Mpox infection of stromal cells and macrophages of macaque with endometriosis

Mpox infection of stromal cells and macrophages of macaque with endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Mpox infection of stromal cells and macrophages of macaque with endometriosis Amanda Martinot, Joshua Hall, Claire Lyons, Jingyi Li, Gisela Martinez-Romero, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4103434/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The mpox outbreak of 2022-2023 represented a new global health challenge and recognition of mpox as a sexually transmitted disease. The majority of cases were reported in men who have sex with men (MSM), but women are also susceptible, especially during pregnancy. We evaluated the reproductive tracts of a subset macaques from a large rechallenge study of mpox infection with virus from the 2022 outbreak and identified intraabdominal mpox replication associated with endometriosis. Mpox virus (MPXV) was found not only in skin, but in the cervix, the uterus, and periovarian endometriotic lesions of the affected macaque. Mpox replication preferentially targeted vimentin-positive poorly differentiated endometriotic stromal tissue and infiltrating macrophages in the reproductive tract. Mpox tropism for stromal cells and macrophages has broad implications for mpox pathogenesis and associated clinical syndromes. In addition, women with endometriosis may be at heightened risk for adverse outcomes associated with mpox infection. The rhesus macaque provides rare insight into this disease and the potential complications of mpox infection in the context of genitourinary tract disease. Health sciences/Pathogenesis Health sciences/Diseases Health sciences/Diseases/Infectious diseases Health sciences/Diseases/Infectious diseases/Viral infection Biological sciences/Microbiology/Pathogens Figures Figure 1 Figure 2 Introduction Mpox (formerly monkeypox) is a zoonotic disease caused by the monkeypox virus (MPXV), member of the Poxviridae family, which includes other viruses of significant public health importance including smallpox and vaccinia viruses 1 . In people, infection causes fever, pustular rashes, and lymphadenopathy, and less commonly pneumonia, encephalitis, ocular lesions, and death 1 . The virus spreads via respiratory droplets, direct contact with mucocutaneous lesions, vertically during pregnancy, and likely via sexual transmission, given high viral loads in seminal fluid and semen 1,2 . While a majority of cases in the 2022 outbreak were reported in MSM, women are equally susceptible to the virus 1,3 . A number of cases in women were identified during pregnancy 4 , and asymptomatically, therefore lesions in women are less well described. Here we present data on viral replication in the reproductive tracts of rhesus macaques experimentally challenged with mpox and report a case of mpox replication associated with endometriosis in a female rhesus macaque. Macaques have similar endometrial physiology to humans 5 and frequently develop spontaneous endometriosis 6,7 with comparable health and fertility sequelae 8 . In both women and macaques, endometriosis is characterized by proliferation of endometrial tissue outside the uterus, causing pain, peritoneal adhesions, and even complete infertility 9 . Endometriosis is estimated to affect up to 10% of reproductive-aged women, but it is likely underdiagnosed due to symptom heterogeneity and requirement for surgical visualization of lesions for definitive diagnosis 9 . Given the possibility of sexual transmission of mpox via semen, the uterus could be a prime target for infection. We previously reported that primary infection with mpox provided robust protection against mpox rechallenge 10 . As part of this rechallenge study, two female rhesus macaques and one male macaque were challenged intravenously with mpox as previously naïve controls, euthanized 10 days following challenge, and necropsied for tissue evaluation. At necropsy, gross lesions in the reproductive tract of a female macaque (Animal 1) were reported as “chocolate cysts” that expanded the uterus with proliferative tissue obscuring the oviducts and ovaries, consistent with endometriosis. No reproductive lesions were noted in the other female macaque (Animal 2) or the male macaque (Animal 3). As previously described, all naïve, mpox infected macaques developed classic vesicular skin lesions 10 . Tissues from the reproductive tracts from mpox infected animals were screened for MPXV by immunohistochemistry (IHC) and the only animal with viral replication in reproductive tissues was the female macaque with endometriosis (Animal 1, Table 1 ). Table 1: Mpox IHC in reproductive tissues as compared to skin and tonsil of rhesus macaques. Skin Tonsil Cervix Uterus Ovary Penis Testis Prostate dpc* Animal 1 +++ + + + +++ N/A N/A N/A 10 Animal 2 +++ + Neg Neg Neg N/A N/A N/A 10 Animal 3 ++ + N/A N/A N/A Neg Neg Neg 10 *dpc=days post challenge Mpox replication targets vimentin-positive stroma and poorly differentiated glandular epithelium in endometriotic lesions. Endometriosis in Animal 1 was best appreciated in evaluation of the ovarian and oviduct tissue ( Fig. 1, Suppl. Fig. 1 ). As compared to normal oviduct and ovarian tissue ( Suppl. Fig. 2 ), histopathologic evaluation of tissue from Animal 1 showed replacement of normal oviduct by numerous endometrial glands (Fig. 1a) within loosely arranged, myxoid or collagenous stroma that expanded the serosa, peri-ovarian tissue and obliterated the oviduct ( Fig. 1a, inset) . Ectopic endometrial glands often contained blood and cellular debris and were lined by low cuboidal to pseudostratified columnar epithelium that was diffusely and strongly positive for cytokeratin and variably positive for vimentin by IHC ( Suppl. Fig. 2). These features are consistent with mixed differentiation endometriosis in rhesus macaques 11 . Multiple foci within stromal regions of poorly differentiated endometriosis had extensive necrosis ( Fig. 1b ), with mixed inflammatory infiltrates of macrophages, lymphocytes, and plasma cells, with foci of neutrophils and necrotic cellular debris, and prominent intranuclear inclusions consistent with MPXV ( Fig. 1b, inset ). Both glands and stroma in regions of endometriosis were diffusely immunoreactive for CD10 ( Fig. 1c ), a diagnostic marker for human endometriosis 12 . Mpox immunoreactivity was observed in both glands ( Suppl. Fig. 3 ) and stroma in regions of poorly differentiated endometriosis by IHC ( Fig. 1d, inset;e ) and correspond to regions with extensive necrosis and mixed inflammatory infiltrates ( Fig. 1a,b ). Within the stroma immunoreactive for mpox were numerous smooth muscle actin (SMA)-positive vessels ( Fig. 1f ), extensive vimentin-positive areas ( Fig. 1g ), many CD68-positive macrophages ( Fig. 1h ), and few cytokeratin-positive cells ( Fig. 1i ). Mpox replication in then endocervix is associated with intraepithelial macrophages. Additional reproductive tract tissues including cervix, uterus, and ovary from both mpox challenged female macaques and prostate and testis from the male mpox challenged macaque were screened by IHC for MPXV. Animal 1 also had evidence of mpox virus in the endocervix ( Fig.2a ). No evidence of virus was detected via IHC or ISH in the evaluated tissues of the reproductive organs of Animal 2 (non-endometriosis female) or in the testis and prostate from the male macaque ( Table 1 ). Mpox infection of the endocervix was confirmed by in situ hybridization for vRNA ( Fig. 2b,c ). Epithelial cells of the cervical glands were focally strongly positive for mpox vRNA, while cervical stroma had rare positivity. Mpox vRNA positive signal corresponded to vimentin immunoreactivity in cervical stroma ( Fig. 2d ). Mpox vRNA signal in the endocervical glands corresponded to positivity for CD68 (macrophages) by IHC ( Fig. 2e ) suggesting that mpox replication in the cervix was associated with focal cervicitis in Animal 1. To further characterize mpox infected cells in endometriosis, we performed cyclic tissue immunofluorescence (CyCIF) using a targeted panel of pan-cytokeratin, vimentin, and SMA, followed by ISH for MPXV RNA and cytokeratin 10 (CK10) ( Fig. 2f-h ). Infected cervical epithelial cells were pan-cytokeratin positive while infected cervical stroma was positive for vimentin ( Fig. 2g,h ). Squamous epithelium of the vagina (CK10) was negative for mpox ( Fig. 2b,f ). MPXV can productively infect and replicate within diverse cell types, including oral and respiratory epithelium and antigen-presenting immune cells, including monocytes, macrophages, B cells, and dendritic cells 2 . Early studies of the pathogenesis of high dose aerosolized mpox Zaire (Mpox-Z) in cynomolgus macaques highlighted a role for the mononuclear phagocyte system in virus distribution and reported that mpox infection was associated with ovarian, uterine, and testicular inflammation 13 . Endometriosis lesions include multiple components of normal uterine tissue, including glandular epithelium and stroma. In Animal 1, regions of glandular epithelium within endometriotic tissue stained strongly for both cytokeratin and vimentin consistent with a de-differentiated state. These same regions were intensely positive for mpox viral protein. Given the strong predilection of mpox for squamous epithelium, it is perhaps not surprising that the endometriotic glandular epithelium would be permissive to mpox replication. More interesting was the prominent mpox replication in the stromal portions of the poorly differentiated endometriosis that were extensively positive for vimentin. It is well-known that Vaccinia virus, a prototypical poxvirus, associates with vimentin intermediate filaments during assembly 14 . Indeed, we previously reported mpox replication in muscle and adipocytes underlying skin lesions during primary infection with mpox highlighting broad cell tropism for mpox including mesenchymal tissues 10 . Notably, endometriotic stroma and endocervical glands that were immunopositive for mpox also contained numerous CD68 positive macrophages. Tissue resident antigen-presenting cells such as dendritic cells and macrophages are well-described in the genitourinary tract of people, but their role in the pathogenesis of many sexually transmitted diseases, including mpox, is understudied 15 . MPXV replication has been shown in human macrophage cell lines 16 and mpox replication in Kupffer cells of the liver in cynomolgus macaques has been reported 13 . Mpox replication in Hofbauer cells, fetal macrophages of the placenta, was reported in a case from a previous outbreak in the Democratic Republic of Congo 17 . Recently, macrophages were proposed as one potential source of mpox infection in the brain 18 . Our finding of macrophage associated mpox replication in reproductive tissues from macaques infected with mpox from the 2022 outbreak, supports the potential role of macrophages in the pathogenesis of other mpox associated clinical syndromes, including neurological disease. Our previous work showed that viral loads in blood peak at 10 days following infection and typically clear by 28 days irrespective of challenge route 10 . Further study on tissue distribution and persistence of MPVX in tissues as compared to plasma would inform whether macrophages or other cell types contribute to prolonged tissue viremia in certain clinical settings. To date, reports of mpox lesions in the reproductive tract of women have been limited to descriptions of vulvar epithelial lesions and a single report of mpox lesions on the external cervical os in women, composed of squamous epithelium 19 , however, assessing uterine tissue in human patients requires invasive methods that are rarely performed. Two prior studies of cynomolgus macaques ( Macaca fascicularis ) experimentally infected by aerosolized MPXV reported superficial lesions in the vagina and uterus, with rare necrotic foci in uterine stroma and myometrium 13,20 . To our knowledge, there are no reports of mpox in endometrial tissue in women or in women affected by endometriosis. This report of MPXV in the endocervix as well as poorly differentiated endometriotic tissue extending to the ovary and fallopian tubes in a mpox infected rhesus macaques suggests that women with endometriosis may be susceptible to similar lesions. Given up to 40% of mpox infected individuals are also HIV positive, associated immune dysfunction may heighten the risk of developing serious sequelae with viral spread 21 . Here we report of a case of mpox replication in endometriotic tissue of an experimentally infected rhesus macaque. This finding highlights the potential of MPXV to replicate in reproductive tissues, including ectopic endometrial tissue of women. Further study of the prevalence of mpox in women with endometriosis and the pathogenesis of mpox infection in non-epithelial tissues is warranted. It is important for clinicians to be aware of the potential for mpox to spread intraabdominally in high-risk or mpox-exposed women with endometriosis. Declarations Author Contribution J.M.H performed the pathology experiments and wrote the first draft of the manuscript. T. H. and J.L. prepared the tissues. C.E.L, J.L., G.M-R, A.J.M. reviewed the histopathology and edited the manuscript. A.C.collected tissue samples and performed gross pathology assessments. D.H.B. and A.J.M designed the study. All authors reviewed the manuscript. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request. References Hatami, H. et al. Demographic, Epidemiologic, and Clinical Characteristics of Human Monkeypox Disease Pre- and Post-2022 Outbreaks: A Systematic Review and Meta-Analysis. Biomedicines 11 , 957 (2023). Lum, F.-M. et al. Monkeypox: disease epidemiology, host immunity and clinical interventions. Nat. Rev. Immunol. 22 , 597–613 (2022). Dashraath, P. et al. Monkeypox in pregnancy: virology, clinical presentation, and obstetric management. Am. J. Obstet. Gynecol. 227 , 849-861.e7 (2022). Clemente, N. S. et al. Paediatric, maternal, and congenital mpox: a systematic review and meta-analysis. Lancet Glob. Heal. (2024) doi:10.1016/s2214-109x(23)00607-1. Kirejczyk, S. et al. Urogenital Lesions in Nonhuman Primates at 2 National Primate Research Centers. Vet. Pathol. 58 , 147–160 (2021). Atkins, H. M. et al. Decidualization of Endometriosis in Macaques. Vet. Pathol. 53 , 1252–1258 (2016). Chaffee, B. K. et al. Spontaneous Reproductive Tract Lesions in Aged Captive Chimpanzees. Vet Pathol JO - 53 , 425–435 (2016). D’Hooghe, T. M. et al. Nonhuman Primate Models for Translational Research in Endometriosis. Reprod. Sci. 16 , 152–161 (2009). Zondervan, K. T., Becker, C. M. & Missmer, S. A. Endometriosis. N. Engl. J. Med. 382 , 1244–1256 (2020). Aid, M. et al. Mpox infection protects against re-challenge in rhesus macaques. Cell 186 , 4652-4661.e13 (2023). Gruber-Dujardin, E., Bleyer, M. & Mätz-Rensing, K. Morphological and immunohistochemical characterization of spontaneous endometriosis in rhesus macaques (Macaca mulatta). Primate Biol. 4 , 77–91 (2017). Sumathi, V. P. & McCluggage, W. G. CD10 is useful in demonstrating endometrial stroma at ectopic sites and in confirming a diagnosis of endometriosis. J. Clin. Pathol. 55 , 391–392 (2002). Zaucha, G. M., Jahrling, P. B., Geisbert, T. W., Swearengen, J. R. & Hensley, L. The Pathology of Experimental Aerosolized Monkeypox Virus Infection in Cynomolgus Monkeys (Macaca fascicularis). Lab Invest 81 , 1581–1600 (2001). Risco, C. et al. Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly. J. Virol. 76 , 1839–1855 (2002). Iijima, N., Thompson, J. M. & Iwasaki, A. Dendritic cells and macrophages in the genitourinary tract. Mucosal Immunol. 1 , 451–459 (2008). Davies, M. L. et al. A systemic macrophage response is required to contain a peripheral poxvirus infection. PLoS Pathog. 13 , e1006435 (2017). Pittman, P. R. et al. Clinical characterization and placental pathology of mpox infection in hospitalized patients in the Democratic Republic of the Congo. PLOS Neglected Trop. Dis. 17 , e0010384 (2023). Sepehrinezhad, A., Ahmadabad, R. A. & Sahab-Negah, S. Monkeypox virus from neurological complications to neuroinvasive properties: current status and future perspectives. J. Neurol. 270 , 101–108 (2023). Ramírez, M. et al. Mpox (Monkeypox) Presenting as Cervical and Vulvar Disease. Obstet. Gynecol. 141 , 613–617 (2023). Nalca, A. et al. Experimental Infection of Cynomolgus Macaques (Macaca fascicularis) with Aerosolized Monkeypox Virus. PLoS ONE 5 , e12880 (2010). Mitjà, O. et al. Mpox in people with advanced HIV infection: a global case series. Lancet 401 , 939–949 (2023). Lin, J.-R., Fallahi-Sichani, M., Chen, J. Y. & Sorger, P. K. Cyclic Immunofluorescence (CycIF), A Highly Multiplexed Method for Single‐cell Imaging. Current Protocols in Chemical Biology 8 , 251–264 (2016). Methods Animal Infections Animal studies were described in 10 . Briefly, 3 rhesus macaques ( Macaca mulatta ) - two female, one male – were inoculated intravenously with mpox (MPXV/USA/MA001/2022; lineage B.1, clade 2b; BEI NR-58622; 10 6 TCID50 (10 8 plaque-forming unit [PFU]); intravenous). On day 10 post-infection, a full necropsy with description of gross lesions and collection of major organs was performed. All animal studies were approved by the institutional animal care and use committee of Bioqual, Inc. in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals; The Guide for the Care and Use of Laboratory Animals; the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training; and ARRIVE guidelines. Histopathology Tissues were fixed in 4% paraformaldehyde for 24 hours, transferred to 70% ethanol, and paraffin embedded and blocks sectioned at 5 mm for routine hematoxylin and eosin staining (H&E) and for IHC and ISH. Tissue pathology was independently assessed by three veterinary pathologists (AJM, GMR, CEL). Immunohistochemistry Immunohistochemical staining was performed on selected formalin-fixed, paraffin-embedded sections of reproductive tissues using standard techniques. The slides were baked for 30 minutes at 60ºC, deparaffinized through xylene, 100% ethanol, 95% ethanol, and 1x tris-buffered saline (TBS). Heat-induced epitope retrieval was performed with a 10% citrate buffer (Sigma-Aldrich, C9999) for all antibodies. To detect mpox, primary mouse anti-Vaccinia antibody (Santa Cruz, SC-58210) was applied at 1:100 followed by mouse Mach-2 HRP-Polymer (Biocare MHRP520) for 30 min, then Nova-Red (Vector, SK4800) for 10 min, and counterstained with hematoxylin followed by bluing using 0.25% ammonia water. For CD10 (1:4000; SinoBiological, 90177-C07H), primary antibody was applied for 60 min by rabbit Mach-2 HRP-Polymer (Biocare, RHRP520) for 30 min, then 3,3’-diaminobenzidine (DAB,Cell Marque 957D-30). The slides were counterstained using 50% hematoxylin solution (BioCare CATHE-MM). Vaccinia and CD10 IHC was performed using a Biocare intelliPATH autostainer. For cytokeratin (1:140, clone AE1/AE3, Dako M3515), CD68 (1:410, clone KP1, Dako M0814), alpha smooth muscle actin (1:1000; clone 1A4, Dako M0851), and vimentin (1:162; clone 3B4, ProGen, 61013) primary antibodies were diluted in Da Vinci Green Diluent (BioCare PD900M) and incubated for 30 min at room temperature, slides were then washed and treated with biotinylated horse anti-mouse secondary antibody (Vector Labs BA-2001, diluted 1:200 in Da Vinci Green) for 30 minutes at room temperature. Elite Avidin-Biotin Complex solution (Vector Labs, PK-6100) was applied and incubated for 30 minutes, followed by DAB solution. Matched negative control slides were incubated with Universal Negative Control Serum (BioCare, NC498L). All slides were counterstained using 50% hematoxylin solution (BioCare, CATHE-MM) and were scanned at 20x using a Midi II Scanner (3DHistotech) on default brightfield settings. In situ Hybridization Duplex chromogenic in situ hybridization was performed using the RNAscope 2.5 HD Duplex Detection Kit (ACD Bio 322500) with customized and general probes (1260381-C1, Mmu-KRT10-C1), 1226271-C2 (V-MPXV-OPG124-C2), 461341 (Mmu-POLR2a), 457711-C2 (Mmu-PPIB-C2), 2-plex negative control (320751) following recommended guidelines (Protocol 322500-QCK Rev B) as previously described 10 . Slides were then scanned at 20x using a Midi II Panoramic Scanner (3D Histotech) on default brightfield settings. Cyclic ISH and Fluorescence Microscopy Cyclic dual RNAscope ISH and immunofluorescence staining was also performed on selected cervical tissues from Animal 1 (T451F). Baking, deparaffinization, and rehydration were performed in the same manner as with IHC, with the substitution of 1x phosphate-buffered saline with 0.2% fish skin gelatin (FSG, Aurion 900.033) in place of 1x TBS. Heat-induced epitope retrieval was achieved using the same method as above. The slides were then washed with 1x PBS/FSG twice for 5 minutes. A protein block was performed using Intercept Blocking Buffer (LiCor 927-70001) for 30 minutes. A photochemical bleaching solution (3% H2O2 in 1xPBS with 20mM NaOH) 22 was used prior to antibody application to decrease autofluorescence and between cycles of fluorescence staining. For each application slides were photo bleached for 60 minutes at room temp while illuminated from above and below with LED light panels (Miroco MI-CL008) on full power. Slides were washed twice with 1x PBS/FSG for 5 minutes. In a lightproof humidity chamber, the slides were protein blocked with Intercept for 30 minutes. For cycle 1, primary antibodies cytokeratin AE1/AE3 (1:140) and SMA (1:100) with Hoechst 1:10,000). For cycle 2, primary antibody vimentin 1:50 with Hoechst 1:10,000 incubated overnight at 4ºC. For both cycles, secondary antibodies against mouse IgG1 conjugated with AlexaFluor 647 (1:1,000, Jackson Immunoresearch 115-605-205) and mouse IgG2a conjugated with AlexaFlour 488 (1:1,000, JIR 115-545-206) were applied and incubated sequentially at room temperature for 30 minutes each. All primary and secondary antibodies were diluted in Intercept. After each cycle slides were coverslipped with 80% glycerol solution and scanned at 20x using a Midi II Scanner in fluorescence mode. Between cycles coverslips were removed by soaking in 1xPBS/FSG for approximately 10 minutes. For cycle 3, dual-plex RNAscope was performed as listed above followed by detection with the Multiplex-Fluorescent reagent kit V2 (323100) as recommended. Slide Analysis HALO (v3.6, Indica Labs) was used to fuse serially obtained fluorescence scans with the HALO registration module within the HiPlex FL Module (v4.2). The fused image underwent thresholding for each fluorescent color based on isotype control slides and according to chromogenic IHC identification of staining pattern and intensity. Additional Declarations No competing interests reported. Supplementary Files 240228Suppl.Fig.1EndometriosisFeatures.pdf Suppl. Figure 1: Endometriosis in periovarian tissue in a rhesus macaque. H&E staining from Animal 1 showing peri-ovarian tissue is expanded by myxoid and collagenous tissue and endometrial glands, with b) numerous hemosiderin-laden macrophages (inset, asterisks), lymphoplasmacytic inflammation (arrowhead), and neutrophils (inset, arrows). c) Endometrial glands within the periovarian tissue are supported by a highly cellular stroma with variable amounts of hemorrhage. d, Low magnification view of endometriotic tissues extending from the serosal surface of the oviduct showing regions of necrosis (#), endometrial glands in poorly differentiated regions of endometriosis (asterisks), and well-differentiated endometrial glands (arrow). e) Region of necrosis (inset) within endometriotic tissue rimmed by hemosiderin laden macrophages (arrow) . f) Higher magnification of endometriotic stroma (f, inset) showing extensive necrosis admixed with lymphocytes and plasma cells and necrotic cellular debris (g) with poxviral inclusion (arrow); see also Figure 1. 240228Suppl.Fig.2NormalRepro.pdf Suppl. Figure 2: Normal rhesus macaque ovary, uterus, and cervix. H&E staining from Animal 2 showing a) cross-section of ovary (left) with follicles in various stages of development and oviduct (right) with branching folds lined by columnar epithelium (inset), b) cross-section of uterus in the proliferative phase of the reproductive cycle. Endometrial glands are lined by columnar cells with basilar nuclei and prominent nucleoli (inset), c) section of cervix with endocervical glands lined with columnar epithelium and a moderate amount of thin luminal mucus (inset). 240228Suppl.Fig.3EndometriosisIHCv2.pdf Suppl. Figure 3: IHC characterization of mixed endometriosis in a rhesus macaque. Periovarian glandular and stromal endometriosis in a rhesus macaque (Animal 1) showing endometriotic glands in regions of poor differentiation (inset) that are strongly positive for a) CD10, b) Mpox, c) smooth muscle actin (SMA), and d) pan-cytokeratin (CK A/E). Epithelial cells of the endometrial glands in regions of poor differentiation (inset, solid) are strongly positive for vimentin (e) while in regions of well-differentiated endometriosis (inset, dotted), glandular epithelium is negative for vimentin (f). Cite Share Download PDF Status: Published Journal Publication published 20 Sep, 2024 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 24 May, 2024 Reviews received at journal 22 May, 2024 Reviews received at journal 08 May, 2024 Reviewers agreed at journal 08 May, 2024 Reviewers agreed at journal 02 May, 2024 Reviewers invited by journal 13 Apr, 2024 Editor assigned by journal 09 Apr, 2024 Editor invited by journal 27 Mar, 2024 Submission checks completed at journal 27 Mar, 2024 First submitted to journal 14 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4103434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":285679611,"identity":"b30ed406-7a8f-4068-b6c7-42a93d21913b","order_by":0,"name":"Amanda Martinot","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie3NP0vDQBzG8ScE2uWKa0pofQsXAnUp+lZ6BJrFoCBIBsGTQl1CXSv1z1uoi6spB06Hbi5ZKgWnLqVQMoiaw2xy6epwX7j7wXGfO8Bk+ofV1EYAxouZFqsNWHN1aHEN2SnJCYetCPUJbI9XkSb/JeeKFFGWbCP0TXzMl2eIriavM3Ecf4ZPTlB/z9FtTVMNyfp73s0zovFLADGWNEqcwBoQ9H096XVcUkPEpQ3RGCpyNBsAgunIQRZuXPIFdl+SkBS/XOT41hKaHXbcxhBsWpKeIpwgrSKnzduRwx6kTQWRvpfIhTUhNPCvtSR8dJabLruT1mJN4vZu/TLAKo/3WyMNKXP+PFV53WQymUxb+gEMHGE8vd9vhAAAAABJRU5ErkJggg==","orcid":"","institution":"Tufts University","correspondingAuthor":true,"prefix":"","firstName":"Amanda","middleName":"","lastName":"Martinot","suffix":""},{"id":285679614,"identity":"481b2f72-67d5-4c23-9cdb-d7e401a48a25","order_by":1,"name":"Joshua Hall","email":"","orcid":"","institution":"Tufts University","correspondingAuthor":false,"prefix":"","firstName":"Joshua","middleName":"","lastName":"Hall","suffix":""},{"id":285679616,"identity":"0d5e8965-bdbb-4304-85d0-7298a00d86c9","order_by":2,"name":"Claire Lyons","email":"","orcid":"","institution":"Tufts University","correspondingAuthor":false,"prefix":"","firstName":"Claire","middleName":"","lastName":"Lyons","suffix":""},{"id":285679617,"identity":"06525861-659c-4b46-93b3-4ac0c6858aef","order_by":3,"name":"Jingyi Li","email":"","orcid":"","institution":"Tufts University","correspondingAuthor":false,"prefix":"","firstName":"Jingyi","middleName":"","lastName":"Li","suffix":""},{"id":285679618,"identity":"05957b8d-8adb-4365-964f-eab30b3ed74c","order_by":4,"name":"Gisela Martinez-Romero","email":"","orcid":"","institution":"Tufts University","correspondingAuthor":false,"prefix":"","firstName":"Gisela","middleName":"","lastName":"Martinez-Romero","suffix":""},{"id":285679619,"identity":"263e5658-b382-4c3f-ad16-c22c064f0766","order_by":5,"name":"Tammy Hayes","email":"","orcid":"","institution":"Tufts University","correspondingAuthor":false,"prefix":"","firstName":"Tammy","middleName":"","lastName":"Hayes","suffix":""},{"id":285679620,"identity":"3f19e6fd-9313-48f4-a533-8ed40ee98026","order_by":6,"name":"Anthony Cook","email":"","orcid":"","institution":"BIOQUAL Inc.","correspondingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"","lastName":"Cook","suffix":""},{"id":285679621,"identity":"d4c82007-6b1e-4425-b687-8ef5b79f6851","order_by":7,"name":"Dan Barouch","email":"","orcid":"","institution":"Beth Israel Deaconess Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Dan","middleName":"","lastName":"Barouch","suffix":""}],"badges":[],"createdAt":"2024-03-14 21:29:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4103434/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4103434/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-024-73012-8","type":"published","date":"2024-09-20T15:57:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53885931,"identity":"b6bc3d15-6c08-4a99-85d9-307819a4a271","added_by":"auto","created_at":"2024-04-01 19:16:18","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2066468,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMpox replication in periovarian glandular and stromal endometriosis in a rhesus macaque. \u003c/strong\u003ea) H\u0026amp;E staining of ovary and oviduct from mpox-infected rhesus macaque with endometriosis (Animal 1), b) higher magnification of inset (a) showing necrosis admixed with macrophages and rare neutrophils with poxviral inclusion (inset, arrow). Serial sections from (a) showing IHC for c) CD10 and d) mpox showing mpox in endometrial glands and stroma (box). High magnification of IHC signal in stroma (d, box) for mpox (e) smooth muscle actin (SMA) (f), vimentin (g), CD68 (h), and pan-cytokeratin (CK A/E, i) showing strong positivity for vimentin and CD68 in regions of stromal mpox immunoreactivity. IHC signal= brown or red.\u003c/p\u003e","description":"","filename":"240228Fig.1OviductEndometriosis.png","url":"https://assets-eu.researchsquare.com/files/rs-4103434/v1/0383019b319782271374b079.png"},{"id":53885933,"identity":"e01b48e8-4d39-409a-9ba7-6644bf20ee98","added_by":"auto","created_at":"2024-04-01 19:16:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1566153,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIHC and ISH shows mpox replication in endocervical gland macrophages and stroma of the rhesus macaque endocervix. \u003c/strong\u003eSerial sections of cervix from mpox-infected rhesus macaque with endometriosis (Animal 1) showing a) endocervical glands and stroma (H\u0026amp;E) positive for b,c) mpox vRNA by ISH (red), CK10 (teal), d) vimentin positive stroma, e) and CD68 positive cervical glands by IHC (brown). f-h) Cyclic IHC and ISH on cervix (IF: DNA=blue, vimentin=green; SMA= yellow; CK A/E, white; ISH: DNA=blue CK10=teal, mpox=red).\u003c/p\u003e","description":"","filename":"240223Fig.2CervixCyCIF.png","url":"https://assets-eu.researchsquare.com/files/rs-4103434/v1/2b444a5a02929582c993b1ca.png"},{"id":65431802,"identity":"6adb3211-58ae-4cdd-a1c2-7e56ef09e334","added_by":"auto","created_at":"2024-09-27 11:59:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4388400,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4103434/v1/3d341551-8bbc-40e0-9a56-9ad25c23a286.pdf"},{"id":53885936,"identity":"3b77dbed-f1e6-4188-b069-33172a626e7f","added_by":"auto","created_at":"2024-04-01 19:16:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1713647,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppl. Figure 1\u003c/strong\u003e: \u003cstrong\u003eEndometriosis in periovarian tissue in a rhesus macaque.\u003c/strong\u003e H\u0026amp;E staining from Animal 1 showing peri-ovarian tissue is expanded by myxoid and collagenous tissue and endometrial glands, with b) numerous hemosiderin-laden macrophages (inset, asterisks), lymphoplasmacytic inflammation (arrowhead), and neutrophils (inset, arrows). c) Endometrial glands within the periovarian tissue are supported by a highly cellular stroma with variable amounts of hemorrhage. d, Low magnification view of endometriotic tissues extending from the serosal surface of the oviduct showing regions of necrosis (#), endometrial glands in poorly differentiated regions of endometriosis (asterisks), and well-differentiated endometrial glands (arrow). e) Region of necrosis (inset) within endometriotic tissue rimmed by hemosiderin laden macrophages (arrow) . f) Higher magnification of endometriotic stroma (f, inset) showing extensive necrosis admixed with lymphocytes and plasma cells and necrotic cellular debris (g) with poxviral inclusion (arrow); see also Figure 1.\u003c/p\u003e","description":"","filename":"240228Suppl.Fig.1EndometriosisFeatures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4103434/v1/560c038e6f5cad82910e4609.pdf"},{"id":53885932,"identity":"2105ba49-6c97-4df3-aa88-6a7dc5bd7966","added_by":"auto","created_at":"2024-04-01 19:16:18","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":737262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppl. Figure 2:\u003c/strong\u003e \u003cstrong\u003eNormal rhesus macaque ovary, uterus, and cervix.\u003c/strong\u003e H\u0026amp;E staining from Animal 2 showing a) cross-section of ovary (left) with follicles in various stages of development and oviduct (right) with branching folds lined by columnar epithelium (inset), b) cross-section of uterus in the proliferative phase of the reproductive cycle. Endometrial glands are lined by columnar cells with basilar nuclei and prominent nucleoli (inset), c) section of cervix with endocervical glands lined with columnar epithelium and a moderate amount of thin luminal mucus (inset).\u003c/p\u003e","description":"","filename":"240228Suppl.Fig.2NormalRepro.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4103434/v1/ff8b018ed575b9d22206ce6d.pdf"},{"id":53885937,"identity":"e03db484-cdee-4682-a346-d57c68fa6925","added_by":"auto","created_at":"2024-04-01 19:16:20","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":17736349,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuppl. Figure 3:\u003c/strong\u003e \u003cstrong\u003eIHC characterization of mixed endometriosis in a rhesus macaque\u003c/strong\u003e. Periovarian glandular and stromal endometriosis in a rhesus macaque (Animal 1) showing endometriotic glands in regions of poor differentiation (inset) that are strongly positive for a) CD10, b) Mpox, c) smooth muscle actin (SMA), and d) pan-cytokeratin (CK A/E). Epithelial cells of the endometrial glands in regions of poor differentiation (inset, solid) are strongly positive for vimentin (e) while in regions of well-differentiated endometriosis (inset, dotted), glandular epithelium is negative for vimentin (f).\u003c/p\u003e","description":"","filename":"240228Suppl.Fig.3EndometriosisIHCv2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4103434/v1/be52e89bf467fbb26377b56d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mpox infection of stromal cells and macrophages of macaque with endometriosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMpox (formerly monkeypox) is a zoonotic disease caused by the monkeypox virus (MPXV), member of the \u003cem\u003ePoxviridae\u0026nbsp;\u003c/em\u003efamily, which includes other viruses of significant public health importance including smallpox and vaccinia viruses\u003csup\u003e1\u003c/sup\u003e. In people, infection causes fever, pustular rashes, and lymphadenopathy, and less commonly pneumonia, encephalitis, ocular lesions, and death\u003csup\u003e1\u003c/sup\u003e. The virus spreads via respiratory droplets, direct contact with mucocutaneous lesions, vertically during pregnancy, and likely via sexual transmission, given high viral loads in seminal fluid and semen\u003csup\u003e1,2\u003c/sup\u003e. \u0026nbsp;While a majority of cases in the 2022 outbreak were reported in MSM, women are equally susceptible to the virus\u003csup\u003e1,3\u003c/sup\u003e. A number of cases in women were identified during pregnancy\u003csup\u003e4\u003c/sup\u003e, and asymptomatically, therefore lesions in women are less well described. Here we present data on viral replication in the reproductive tracts of rhesus macaques experimentally challenged with mpox and report a case of mpox replication associated with endometriosis in a female rhesus macaque.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMacaques have similar endometrial physiology to humans\u003csup\u003e5\u003c/sup\u003e and frequently develop spontaneous endometriosis \u003csup\u003e6,7\u003c/sup\u003e with comparable health and fertility sequelae\u003csup\u003e8\u003c/sup\u003e. In both women and macaques, endometriosis is characterized by proliferation of endometrial tissue outside the uterus, causing pain, peritoneal adhesions, and even complete infertility\u003csup\u003e9\u003c/sup\u003e. Endometriosis is estimated to affect up to 10% of reproductive-aged women, but it is likely underdiagnosed due to symptom heterogeneity and requirement for surgical visualization of lesions for definitive diagnosis\u003csup\u003e9\u003c/sup\u003e. Given the possibility of sexual transmission of mpox via semen, the uterus could be a prime target for infection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe previously reported that primary infection with mpox provided robust protection against mpox rechallenge\u003csup\u003e10\u003c/sup\u003e. \u0026nbsp;As part of this rechallenge study, two female rhesus macaques and one male macaque were challenged intravenously with mpox as previously naïve controls, euthanized 10 days following challenge, and necropsied for tissue evaluation. At necropsy, gross lesions in the reproductive tract of a female macaque (Animal 1) were reported as “chocolate cysts” that expanded the uterus with proliferative tissue obscuring the oviducts and ovaries, consistent with endometriosis. No reproductive lesions were noted in the other female macaque (Animal 2) or the male macaque (Animal 3). As previously described, all naïve, mpox infected macaques developed classic vesicular skin lesions\u003csup\u003e10\u003c/sup\u003e. \u0026nbsp;Tissues from the reproductive tracts from mpox infected animals were screened for MPXV by immunohistochemistry (IHC) and the only animal with viral replication in reproductive tissues was the female macaque with endometriosis\u003cstrong\u003e\u0026nbsp;(Animal 1, Table 1\u003c/strong\u003e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1: Mpox IHC in reproductive tissues as compared to skin and tonsil of rhesus macaques.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eSkin\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTonsil\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eCervix\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eUterus\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eOvary\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003ePenis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eTestis\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eProstate\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003edpc*\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnimal 1\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnimal 2\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+++\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e\u003cstrong\u003eAnimal 3\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e++\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eN/A\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003eNeg\u003c/p\u003e\n \u003c/td\u003e\u003ctd valign=\"bottom\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003e*dpc=days post challenge\u003c/strong\u003e\u003c/p\u003e\n\n\n\n\n\n"},{"header":"Mpox replication targets vimentin-positive stroma and poorly differentiated glandular epithelium in endometriotic lesions.","content":"\u003cp\u003eEndometriosis in Animal 1 was best appreciated in evaluation of the ovarian and oviduct tissue (\u003cstrong\u003eFig. 1, Suppl. Fig. 1\u003c/strong\u003e). As compared to normal oviduct and ovarian tissue (\u003cstrong\u003eSuppl. Fig. 2\u003c/strong\u003e), histopathologic evaluation of tissue from Animal 1 showed replacement of normal oviduct by numerous endometrial glands \u003cstrong\u003e(Fig. 1a)\u003c/strong\u003e within loosely arranged, myxoid or collagenous stroma that expanded the serosa, peri-ovarian tissue and obliterated the oviduct \u003cstrong\u003e(\u003c/strong\u003e\u003cstrong\u003eFig. 1a, inset)\u003c/strong\u003e.\u0026nbsp;Ectopic endometrial glands often contained blood and cellular debris and were lined by low cuboidal to pseudostratified columnar epithelium that was diffusely and strongly positive for cytokeratin and variably positive for vimentin by IHC (\u003cstrong\u003eSuppl.\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFig. 2).\u003c/strong\u003e\u0026nbsp; These features are consistent with mixed differentiation endometriosis in rhesus macaques\u003csup\u003e11\u003c/sup\u003e. \u0026nbsp;Multiple foci within stromal regions of poorly differentiated endometriosis had extensive necrosis (\u003cstrong\u003eFig. 1b\u003c/strong\u003e), with mixed inflammatory infiltrates of macrophages, lymphocytes, and plasma cells, with foci of neutrophils and necrotic cellular debris, and prominent intranuclear inclusions consistent with MPXV (\u003cstrong\u003eFig. 1b, inset\u003c/strong\u003e). \u0026nbsp;Both glands and stroma in regions of endometriosis were diffusely immunoreactive for CD10 (\u003cstrong\u003eFig. 1c\u003c/strong\u003e), a diagnostic marker for human endometriosis\u003csup\u003e12\u003c/sup\u003e.\u0026nbsp;Mpox immunoreactivity was observed in both glands (\u003cstrong\u003eSuppl. Fig. 3\u003c/strong\u003e) and stroma in regions of poorly differentiated endometriosis by IHC (\u003cstrong\u003eFig. 1d, inset;e\u003c/strong\u003e) and correspond to regions with extensive necrosis and mixed inflammatory infiltrates (\u003cstrong\u003eFig. 1a,b\u003c/strong\u003e).\u0026nbsp;Within the stroma immunoreactive for mpox were numerous smooth muscle actin (SMA)-positive vessels (\u003cstrong\u003eFig. 1f\u003c/strong\u003e), extensive vimentin-positive areas (\u003cstrong\u003eFig. 1g\u003c/strong\u003e), many CD68-positive macrophages (\u003cstrong\u003eFig. 1h\u003c/strong\u003e), and few cytokeratin-positive cells (\u003cstrong\u003eFig. 1i\u003c/strong\u003e).\u0026nbsp;\u003c/p\u003e"},{"header":"Mpox replication in then endocervix is associated with intraepithelial macrophages. ","content":"\u003cp\u003eAdditional reproductive tract tissues including cervix, uterus, and ovary from both mpox challenged female macaques and prostate and testis from the male mpox challenged macaque were screened by IHC for MPXV. Animal 1 also had evidence of mpox virus in the endocervix (\u003cstrong\u003eFig.2a\u003c/strong\u003e). No evidence of virus was detected via IHC or ISH in the evaluated tissues of the reproductive organs of Animal 2 (non-endometriosis female) or in the testis and prostate from the male macaque (\u003cstrong\u003eTable 1\u003c/strong\u003e). Mpox infection of the endocervix was confirmed by in situ hybridization for vRNA (\u003cstrong\u003eFig. 2b,c\u003c/strong\u003e). Epithelial cells of the cervical glands were focally strongly positive for mpox vRNA, while cervical stroma had rare positivity. Mpox vRNA positive signal corresponded to vimentin immunoreactivity in cervical stroma (\u003cstrong\u003eFig. 2d\u003c/strong\u003e). Mpox vRNA signal in the endocervical glands corresponded to positivity for CD68 (macrophages) by IHC (\u003cstrong\u003eFig. 2e\u003c/strong\u003e) suggesting that mpox replication in the cervix was associated with focal cervicitis in Animal 1.\u0026nbsp;To further characterize mpox infected cells in endometriosis, we performed cyclic tissue immunofluorescence (CyCIF) using a targeted panel of pan-cytokeratin, vimentin, and SMA,\u0026nbsp;followed by ISH for MPXV RNA and cytokeratin 10 (CK10) (\u003cstrong\u003eFig. 2f-h\u003c/strong\u003e).\u0026nbsp;Infected cervical epithelial cells were pan-cytokeratin positive while infected cervical stroma was positive for vimentin (\u003cstrong\u003eFig. 2g,h\u003c/strong\u003e). Squamous epithelium of the vagina (CK10) was negative for mpox (\u003cstrong\u003eFig. 2b,f\u003c/strong\u003e).\u003c/p\u003e\u003cp\u003eMPXV can productively infect and replicate within diverse cell types, including oral and respiratory epithelium and antigen-presenting immune cells, including monocytes, macrophages, B cells, and dendritic cells\u003csup\u003e2\u003c/sup\u003e.\u003cem\u003e\u0026nbsp;\u003c/em\u003eEarly studies of the pathogenesis of high dose aerosolized mpox Zaire (Mpox-Z)\u0026nbsp;in cynomolgus macaques highlighted a role for the mononuclear phagocyte system in virus distribution and reported that mpox infection was associated with ovarian, uterine, and testicular inflammation\u003csup\u003e13\u003c/sup\u003e. \u0026nbsp;Endometriosis lesions include multiple components of normal uterine tissue, including glandular epithelium and stroma. In Animal 1, regions of glandular epithelium within endometriotic tissue stained strongly for both cytokeratin and vimentin consistent with a de-differentiated state. These same regions were intensely positive for mpox viral protein. Given the strong predilection of mpox for squamous epithelium, it is perhaps not surprising that the endometriotic glandular epithelium would be permissive to mpox replication. More interesting was the prominent mpox replication in the stromal portions of the poorly differentiated endometriosis that were extensively positive for vimentin. It is well-known that Vaccinia virus, a prototypical poxvirus, associates with vimentin intermediate filaments during assembly\u003csup\u003e14\u003c/sup\u003e. Indeed, we previously reported mpox replication in muscle and adipocytes underlying skin lesions during primary infection with mpox highlighting broad cell tropism for mpox including mesenchymal tissues \u003csup\u003e10\u003c/sup\u003e. Notably, endometriotic stroma and endocervical glands that were immunopositive for mpox also contained numerous CD68 positive macrophages. \u0026nbsp;Tissue resident antigen-presenting cells such as dendritic cells and macrophages are well-described in the genitourinary tract of people, but their role in the pathogenesis of many sexually transmitted diseases, including mpox, is understudied\u003csup\u003e15\u003c/sup\u003e. MPXV replication has been shown in human macrophage cell lines\u003csup\u003e16\u003c/sup\u003e\u0026nbsp; and mpox replication in Kupffer cells of the liver in cynomolgus macaques has been reported\u003csup\u003e13\u003c/sup\u003e. \u0026nbsp;Mpox replication in Hofbauer cells, fetal macrophages of the placenta, was reported in a case from a previous outbreak in the Democratic Republic of Congo\u003csup\u003e17\u003c/sup\u003e. \u0026nbsp;Recently, macrophages were proposed as one potential source of mpox infection in the brain\u003csup\u003e18\u003c/sup\u003e. Our finding of macrophage associated mpox replication in reproductive tissues from macaques infected with mpox from the 2022 outbreak, supports the potential role of macrophages in the pathogenesis of other mpox associated clinical syndromes, including neurological disease. Our previous work showed that viral loads in blood peak at 10 days following infection and typically clear by 28 days irrespective of challenge route\u003csup\u003e10\u003c/sup\u003e. Further study on tissue distribution and persistence of MPVX in tissues as compared to plasma would inform whether macrophages or other cell types contribute to prolonged tissue viremia in certain clinical settings.\u003c/p\u003e\u003cp\u003eTo date, reports of mpox lesions in the reproductive tract of women have been limited to descriptions of vulvar epithelial lesions and a single report of mpox lesions on the external cervical os in women, composed of squamous epithelium\u0026nbsp;\u003csup\u003e19\u003c/sup\u003e, however, assessing uterine tissue in human patients requires invasive methods that are rarely performed. Two prior studies of cynomolgus macaques (\u003cem\u003eMacaca fascicularis\u003c/em\u003e) experimentally infected by aerosolized MPXV reported superficial lesions in the vagina and uterus, with rare necrotic foci in uterine stroma and myometrium\u003csup\u003e13,20\u003c/sup\u003e. To our knowledge, there are no reports of mpox in endometrial tissue in women or in women affected by endometriosis. This report of MPXV in the\u0026nbsp;endocervix\u0026nbsp;as well as poorly differentiated\u0026nbsp;endometriotic tissue extending to the ovary and fallopian tubes in a mpox infected rhesus macaques suggests that women with endometriosis may be susceptible to similar lesions. Given up to 40% of mpox infected individuals are also HIV positive, associated immune dysfunction may heighten the risk of developing serious sequelae with viral spread\u003csup\u003e21\u003c/sup\u003e. Here we report of a case of mpox replication in endometriotic tissue of an experimentally infected rhesus macaque. \u0026nbsp; This finding highlights the potential of MPXV to replicate in reproductive tissues, including ectopic endometrial tissue of women. Further study of the prevalence of mpox in women with endometriosis and the pathogenesis of mpox infection in non-epithelial tissues is warranted. \u0026nbsp;It is important for clinicians to be aware of the potential for mpox to spread intraabdominally in high-risk or mpox-exposed women with endometriosis.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eJ.M.H performed the pathology experiments and wrote the first draft of the manuscript. T. H. and J.L. prepared the tissues. C.E.L, J.L., G.M-R, A.J.M. reviewed the histopathology and edited the manuscript. A.C.collected tissue samples and performed gross pathology assessments. D.H.B. and A.J.M designed the study. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHatami, H. \u003cem\u003eet al.\u003c/em\u003e Demographic, Epidemiologic, and Clinical Characteristics of Human Monkeypox Disease Pre- and Post-2022 Outbreaks: A Systematic Review and Meta-Analysis. \u003cem\u003eBiomedicines\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e, 957 (2023).\u003c/li\u003e\n\u003cli\u003eLum, F.-M. \u003cem\u003eet al.\u003c/em\u003e Monkeypox: disease epidemiology, host immunity and clinical interventions. \u003cem\u003eNat. Rev. 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M. \u003cem\u003eet al.\u003c/em\u003e Decidualization of Endometriosis in Macaques. \u003cem\u003eVet. Pathol.\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 1252\u0026ndash;1258 (2016).\u003c/li\u003e\n\u003cli\u003eChaffee, B. K. \u003cem\u003eet al.\u003c/em\u003e Spontaneous Reproductive Tract Lesions in Aged Captive Chimpanzees. \u003cem\u003eVet Pathol JO -\u003c/em\u003e \u003cstrong\u003e53\u003c/strong\u003e, 425\u0026ndash;435 (2016).\u003c/li\u003e\n\u003cli\u003eD\u0026rsquo;Hooghe, T. M. \u003cem\u003eet al.\u003c/em\u003e Nonhuman Primate Models for Translational Research in Endometriosis. \u003cem\u003eReprod. Sci.\u003c/em\u003e \u003cstrong\u003e16\u003c/strong\u003e, 152\u0026ndash;161 (2009).\u003c/li\u003e\n\u003cli\u003eZondervan, K. T., Becker, C. M. \u0026amp; Missmer, S. A. Endometriosis. \u003cem\u003eN. Engl. J. Med.\u003c/em\u003e \u003cstrong\u003e382\u003c/strong\u003e, 1244\u0026ndash;1256 (2020).\u003c/li\u003e\n\u003cli\u003eAid, M. \u003cem\u003eet al.\u003c/em\u003e Mpox infection protects against re-challenge in rhesus macaques. \u003cem\u003eCell\u003c/em\u003e \u003cstrong\u003e186\u003c/strong\u003e, 4652-4661.e13 (2023).\u003c/li\u003e\n\u003cli\u003eGruber-Dujardin, E., Bleyer, M. \u0026amp; M\u0026auml;tz-Rensing, K. Morphological and immunohistochemical characterization of spontaneous endometriosis in rhesus macaques (Macaca mulatta). \u003cem\u003ePrimate Biol.\u003c/em\u003e \u003cstrong\u003e4\u003c/strong\u003e, 77\u0026ndash;91 (2017).\u003c/li\u003e\n\u003cli\u003eSumathi, V. P. \u0026amp; McCluggage, W. G. CD10 is useful in demonstrating endometrial stroma at ectopic sites and in confirming a diagnosis of endometriosis. \u003cem\u003eJ. Clin. Pathol.\u003c/em\u003e \u003cstrong\u003e55\u003c/strong\u003e, 391\u0026ndash;392 (2002).\u003c/li\u003e\n\u003cli\u003eZaucha, G. M., Jahrling, P. B., Geisbert, T. W., Swearengen, J. R. \u0026amp; Hensley, L. The Pathology of Experimental Aerosolized Monkeypox Virus Infection in Cynomolgus Monkeys (Macaca fascicularis). \u003cem\u003eLab Invest\u003c/em\u003e \u003cstrong\u003e81\u003c/strong\u003e, 1581\u0026ndash;1600 (2001).\u003c/li\u003e\n\u003cli\u003eRisco, C. \u003cem\u003eet al.\u003c/em\u003e Endoplasmic Reticulum-Golgi Intermediate Compartment Membranes and Vimentin Filaments Participate in Vaccinia Virus Assembly. \u003cem\u003eJ. Virol.\u003c/em\u003e \u003cstrong\u003e76\u003c/strong\u003e, 1839\u0026ndash;1855 (2002).\u003c/li\u003e\n\u003cli\u003eIijima, N., Thompson, J. M. \u0026amp; Iwasaki, A. Dendritic cells and macrophages in the genitourinary tract. \u003cem\u003eMucosal Immunol.\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e, 451\u0026ndash;459 (2008).\u003c/li\u003e\n\u003cli\u003eDavies, M. L. \u003cem\u003eet al.\u003c/em\u003e A systemic macrophage response is required to contain a peripheral poxvirus infection. \u003cem\u003ePLoS Pathog.\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e, e1006435 (2017).\u003c/li\u003e\n\u003cli\u003ePittman, P. R. \u003cem\u003eet al.\u003c/em\u003e Clinical characterization and placental pathology of mpox infection in hospitalized patients in the Democratic Republic of the Congo. \u003cem\u003ePLOS Neglected Trop. Dis.\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e, e0010384 (2023).\u003c/li\u003e\n\u003cli\u003eSepehrinezhad, A., Ahmadabad, R. A. \u0026amp; Sahab-Negah, S. Monkeypox virus from neurological complications to neuroinvasive properties: current status and future perspectives. \u003cem\u003eJ. Neurol.\u003c/em\u003e \u003cstrong\u003e270\u003c/strong\u003e, 101\u0026ndash;108 (2023).\u003c/li\u003e\n\u003cli\u003eRam\u0026iacute;rez, M. \u003cem\u003eet al.\u003c/em\u003e Mpox (Monkeypox) Presenting as Cervical and Vulvar Disease. \u003cem\u003eObstet. Gynecol.\u003c/em\u003e \u003cstrong\u003e141\u003c/strong\u003e, 613\u0026ndash;617 (2023).\u003c/li\u003e\n\u003cli\u003eNalca, A. \u003cem\u003eet al.\u003c/em\u003e Experimental Infection of Cynomolgus Macaques (Macaca fascicularis) with Aerosolized Monkeypox Virus. \u003cem\u003ePLoS ONE\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, e12880 (2010).\u003c/li\u003e\n\u003cli\u003eMitj\u0026agrave;, O. \u003cem\u003eet al.\u003c/em\u003e Mpox in people with advanced HIV infection: a global case series. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e401\u003c/strong\u003e, 939\u0026ndash;949 (2023).\u003c/li\u003e\n\u003cli\u003eLin, J.-R., Fallahi-Sichani, M., Chen, J. Y. \u0026amp; Sorger, P. K. Cyclic Immunofluorescence (CycIF), A Highly Multiplexed Method for Single‐cell Imaging. \u003cem\u003eCurrent Protocols in Chemical Biology\u003c/em\u003e \u003cstrong\u003e8\u003c/strong\u003e, 251\u0026ndash;264 (2016).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal Infections\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal studies were described in\u003csup\u003e10\u003c/sup\u003e. Briefly, 3 rhesus macaques (\u003cem\u003eMacaca mulatta\u003c/em\u003e) - two female, one male \u0026ndash; were inoculated intravenously with mpox (MPXV/USA/MA001/2022; lineage B.1, clade 2b; BEI NR-58622; 10\u003csup\u003e6\u003c/sup\u003e TCID50 (10\u003csup\u003e8\u003c/sup\u003e plaque-forming unit [PFU]); intravenous). On day 10 post-infection, a full necropsy with description of gross lesions and collection of major organs was performed. \u0026nbsp; All animal studies were approved by the institutional animal care and use committee of Bioqual, Inc.\u0026nbsp;in accordance with\u0026nbsp;the Public Health Service Policy on Humane Care and Use of Laboratory Animals; The Guide for the Care and Use of Laboratory Animals; the U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training; and ARRIVE guidelines. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistopathology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were fixed in 4% paraformaldehyde for 24 hours, transferred to 70% ethanol, and paraffin embedded and blocks sectioned at 5 mm for routine hematoxylin and eosin staining (H\u0026amp;E) and for IHC and ISH.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eTissue pathology was independently assessed by three veterinary pathologists (AJM, GMR, CEL).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining was performed on selected formalin-fixed, paraffin-embedded sections of reproductive tissues using standard techniques. The slides were baked for 30 minutes at 60\u0026ordm;C, deparaffinized through xylene, 100% ethanol, 95% ethanol, and 1x tris-buffered saline (TBS). Heat-induced epitope retrieval was performed with a 10% citrate buffer (Sigma-Aldrich, C9999) for all antibodies. To detect mpox, primary mouse anti-Vaccinia antibody (Santa Cruz, SC-58210) was applied at 1:100 followed by mouse Mach-2 HRP-Polymer (Biocare MHRP520) for 30 min, then Nova-Red (Vector, SK4800) for 10 min, and counterstained with hematoxylin followed by bluing using 0.25% ammonia water.\u0026nbsp;For CD10 (1:4000; SinoBiological,\u0026nbsp;90177-C07H), primary antibody was applied for 60 min\u0026nbsp;by rabbit Mach-2 HRP-Polymer (Biocare, RHRP520) for 30 min, then\u0026nbsp;3,3\u0026rsquo;-diaminobenzidine (DAB,Cell Marque 957D-30).\u0026nbsp;The slides were counterstained using 50% hematoxylin solution (BioCare CATHE-MM). Vaccinia and CD10 IHC was performed using a Biocare intelliPATH autostainer. For\u0026nbsp;cytokeratin (1:140, clone AE1/AE3, Dako M3515), CD68 (1:410, clone KP1, Dako M0814), alpha smooth muscle actin (1:1000; clone 1A4, Dako M0851), and vimentin (1:162; clone 3B4, ProGen, 61013) primary antibodies were diluted in Da Vinci Green Diluent (BioCare PD900M) and incubated for 30 min at room temperature, slides were then washed and treated with biotinylated horse anti-mouse secondary antibody (Vector Labs BA-2001, diluted 1:200 in Da Vinci Green) for 30 minutes at room temperature. Elite Avidin-Biotin Complex solution (Vector Labs, PK-6100) was applied and incubated for 30 minutes, followed by DAB solution. Matched negative control slides were incubated with Universal Negative Control Serum (BioCare, NC498L). All slides were counterstained using 50% hematoxylin solution (BioCare, CATHE-MM) and were scanned at 20x using a Midi II Scanner (3DHistotech) on default brightfield settings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn situ Hybridization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDuplex chromogenic in situ hybridization was performed using the RNAscope 2.5 HD Duplex Detection Kit (ACD Bio 322500) with customized and general probes (1260381-C1, Mmu-KRT10-C1),\u0026nbsp;1226271-C2\u0026nbsp;(V-MPXV-OPG124-C2), 461341 (Mmu-POLR2a), 457711-C2 (Mmu-PPIB-C2), 2-plex negative control (320751) following recommended guidelines (Protocol 322500-QCK Rev B) as previously described\u003csup\u003e10\u003c/sup\u003e. Slides were then scanned at 20x using a Midi II Panoramic Scanner (3D Histotech) on default brightfield settings.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCyclic ISH and Fluorescence Microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCyclic dual RNAscope ISH and immunofluorescence staining was also performed on selected\u0026nbsp;cervical\u0026nbsp;tissues from Animal 1 (T451F). \u0026nbsp; Baking, deparaffinization, and rehydration were performed in the same manner as with IHC,\u0026nbsp;with the substitution of\u0026nbsp;1x phosphate-buffered saline with 0.2% fish skin gelatin (FSG, Aurion 900.033) in place of 1x TBS. Heat-induced epitope retrieval was achieved using the same method as above. The slides were then washed with 1x PBS/FSG twice for 5 minutes. A protein block was performed using Intercept Blocking Buffer (LiCor 927-70001) for 30 minutes. A photochemical bleaching solution (3% H2O2 in 1xPBS with 20mM NaOH) \u003csup\u003e22\u003c/sup\u003e was used prior to antibody application to decrease autofluorescence and between cycles of fluorescence staining. For each application slides were photo bleached for 60 minutes at room temp while illuminated from above and below with LED light panels (Miroco MI-CL008) on full power. Slides were washed twice with 1x PBS/FSG for 5 minutes. In a lightproof humidity chamber, the slides were protein blocked with Intercept for 30 minutes.\u0026nbsp;For cycle 1, primary antibodies\u0026nbsp;cytokeratin AE1/AE3 (1:140) and SMA (1:100) with Hoechst 1:10,000). \u0026nbsp;For\u0026nbsp;cycle 2, primary\u0026nbsp;antibody vimentin 1:50 with Hoechst 1:10,000 incubated overnight at 4\u0026ordm;C. For both cycles, secondary antibodies against mouse IgG1 conjugated with AlexaFluor 647 (1:1,000, Jackson Immunoresearch 115-605-205) and mouse IgG2a conjugated with AlexaFlour 488 (1:1,000, JIR 115-545-206) were applied and incubated sequentially at room temperature for 30 minutes each. All primary and secondary antibodies were diluted in Intercept. \u0026nbsp; After each cycle slides were coverslipped with 80% glycerol solution and scanned at 20x using a Midi II Scanner in fluorescence mode. \u0026nbsp;Between cycles coverslips were removed by soaking in 1xPBS/FSG for approximately 10 minutes. For cycle 3, dual-plex RNAscope was performed as listed above followed by detection with the Multiplex-Fluorescent reagent kit V2 (323100) as recommended.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSlide Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHALO (v3.6, Indica Labs) was used to fuse serially obtained fluorescence scans with the HALO registration module within the HiPlex FL Module (v4.2). The fused image underwent thresholding for each fluorescent color based on isotype control slides and according to chromogenic IHC identification of staining pattern and intensity.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4103434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4103434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe mpox outbreak of 2022-2023 represented a new global health challenge and recognition of mpox as a sexually transmitted disease. The majority of cases were reported in men who have sex with men (MSM), but women are also susceptible, especially during pregnancy. We evaluated the reproductive tracts of a subset macaques from a large rechallenge study of \u0026nbsp;mpox infection with virus from the 2022 outbreak and identified intraabdominal mpox replication associated with endometriosis. \u0026nbsp;Mpox virus (MPXV) was found not only in skin, but in the cervix, the uterus, and periovarian endometriotic lesions of the affected macaque. Mpox replication preferentially targeted vimentin-positive poorly differentiated endometriotic stromal tissue and infiltrating macrophages in the reproductive tract. Mpox tropism for stromal cells and macrophages has broad implications for mpox pathogenesis and associated clinical syndromes. In addition, women with endometriosis may be at heightened risk for adverse outcomes associated with mpox infection. The rhesus macaque provides rare insight into this disease and the potential complications of mpox infection in the context of genitourinary tract disease.\u003c/p\u003e","manuscriptTitle":"Mpox infection of stromal cells and macrophages of macaque with endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 19:15:54","doi":"10.21203/rs.3.rs-4103434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-24T05:09:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-22T19:54:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-08T21:59:42+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3c4ff2f9-3063-489c-ad22-4c9c49cee97b","date":"2024-05-08T19:56:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"917234992205208763982289323727199632","date":"2024-05-02T18:51:57+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-13T15:42:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-09T09:35:39+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-27T14:32:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-27T14:31:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-03-14T21:20:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"244acf1f-7d76-4d0b-a51b-a953ae4870d4","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":30073749,"name":"Health sciences/Pathogenesis"},{"id":30073750,"name":"Health sciences/Diseases"},{"id":30073751,"name":"Health sciences/Diseases/Infectious diseases"},{"id":30073752,"name":"Health sciences/Diseases/Infectious diseases/Viral infection"},{"id":30073753,"name":"Biological sciences/Microbiology/Pathogens"}],"tags":[],"updatedAt":"2024-09-27T10:49:27+00:00","versionOfRecord":{"articleIdentity":"rs-4103434","link":"https://doi.org/10.1038/s41598-024-73012-8","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2024-09-20 15:57:18","publishedOnDateReadable":"September 20th, 2024"},"versionCreatedAt":"2024-04-01 19:15:54","video":"","vorDoi":"10.1038/s41598-024-73012-8","vorDoiUrl":"https://doi.org/10.1038/s41598-024-73012-8","workflowStages":[]},"version":"v1","identity":"rs-4103434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4103434","identity":"rs-4103434","version":["v1"]},"buildId":"WvIrzKhiLBfengagbw6Ux","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}