Yes   0/9 (0%) 1/11 (9%) 1/13 (8%) 2/33 (6%) \n \nPain in this cycle           0 . 0 6  \n Yes   5/9 (56%) 10/11 (91%) 13/13 (100%)\n*  28/33 (85%) \n  None  4/9 (44%) 1/11 (9%) 0/13 (0%) 5/33 (15%) \n  Mild  2/9 (22%) 3/11 (23%) 3/13 (23%) 8/33 (24%) \n  Moderate 3/9 (33%) 6/11 (55%) 4/13 (31%) 13/33 (40%) \n  Severe  0/9 (0%) 1/11 (9%) 6/13 (46%) 7/33 (21%) \n \nPain medication in this cycle \n(Midol, Advil, Tylenol, Naproxen, Hydromorphone 2mg/Baclofen/diazepam/Ketamine 8/10/15mg)  \n Yes   1/9 (11%) 6/11 (55%) 10/13 (77%) 17/33 (52%)  0.04 \n--------------------------------------------------------------------------------------------------------------------------------------------------- \nNo comparisons  of CTRL vs. Sx  were significant for the above characteristics, except pain (yes/no): \n* P=0.008\n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n30 \n \nReferences \n1. Inte rna tional working group of Aagl EE, Wes, Tomasset ti C, Johnso n NP, Petroz za  J, Abra o MS, e t \nal. An In ter nati onal Terminol ogy for Endometriosis , 2021. J Minim Invasive Gyn ecol. 2021;28(11):1849-\n59. \n2. Jensen JR, Coddingto n C.C. Evolving Spectrum: The Path ogenesisof Endome triosis.  Clinical \nobste trics and gynecology. 2010(2):379- 88. \n3. Zondervan KT, Becker CM, Koga K, Missmer SA, Taylor RN , Vigano P. Endometri o sis. Nat R ev Dis \nPrimers. 2018;4(1):9. \n4. Halme J, Hammond M G, Hulka JF, Raj SG,  Talbert LM. R etr ograd e menstru atio n in healthy \nwomen and in pati ents with end omet rio sis. Obste t Gynecol . 1984;64(2):151-4. \n5. Zondervan KT, Becker CM, Missmer S A. E ndometri osis. N Engl J Me d. 2020;382(13):1244-56. \n6. Brosens I , Brosens JJ, B enagian o G. The e utopic endom etrium in e ndome triosis: a re the ch anges \nof clinical significance? Reprod Biome d O nline. 2012;24(5):496-502. \n7. Bulun SE. Endomet riosis. N Engl J Med. 2 009;360(3):268-79. \n8. Vallve-Juanico J, Housh dara n S, Giudice L C. The endomet rial immune envir onmen t of women \nwith endome triosis. Hum R eprod Up dat e . 2019;25(5):564-91. \n9. Liu H, Lang JH. Is abnormal eutop ic endo metrium th e cause of endom etri osis? The role of \neutopic en domet rium in path ogenesis of  endomet riosis. M ed Sci Monit . 2011;17(4):RA92-9. \n10. van der Molen R G, Schu tte n JH, van Cran enbro ek B, te r Mee r M, Donckers J , Scho lten RR, et al . \nMenstr ual blood close ly resembles t he u terin e immune micro-environme nt and i s clearly distinct from \nperiphe ral bloo d. Hum Rep rod. 2014;29( 2):303-14. \n11. Warr en LA, Shih A, R ent eira SM , Seckin T, Blau B, Simpfendo rfer K, et a l. Ana lysis of menstrual \neffluent: diagnostic pot enti al for endom etrios is. Mol Me d. 2018;24(1):1.  \n12. Schmitz T, Hoffmann V, Olliges E, Bobinger A, Popovici R, Nöß ner E, e t al. R educe d frequency of \nperforin-posi tive CD8+ T cells in menstrual effluent of endome triosis pa tien ts co mpared to h eal thy \ncontrols. MedRXiv. 2021.  \n13. Hosseini S, Shokri F, Tokhmechy R, Savad i-Shiraz E, Jeddi-Teh rani M, R ahbari M, et  al. Menst rual \nblood contai ns immune cells with inflammatory and an ti-inflammatory pro per tie s. J Obst et Gynaecol \nRes. 2015;41(11):1803-12. \n14. Sabbaj S, Hel Z, Richte r HE, Mest ecky J, G oepfer t PA. Mens trual blo od as a pot enti al source of \nendome trial de rived CD3+ T cells. PLoS One. 2011;6(12):e28894.  \n15. Nayyar A, Sal eem MI, Yilmaz M , DeFranc o M, Klein G, Elmaliki KM, e t al. M enstru al Effluent \nProvides a Novel Diagnostic Window on t he Pathogen esis of Endometriosis . Fron ti ers in Repr oductive \nHealth . 2020;2(3). \n16. W G McCluggage VPS, P Maxwell. CD10 i s a sensitive and diagnos tically useful \nimmunohistochemical marke r of normal. pdf>.  \n17. Potlog-Nahari C, Feldma n AL, Str at ton P, Koziol DE, Segars J, Me rino M J, et al. CD10 \nimmunohistochemical st aining enhanc es the histo logical det ection of endomet rio sis. Fertil S teril . \n2004;82(1):86-92. \n18. Chen J, Cheung F, Shi R, Zhou H, Lu W, Consortium CHI. PBMC fixa tion and pr oce ssing for \nChromium single-cell RNA seque ncing. J Transl Med. 2018;16(1):198.  \n19. Kang HM, Subramaniam M, Ta rg S, Nguyen M, Maliskova L, McCarthy E, e t al. Mu ltiple xed \ndrople t single-cell RNA-se quencing using natur al genetic vari ation . Na tur e biot ech nology. 2017;36(1):89-\n94. \n20. Hao Y, Hao S, And ersen- Nissen E, Mauck WM, 3rd, Zheng S, Bu tle r A, e t al. In tegr a ted analysis of \nmultimodal single-cell da ta. Cell. 2021;18 4(13):3573-87 e29.  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n31 \n \n21. Hafemeiste r C, Satija R. Normaliz ation a n d variance stabiliz atio n of single-cell RNA -seq data \nusing regularize d negative bin omial regr ession. G enome Bi ol. 2019;20(1):296.  \n22. Korsunsky I, Millard N, F an J, Slowikowski K, Zhang F, Wei K, et al. Fas t, sensi tive a nd accurate \nintegra tion of single-cell dat a with Harm ony. Nat M eth ods. 2019;16(12):1289-96. \n23. Wang F, Qualls AE, Ma rques-Fe rnand ez L, Colucci F. Biology and pathology of the uterin e \nmicroenvironmen t and its na tur al killer c ells. Cell Mol Immunol . 2021;18(9):2101-13. \n24. Queckborne r S, von Gro thusen C, Boggav arapu NR , Francis RM , Davies LC, Gemzel l-Danielsson K. \nStromal He te rogen eity in the Huma n Proliferative Endome trium-A Single-Cell R NA  Sequencing Study. J \nPers Med. 2021;11(6).  \n25. Andre att a M, Corri a-Osorio J, Mull er S, Cubas R, Coukos G, Carmona S J. In ter pre t ation of T cell \nstat es from single-cell transcrip tomics da ta using refer ence a tlases . Nat Commun. 2021;12(1):2965. \n26. Vento-Tormo R, Efremova M, B ot ting RA,  Turco MY, Vento-Tormo M, M eyer KB, e t al. Single-cell \nreconst ruction of th e ea rly matern al-feta l interface in humans . Na tur e. 2018;563(7731):347-53. \n27. Dinh HQ, Lin X, Abbasi F, Nameki R, Har o M, Olingy CE, et al. Single-cell t ranscrip to mics identifies \ngene exp ression n etworks driving differe ntiati on and tumo rigenesis in th e human  fallopian tube . Cell \nRep. 2021;35(2):108978.  \n28. Lee RD, Munro SA , Knutson TP, LaRue RS , Heltem es-Harris LM, Fa rrar M A. Single- cell analysis \nidentifies dynamic gene e xpr ession ne tworks that gover n B cell developm ent and transforma tion . Na t \nCommun. 2021;12(1):6843. \n29. Collin M, McGovern N, Haniffa M. Huma n dendritic cell subs ets. Immunology. 2013;140(1):22-\n30. \n30. Chambers SEJ, Pathak V, Pedrini E, So ret L, Gendro n N, Gu erin CL, et al . Curren t c oncepts on \nendoth elial st em cells definition , locati o n, and markers . Stem Cells Transl M ed. 2 021;10 Suppl 2:S54-\nS61. \n31. Garcia-Al onso L, Handfield LF, Rob erts K, Nikolakopoulo u K, Fernand o RC, Gar dner  L, et al. \nMapping the tempor al and spa tial dyna mics of the human endome trium in vivo and in vitro. Nat Gen et. \n2021;53(12):1698-711.  \n32. Barraga n F, Irwin JC, Bal ayan S, Erikson DW, Chen JC, Houshda ran S, e t al. Human Endometri al \nFibroblasts Der ived from Mesenchymal Progenito rs Inh erit Proges ter one R esistan ce and Acquire an \nInflammatory Pheno type in th e Endomet rial Niche in Endome triosis . Biol Re prod . 2016;94(5):118. \n33. Satt erfield MC, Hayashi K, Song G , Black SG, Baz er FW , Spence r TE. Progeste rone regulat es \nFGF10, MET, I GFBP1, and I GFBP3 in the e ndometri um of the ovine ut erus. Biol Re prod. 2008;79(6):1226-\n36. \n34. Young CH, Snow B, DeVore SB, Mohand a ss A, Nemmara VV, Thompson PR, et al . Progester one \nstimulates his tone ci trullina tion t o increa se IGFBP1 e xpressio n in ute rine cells. R e production . \n2021;162(2):117-27.  \n35. Ujvari D, Jakson I, Babayeva S, S alamon D, Rethi B, Gi dlof S, et al . Dysregulation o f In Vitro \nDecidualizatio n of Human Endometr ial St romal Cells by Insulin via Transcription al Inhibitio n of Forkhead \nBox Prot ein O1. PLoS On e. 2017;12(1):e0 171004. \n36. Fei W, Kijima D, Hashimoto M, Hashimur a M, Oguri Y, Kajita S, e t al. A functional r ole of LEFTY \nduring progest eron e th erapy for end ome trial carcinom a. Cell Commun Signal. 2017;15(1):56. \n37. Takano M, Lu Z, Goto T, Fusi L, Higham J, Francis J, e t al. Tra nscription al cross talk betwee n the \nforkhead tr anscripti on factor forkh ead b ox O1A an d the p rogest eron e rece pto r coordina tes cell cycle \nregulati on and differen tia tion in human endome trial st romal cells. M ol Endocrin ol. 2007;21(10):2334-\n49. \n38. Ono YJ, Ter ai Y, Tanabe A , Hayashi A, Hay ashi M, Yamashita Y, e t al. Decor in induc ed by \nprogest eron e plays a crucial role in supp r essing endomet riosis. J End ocrinol. 2014 ; 223(2):203-16.  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n32 \n \n39. Halari CD, Nandi P, Jeyar ajah MJ , Renaud  SJ, Lala PK. Decorin producti on by the h uman decidua: \nrole in decidu al cell matur ation . Mol Hu m Reprod. 2020;26(10):784-96.  \n40. Tamm-Rosenstein K, Simm J, Suho rutsh e nko M, Salumets A , Me tsis M. Changes in  the \ntranscrip tome of th e human end ometr ial  Ishikawa cancer cell line induc ed by estr ogen, proges ter one, \ntamoxifen , and mifeprist one (RU486) as detec ted by RN A-sequencing. PLoS On e. 2013;8(7):e68907. \n41. Salgado RM FR, , Zorn TMT. M odula tion of small leucine-rich prot eoglycans (SLRP s) expression in \nthe mouse u terus by est radiol a nd proge steron e. R eproduc tive Biology and Endoc rinology. 2011;9(9):22. \n42. LUCARIELLO A TE, BOCCIA O, PERNA A, S ELLITTO C, CASTAL D MA, DE FALCO M,  D E LUCA A,  \nCOBELLIS L. Small Leucine Rich Proteogly cans Are Differently Distribu ted in Norm al and Pathological \nEndometrium . in vivo. 2015(29):217-22.  \n43. San Mar tin S MS-SM, Fe rrei ra S,  de Olive ira F, Aplin F JD, P. Abrahamsohn P , Zorn TMT. Small \nleucine-rich pro teoglycans (SLRPs) in uterine tissues du ring pregnancy in mice. R e production . \n2003(125):585–95. \n44. Do Carmo S, Seguin D, Milne R, Rassar t E. Modula tion of apolipo pro tein D and ap olipopro tein E \nmRNA ex pression by growth a rres t and i dentificati on of key elements in the pro moter . J Biol Chem. \n2002;277(7):5514-23.  \n45. KAO LC TS, LOBO S,  IMAN I B , YAN G JP,  GERMEYER A,  OSTEEN K, RTAYLOR RN , B . A. LESSEY BA, \nGIUDICE LC. Global G ene Profiling in Hu man Endometrium dur ing the Win dow of Implantati on. \nEndocrinology \n143:2119–38.  \n46. Altmae S, Ko el M, Vosa U, Adl er P, Suhor utsenko M, Laisk-Podar T, et a l. Me ta-sig natur e of \nhuman endome trial r ecep tivity: a met a-a nalysis and validation study of tr anscript omic biomarkers. Sci \nRep. 2017;7(1):10077.  \n47. Omar M, Laknaur A, Al-Hendy A , Yang Q. Myometrial p rogest eron e hyper-r espons iveness \nassociated with inc reas ed risk of human uterin e fibroids. B MC Womens Heal th. 2 019;19(1):92. \n48. Rytkonen KT, Erkenbrack EM, Pout anen M, Elo LL, Pavlicev M, Wagner GP. Decidualizati on of \nHuman Endometri al Str omal Fibrobl asts i s a Multiphasic Process Involving Distinct  Transcription al \nPrograms. Repr od Sci. 2019;26(3):323-36. \n49. Giudice LC, Milkowski DA, Lamson G, Rosenfeld RG, Irwin JC. Insuli n-like growth fa ctor binding \nprote ins in human endom etrium : ster oid -dependen t messenge r ribonucl eic acid e xpression a nd pro tein \nsynthesis. J Clin Endocrinol M eta b. 1991; 72(4):779-87.  \n50. Tarantin o S, Verhage H G, Fazl eabas AT . R egulation of insulin-like growth fact or-bi nding protei ns \nin the babo on (Papio anubis) uterus du ri ng early pregnancy. Endocrin ology. 1992;130(4):2354-62. \n51. Jasienska G , Ellison PT, Galbarczyk A, J asi enski M, Kalemba-Drozdz M, Kapisz ewska M, et al . \nApolipopr ot ein E (ApoE) polymorphism is relat ed to differ ences in pot enti al fertili ty in women: a case of \nantagonistic pleiot ropy? Proc Biol Sci. 20 15;282(1803):20142395. \n52. Garcia A J, Tom C, Guemes M , Polanco G, Mayorga ME, We nd K, et al . ERalpha sign aling \nregulat es MMP3 exp ression t o induce Fa sL cleavage and osteoclas t apop tosis. J B one Mine r Res. \n2013;28(2):283-90. \n53. KELLER NR S-RE, EISENBERG E, OSTEEN KG. Progest eron e Exposu re Preven ts Mat r ix \nMetall opro tein ase-3 (MMP-3) Stimulation by Interl eukin-1a in Human Endomet ri al Stromal Cells.  \nThe Journ al of Clinical Endocrinology & Metab olism Printed in US A. 2000;85:11 – 1619. \n54. Yamashita CM, Dolgonos L, Zemans RL, Young SK, Rober tson J, B riones N, et al. M atri x \nmetallop rot einase 3 is a media tor of pul monary fibrosis. Am J Pathol . 2011;179(4):1733-45. \n55. Luddi A, Marr occo C, Governini L, Sempli ci B, Pavone V, Luisi S, et al. Expr ession o f Matrix \nMetall opro tein ases and Their Inhibit ors i n Endometrium: High Levels in Endome tr iotic Lesions. In t J Mol \nSci. 2020;21(8). \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n33 \n \n56. Young CH SB, DeVore SB, Mohand ass A, Nemmara VV,Thompson PR,  Thyagaraja n B, Navra til \nAM, Cherring ton BD. Proges tero ne stimu lates hist one cit rullina tion to incr ease IG FBP1 expression in \nuterin e cells. R eproduc tion 2021;162:11 7-27. \n57. Chen C, Li C, Liu W, Guo F, Kou X, Sun S, et al. Est rogen-induce d FOS-like 1 regula tes matri x \nmetallop rot einase expr ession and the m otility of human endom etri al and decidu al stromal cells . J Biol \nChem. 2020;295(8):2248-58. \n58. LOCKWOOD CJ KG, HAUSKNECHT VA,PAPP,C, SCHATZ F. Matrix Me tallop rot einase and Mat rix \nMetall opro tein ase Inhibi tor Ex pression i n Endometrial S tromal Cells du ring Progestin-Initia ted \nDecidualizatio n and Menstrua tion-R elat e d Proges tin With drawal. End ocrinology. 1998;139:4607–13.  \n59. Singer CF, Marbai x E, Kokorine I, Lemoin e P, Donnez J, Eeckhout Y, et al . Paracrin e stimulati on of \ninters titial coll agenase (MMP-1) in the h uman endome trium by inte rleukin 1alph a and its dual block by \novarian ste roids. Proc N atl Acad Sci U S A . 1997;94(19):10341-5. \n60. Ghosh K, Capell BC. The Sen escence-Ass ociated S ecre tory Phenotyp e: Critical Effector in Skin \nCancer and Aging. J Invest Derma tol. 201 6;136(11):2133-9. \n61. Basisty N, Kale A , Je on OH, Kuehn emann C, Payne T, Rao C, et al. A pro teomic a tla s of \nsenescence-associa ted sec ret omes for a ging biomarker developm ent . PLoS Biol. 2020;18(1):e3000599. \n62. von Rango U, Alfer J , Kertscha nska S, Ke mp B, Muller-N ewen G, H einrich PC, et al . Int erleukin-11 \nexpr ession: its significance in eu topic an d ectopic human implan tati on. Mol Hum Reprod . \n2004;10(11):783-92.  \n63. Ng B, Cook SA, Schafer S. Int erleukin-11 s ignaling underlies fibrosis, p arenchymal dysfunction, \nand chronic inflammation of th e airway. Exp Mol Med . 2020;52(12):1871-8. \n64. Ng B, Dong J, Viswanathan S, W idjaja AA,  Paleja BS, Adami E, et al . Fibrobl ast-spec ific IL11 \nsignaling drives chronic inflammation in murine fibrotic lung diseas e. FA SEB J. 20 20;34(9):11802-15. \n65. Chen H, Chen H, Liang J, Gu X, Zhou J, Xie  C, et al. TGF-be ta1/IL-11/MEK/ERK signa ling mediates \nsenescence-associa ted pulmo nary fibrosi s in a stress-induced pr ematu re sen esce nce model of Bmi-1 \ndeficiency. Exp Mol Me d. 2020;52(1):130-51. \n66. Dimitriadis E, Stoikos C, Stafford-Bell M, Clark I, Paiva P, Kovacs G, et al. In terl euki n-11, IL-11 \nrecep toralp ha and leuk emia inhibit ory factor ar e dysregulat ed in endom etri um of infertile women with \nendome triosis during th e implant atio n window. J Rep rod Immunol . 2006;69(1):53-64. \n67. Gubbels Bup p MR, J orgensen T N, Kotzin BL. Iden tification of candida te gen es tha t  influence sex \nhormone-dep ende nt dise ase phen otype s in mouse lupus. Gen es Immun. 2008;9( 1):47-56. \n68. Schroder WA, Le TT, Major L, Str ee t S, G a rdner J, Lambley E, et al . A physiological function of \ninflammation-associat ed Ser pinB2 is reg ulation of adap tive immunity. J Immunol.  2010;184(5):2663-70.  \n69. Hsieh HH, Chen YC, Jhan JR , Lin JJ. Th e se rine pro teas e inhibit or serpi nB2 binds an d stabilizes \np21 in senescent cells . J Cell Sci. 2017;13 0(19):3272-81.  \n70. Park SR, Lee JW, Kim SK, Yu WJ, Le e SJ, Ki m D, et al. The impact of fine par ticulat e matte r (PM) \non various beneficial functio ns of human endomet rial st em cells through its key r egulato r SERPINB2. E xp \nMol Med. 2021 ;53(12):1850-65. \n71. Zhang X, Christenson LK, Nothnick WB . R egulation of MMP-9 exp ression and activity in the \nmouse ute rus by estrogen . Mol R eprod D ev. 2007;74(3):321-31. \n72. Ahmad N, Chen S, W ang W, Kapila S . 17beta-estradi ol Induces  MMP-9 and MMP- 13 in TMJ \nFibrochondr ocytes via Estrogen R ecep tor  alpha. J Den t Res. 2018;97(9):1023-30.  \n73. MARBA IX E DJCP, EE CKHOUT Y. Progeste rone r egulat es the ac tivity of collagenase  and rela ted \ngelatinas es A and B in human en domet ri al explan ts. Proc N ati Acad Sci US A  \n1992;89:11789-93. \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n34 \n \n74. Steen por t M, Khan KM, Du B, Barn hard S E, Dannenberg A J, Falcon e DJ. Ma tri x \nmetallop rot einase (MMP)-1 and MMP-3 induce macrophage MMP-9: evidence fo r the rol e of TNF-alpha \nand cyclooxygenase-2. J Immunol. 2009; 183(12):8119-27.  \n75. Su L, Dong Y, Wang Y, Wang Y, Guan B, Lu Y, et al. Pot ential role of sen escent ma crophages in \nradiati on-induced pulmon ary fibrosis. Ce ll Death Dis. 2021;12(6):527. \n76. Hong EJ, Park SH, Choi KC, Leung P C, Jeung EB. Identifica tion of est rogen-r egulat e d genes by \nmicroarray analysis of the u ter us of immatur e rats expos ed to e ndocrine dis rupti ng chemicals. Reprod \nBiol Endocrinol . 2006;4:49.  \n77. Ghezz o F, Ber ta GN, B eccaro M, D' Avolio A, Racca S, Conti G , et al. Calcyclin gene expr ession \nmodulation by medr oxyprogeste rone ac eta te. B iochemical pha rmacology. 1997; 54(2):299-305.  \n78. Xia C, Braunstein Z, Toomey AC, Zhong J, Rao X. S100 Proteins As an Impor tant R e gulator of \nMacrophag e Inflammation . Fron t Immun ol. 2017;8:1908.  \n79. Landi C, Bargagli E, Carleo A, R efini RM, B ennet t D, Bianchi L, et al. Br onchoalveol a r lavage \nprote omic analysis in pulmonary fibrosis associated with syst emic sclerosis: S100 A6 and 14-3-3epsilon as \npoten tial bioma rkers. R heuma tology (Ox ford). 2019;58(1):165-78. \n80. Slomnicki LP, Lesniak, W. S100A6 (calcycl in) deficiency induces senescence-like ch anges in cell \ncycle, morphology and functional char ac teris tics of mouse NIH 3T3 fibroblasts . J Cell Biochem. \n2010;109(3):576-84.  \n81. Haim K, Weitz enfeld P, Meshel T, B en-Ba ruch A. Epide rmal growth facto r and est r ogen act by \nindepend ent pa thways to addi tively pro mote th e rel ease of th e angiogenic chem okine CXCL8 b y breast \ntumor cells. Neopl asia. 2011;13(3):230-43. \n82. Armstrong GM, Maybin JA, Mu rray AA , N icol M, Walker C, Saund ers PTK, et al. En dometri al \napoptosis and n eut rophil infilt rati on duri ng menstruat ion e xhibits spa tial and tem poral dynamics tha t \nare rec apitul ate d in a mouse model . Sci Rep. 2017;7(1):17416.  \n83. Russo RC, Garcia CC, Teixei ra MM, Amar al FA. The CXCL8/IL-8 chemokine family and its \nrecep tors in inflammato ry diseases. E xpe rt review of clinical immunology. 2014;1 0(5):593-619.  \n84. KONN O Rl iY-OH, FUJ IWAR A H',  UCHI IDE I,  SHIBAH ARA H , OKwADA M,  IH ARA T,  SUGAI 'vL4TA \nM, SUZUK1 M. Role of immunore actions and mast cells in path ogenesis of human endome triosis -\nmorphologic study and gene exp ression analysis- \n \nVol. 16 No. 3. HU MAN CELL(Hurn Cell). 2003;16:141 - 9. \n85. Luckow Invitti A, Schor E, Mar tins Parr eir a R, Kopelman A, Kame rgorodsky G, Gon calves GA, e t \nal. Inflammat ory cytokine profile of cocul tivated p rimary cells from the en domet ri um of women with \nand without endome triosis. Mol Med R e p. 2018;18(2):1287-96. \n86. Acosta JC, O 'Loghlen A , Bani to A, Guijarr o MV, Augert A, Raguz S, e t al. Chemokin e signaling via \nthe CXCR2 recepto r reinforc es senescenc e. Cell. 2008;133(6):1006-18. \n87. Carleton JB, B err et t KC, Gert z J. Mul tiple x Enhancer I nte rfere nce Reveals Collab or ative Control \nof Gene R egulation by Estr ogen Rece pto r  alpha-Bound Enhanc ers. Cell Syst. 2017; 5(4):333-44 e5.  \n88. Heckmann BL, Zhang X, Xie X, Liu J. The G0/G1 switch gene 2 (G0S2): regulating m etabol ism and \nbeyond. Biochim Biop hys Acta. 2013;183 1(2):276-81.  \n89. Barrad as M, G onos ES, Zebed ee Z, Kolet t as E, Petropoulo u C, Delgado MD, et al . I dentificati on of \na candidat e tumor-supp ressor gen e spec ifically activated during Ras-induced s ene scence. Exp Cell Res . \n2002;273(2):127-37.  \n90. Hu WP, Tay SK, Zhao Y. Endometriosis-specific genes identified by re al-time r ever se \ntranscrip tion-polymer ase chain r eaction expr ession profiling of endome triosis ver sus autologous ut erin e \nendome trium. J Clin Endocrin ol Me tab. 2 006;91(1):228-38. \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n35 \n \n91. Andrad e PM, Silva ID, Borra RC, Lima GR ,  Baracat EC. Estr ogen and sel ective es tro gen recep tor \nmodulato r regula tion of insulin-like growth factor binding pr otei n 5 in the ra t ut er us. Gynecol \nEndocrinol. 2002;16(4):265-70.  \n92. Nguyen X-X, Muhammad L, Niete rt PJ, F e ghali-Bostwick C. IGFBP-5 Promotes Fibr osis via \nIncreasing I ts Own Expr ession and That o f Other Pro-fibro tic Media tors . Fron tiers in Endocrinology. \n2018;9.  \n93. Kim KS, Seu YB, Baek SH, Kim MJ, Kim KJ, Kim JH, et al. Inductio n of cellular sen escence by \ninsulin-like growth factor binding pr otei n -5 through a p53-depend ent mech anism.  Mol Biol Cell. \n2007;18(11):4543-52.  \n94. Sanada F, Taniyama Y, Mur atsu J, O tsu R,  Shimizu H, Rakugi H, et al. IGF Bin ding Protein-5 \nInduces Cell Senesc ence. F ront Endoc rin ol (Lausanne). 2018;9:53.  \n95. Salih DAM TG, Holding C,  Szestak TAM , Gonzal ez MI , Carte rEJ , Cobb LJ, , Eisema nn JE, Pell JM. \nInsulin-like growth factor-bin ding prot ein  5 (Igfbp5)  compromises survival, growth, muscle developmen t, \nand fertili ty in mice. PNAS. 2004;101 :4314–9.  \n96. Sheikh MS, Shao ZM, Chen JC, Fontan a J A. Differential regula tion of matri x Gla p r otein (MGP) \ngene exp ression by re tinoic acid and estr ogen in human bre ast carcinoma cells . M ol Cell Endocrinol. \n1993;92(2):153-60. \n97. Dressman MA. W alz TM LC, Barnes L,  Bu chholtz S, I Kwon, M J Ellis MJ,  Polymero poulos MH. \nGenes that co-clust er with es troge n rece ptor alph a in microarr ay analysis of brea st biopsies. The \nPharmacogenomics Journ al. 2001;1 : 135–41.  \n98. Han L, Li X, Zhang G, Xu Z, Gong D, Lu F, e t al. Perica rdial int ersti tial cell se nescenc e responsibl e \nfor pericardi al struc tural r emodeling in id iopathic and pos tsurgical const rictive pe r icarditis. J Thorac \nCardiovasc Surg. 2017;154(3):966-75 e4. \n99. Kumari R, Ja t P. Mechanisms of Cellular S enescence : Cell Cycle Arrest and Senesc e nce \nAssociated S ecre tory Pheno type. Fr ont C ell Dev Biol. 2021;9:645593.  \n100. Stilley JA , Birt JA, Nagel SC, Sutovsky M, S utovsky P, Sharpe-Timms KL. Neutralizin g TIMP1 \nresto res fecundity in a ra t model of end o metriosis and tre ating cont rol ra ts with TIMP1 causes \nanomalies in ovaria n function and emb ryo developmen t. Biol R epro d. 2010;83(2):185-94. \n101. Wang J, J arr et t J, Huang CC, Satche r RL, J r., Levenson AS . Id entificati on of estr oge n-responsive \ngenes involved in breas t cancer me tast ases to th e bone . Clin Exp Met astasis. 2007 ;24(6):411-22. \n102. Kunzmann S, Ot tensmei er B, Sp eer CP, F ehrholz M . Effect of progeste rone on Smad signaling \nand TGF-bet a/Smad-regula ted genes in l ung epitheli al cells. PLoS One . 2018;13(7):e0200661.  \n103. Thweatt R, Lumpkin CK, Jr., Golds tein S. A novel gene encodi ng a smooth muscle prote in is \noverexp ressed in s enescen t human fibro blasts. Bioch emical and biophysical r esea rch communications. \n1992;187(1):1-7. \n104. Vafashoar F, Mousavizad eh K, Poormogh im H, Haghighi A, Pashangzadeh S, Moj ta bavi N. \nProgester one Aggrava tes Lung Fibrosis in  a Mouse Mod el of Systemic Sclerosis. Fr ont Immunol. \n2021;12:742227.  \n105. Schafer MJ, Whit e TA, Iijima K, Haak AJ , Ligresti G, Atkinson EJ , et al . Cellular se ne scence \nmediates fibro tic pulmona ry disease. Nat  Commun. 2017;8:14532. \n106. Kim TH, Yoo JY, Choi KC, Shin JH, Leach RE, Fazleabas AT , et al . Loss of HDAC3 results in \nnonrecep tive end omet rium and female i nfertility . Sci Transl Med. 2019;11(474).  \n107. DeNardo DG , Kim HT, Hilsenbeck S, Cuba V, Tsimelzon A, Brown PH. Glob al gene e xpression \nanalysis of estrogen r ecep tor t ranscrip tio n factor cross talk in br east cance r: ide nti fication of estrog en-\ninduced/activato r pro tein-1-dep enden t genes. M ol Endocrinol . 2005;19(2):362-78 . \n108. Cao W, Mah K, Carroll RS , Slayden OD, Br enner R M. Progest eron e withdr awal up-r egulates \nfibronectin an d integri ns during menstr u ation and r epai r in the r hesus macaque e ndometri um. Hum \nReprod . 2007;22(12):3223-31. \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n36 \n \n109. Chen G, Liu L, Sun J, Zeng L, Cai H, He Y. Foxf2 and Smad6 co-regula tion of collage n 5A2 \ntranscrip tion is involved in the p athog en esis of intraut erin e adhesi on. J Cell Mol Med. 2020;24(5):2802-\n18. \n110. Chan JM, Ho SH, Tai IT. S ecre ted pr ot ein acidic and rich in cysteine-induced cell ul ar senescenc e \nin colorect al cancers in resp onse t o irino t ecan is mediat ed by P53. Carcinogenesis.  2010;31(5):812-9.  \n111. Urushiyama H, Terasaki Y, N agasaka S, T erasaki M, Kunugi S, N agase T, et al . Rol e  of alpha1 and \nalpha2 chains of type IV collagen in e arly fibrotic lesions of idiopa thic inte rsti tial p neumonias and \nmigration of lung fibroblasts . Lab Invest . 2015;95(8):872-85. \n112. Lee Y, Shivashankar GV. Analysis of trans criptional modul es during human fibrobl ast ageing. Sci \nRep. 2020;10(1):19086.  \n113. Teo YV, Rattan avirotkul N , Olova N , Salza no A, Quint anilla A, Ta rra ts N, e t al. Notc h Signaling \nMedia tes Second ary Senesc ence. Cell R e p. 2019;27(4):997-1007 e5. \n114. Matalli otaki C, Mat allio takis M, Rahmiogl u N, Mavroma tidis G, M atalli otakis I, Kou mantakis G, e t \nal. Role of FN1 a nd GREB1 gen e polymor phisms in endometri osis. Mol M ed Rep . 2019;20(1):111-6. \n115. Klemmt PA, Carver JG , Kennedy SH, Koni nckx PR, Mardon HJ . Str omal cells from e ndometri otic \nlesions and endom etri um from women with endome triosis have r educed d ecidu alizatio n capacity. F ertil \nSteril . 2006;85(3):564-72. \n116. Cicinelli E, Trojano G, Mastromau ro M, V imercati A , Marin accio M, Mit ola PC, et a l. Higher \nprevalence of chron ic endome tritis in wo men with endome triosis: a possible e tio pathogen etic link. F ertil \nSteril . 2017;108(2):289-95 e1. \n117. Sapkota Y, St einth orsdo ttir V, Mo rris AP, Fassbende r A, Rahmioglu N, De Vivo I, et al. Me ta-\nanalysis identifies five novel loci associat ed with endome triosis highlighti ng key genes involved in \nhormone me tabolism. Nat Commun. 201 7;8:15539.  \n118. Tomari H, Kawamura T, Asanoma K, Egas hira K, Kawamura K, Honjo K, et al . Contr ibution of \nsenescence in human endome trial s trom al cells during prolifer ative phas e to emb ryo recep tivitydagger. \nBiol Repr od. 2020;103(1):104-13. \n119. Lin X, Dai Y, Tong X, Xu W, Huang Q, Jin X, et al. E xcessive oxid ative str ess in cumulus granulosa \ncells induced cell senesc ence cont ribut es  to endome triosis-associa ted infe rtili ty. R edox Bio l. \n2020;30:101431.  \n120. Yu CX, Song JH, Li YF, Tuo Y, Zheng JJ, Mi ao RJ, e t al. Cor rela tion be twee n replica ti ve senescence \nof endometr ial gland epi thelia l cells in shedding and non-shedding e ndome tria an d endomet riosis cyst \nduring menstru ation . Gynecol Endoc rinol . 2018;34(11):981-6. \n121. Takebayashi A, Kimura F, Kishi Y, Ishida M, Takahashi A, Yamanak a A, e t al. The a ssociation \nbetwee n endome triosis an d chronic end ometri tis. PLoS One . 2014;9(2):e88354.  \n122. Wu D, Kimura F, Zheng L, Ishida M, Niwa Y, Hirata K, e t al. Chronic e ndome tritis m odifies \ndecidualiza tion in human e ndome trial st r omal cells. Rep rod Biol Endocri nol. 2017; 15(1):16. \n123. Miyagaki T, Fujimoto M, Sato S . Regula to ry B cells in human inflammatory and aut oimmune \ndiseases: from mouse mod els to clinical r esearch . Int Immunol. 2015;27(10):495-504. \n124. Sojka DK, Yang L, Yokoyama WM. Ute rin e Natu ral Killer Cells. Fron t Immunol. 201 9;10:960.  \n125. Zhang Y, Wang Y, Wang XH, Zhou WJ, Jin LP, Li MQ. Crosstalk between human end ometria l \nstromal cells and d ecidual NK cells prom otes decidu aliza tion in vitro by upr egulat ing IL25. Mol Med Rep . \n2018;17(2):2869-78.  \n126. Bany BM, Scot t CA, Eckstrum KS. Analysis  of uterine gen e ex pression in in terl eukin -15 knockout \nmice reveals ut erine n atur al killer cells d o not play a major rol e in decidualiz ation  and associated \nangiogenesis. R eprod uction . 2012;143(3):359-75. \n127. Ashkar AA, Black GP, Wei Q, H e H, Liang L, Head JR, et al . Assessment of r equir ements for IL-15 \nand IFN r egulat ory factors in ut erin e NK cell differentia tion an d function during p regnancy. J Immunol . \n2003;171(6):2937-44.  \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint \n\n37 \n \n128. Haiyuan Liu JHL. Is abno rmal eut opic end ometrium t he cause of end omet riosis? T he role of \neutopic en domet rium in path ogenesis of  endomet riosis.  \n129. Jorgens en H, Fedo rcsak P, Isaacson K, Te vonian E, Xiao A, Best e M, e t al. Endom et rial cytokines \nin patien ts with and withou t end omet rio sis evaluated for infe rtili ty. Fer til Ste ril. 2 022. \n130. Seshadri S, Sunka ra SK. N atur al killer cell s in female infertility and recurr ent misca rriage: a \nsystematic review and me ta-analysis. Hu m Reprod Upda te . 2014;20(3):429-38. \n131. As-Sanie S, Black R, Giudice LC, Gray Valb run T, Gupt a J, J ones B, et al . Assessing r esearch gaps \nand unmet ne eds in endom etri osis. Ame rican journal of obst etrics and gynecolog y. 2019;221(2):86-94.  \n132. Kirkland JL, Tchkonia T. Senolytic drugs: f rom discovery to tr anslati on. J Int ern Me d. \n2020;288(5):518-36.  \n133. Wissler G erdes EO , Zhu Y, Tchkonia T, Kirkland JL. Discovery, developmen t, and fu ture \napplication of sen olytics: th eori es and pr edictions. FEB S J. 2020;287(12):2418-27. \n \n . CC-BY-NC-ND 4.0 International licenseIt is made available under a \n is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)\nThe copyright holder for this preprint this version posted June 7, 2022. ; https://doi.org/10.1101/2022.02.10.22270810doi: medRxiv preprint","source_license":"CC0","license_restricted":false}