{"paper_id":"a82b3497-0338-4ac0-bdc2-131cdab7592c","body_text":"The vaginal microbiome drives endometriosis pain \n \nMcKenna L. Pratt1, Zulmary Manjarres1, Katelyn E. Sadler1 \n \n1 Department of Neuroscience, Center for Advanced Pain Studies, University of Texas at Dallas; Richardson, \nTX, USA \n \nCorresponding author contact: katelyn.sadler@utdallas.edu \n \nAbstract \nDespite being one of two cardinal disease symptoms, endometriosis pain is poorly understood. Using a \nvalidated mouse model, we demonstrate that endometriosis-associated vaginal dysbiosis is sufficient to induce \npain in the absence of disease pathology. In addition, intravaginal antibiotic treatment and vaginal microbiome \ntransplant from healthy control animals reverses pain in endometriosis mice.  \n \nMain Text \nPain is one of two cardinal symptoms of endometriosis, a disease defined by the presence of \nendometrial-like tissue outside of the uterus1. Historically, endometriosis pain was exclusively attributed to \nnociceptive signaling in extrauterine lesions. This dogma, however, is challenged by the fact that lesion number \nand size do not correlate with pain reports and lesion removal does not alleviate pain in a subset of patients2,3,4. \nGiven this, additional drivers of endometriosis pain must exist. Previous studies demonstrate that individuals \nwith endometriosis exhibit vaginal dysbiosis5,6. Considering the causal role of gut dysbiosis in other chronic \npain conditions7,8 we investigated the extent to which the vaginal microbiome drives endometriosis pain using \nmouse models.   \nEndometriosis was induced in naïve C57BL/6 female mice through intraperitoneal injection of donor \nuterine tissue9. This procedure resulted in the development of extra-uterine lesions throughout the abdominal \ncavity (Fig. 1a, 1b). Relative to sham controls, endometriosis mice developed persistent abdominal mechanical \nallodynia, a feature also reported in individuals with endometriosis10,11,12,13 Fig. 1c, 1d; Extended Data Fig. 1). \nEndometriosis mice also developed spontaneous pain as indicated by higher facial grimace scores, more \nfrequent abdominal licking, and reduced rearing behavior (Fig. 1e-h). To examine vaginal microbiome \ncontributions to these phenotypes, pain-like behaviors were re-assessed in endometriosis mice following 5 days \nof intravaginal antibiotic administration (Fig. 2a). Elimination of the vaginal microbiome decreased both \nabdominal mechanical allodynia and ongoing pain in endometriosis mice (Fig. 2b-d; Extended Data Fig. 1). \nThese findings suggest that elimination of the dysbiotic vaginal microbiome is sufficient to decrease pain in \nendometriosis.  \nNext, we performed a series of vaginal microbiome transplant (VMT) experiments to assess if \nendometriosis dysbiosis induces pain in the absence of disease pathology (Fig. 2e). Five consecutive days of \nVMT were performed from endometriosis or sham donor mice to naïve C57BL/6 recipients. Resulting changes \nin pain-like behaviors were recorded for up to one week following VMT completion. Endometriosis VMT \nrecipients developed abdominal mechanical allodynia and ongoing pain that mirrored phenotypes observed in \nVMT donors (Fig. 2f-h; Extended Data Fig. 1). These results support the idea that endometriosis-associated \ndysbiosis is sufficient to induce persistent pain, even in the absence of ectopic lesions. In a reciprocal fashion, \nwe finally assessed whether introduction of a healthy vaginal microbiome reverses endometriosis pain. For \nthese studies, endometriosis mice received five consecutive days of VMT from sham controls or a separate \ncohort of mice with endometriosis (Fig. 2i). Sham VMT resulted in a transient alleviation of abdominal \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.21.671604doi: bioRxiv preprint \n\nmechanical allodynia and ongoing pain in endometriosis mice (Fig. 2j-l; Extended Data Fig. 1). Thus, \nintroduction of probiotic vaginal species should be explored as a novel means of treating endometriosis pain.  \nTo our knowledge, this study is the first to provide a direct link between the vaginal microbiome and \nendometriosis pain. Our work demonstrates that both intravaginal antibiotic treatment and VMT from healthy \ncontrol mice temporarily alleviates pain in mice with endometriosis. The transient nature of this pain relief \nsuggests that disease-specific selective pressures are not modified by either treatment. Regardless, these data \nprovide a foundation on which analgesics based on microbial compounds – a new class of therapies for \nendometriosis treatment – can be developed. This is particularly notable given that current first-line treatments \nfor endometriosis pain including hormonal contraceptives and non-steroidal anti-inflammatory drugs fail to \nprovide sufficient pain relief for many patients14,15. Limitations of the current study include species differences \nin the vaginal microbiome; the human vaginal microbiome is almost exclusively colonized by Lactobacilli \nwhereas the mouse vaginal microbiome is more diverse16,17,18,19,20,21. As such, future translational investigations \nshould examine convergent microbial metabolic pathways in the vagina that are disrupted in both mouse models \nand patients with endometriosis.   \n \nAcknowledgements \nThis work was funded by grants from the National Institutes of Health (R00HL155791 to KES) and the Rita \nAllen Foundation (Award in Pain to KES). The authors thank Nurfatihah Zulkifly for assistance with \nspontaneous behavior video scoring.  \n \nAuthor contributions \nMLP, ZM, and KES conceptualized study and developed methodology. MLP and ZM performed the \ninvestigation. MLP and KES are responsible for data visualization. MLP wrote the original manuscript draf; ZM \nand KES editied draft. KES acquired funds and supervised the project.  \n \nCompeting interests \nAuthors declare no competing interests.  \n \n \n \n \n \n \n \n \n \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.21.671604doi: bioRxiv preprint \n\n \nFig. 1 | Murine model of endometriosis. a, Example endometriotic lesions isolated from intraperitoneal cavity or adhered to ovaries. \nb, Total number of lesions per location from N=15 endometriosis mice. c, Abdominal mechanical sensitivity of endometriosis and \nsham mice displayed as a percent of trials with response to the 0.16g von Frey filament from baseline to 56 days post \nendometriosis/sham induction; N=8-10. d, Responses to abdominal stimulation with 0.16, 0.64, and 2.56 g von Frey filaments \nsummed from baseline to day 56 post-sham or endometriosis induction. e, Facial grimace scores, f, bouts of abdominal licking and g, \nbouts of rearing observed in endometriosis and sham mice up to day 56 post endometriosis/sham induction; N=8-16. h, Ongoing pain \nbehaviors summed from up to day 56 post-sham or endometriosis induction. Percent of trials with response, mouse grimace score, \nabdominal licking, and rearing comparisons were performed using 2-way repeated measures ANOV A with post hoc Bonferroni \ncorrection. von Frey AUC was analyzed using a 2-way mixed ANOV A with post hoc Bonferroni correction. Facial grimace, abdominal \nlicking, and rearing AUC were analyzed using individual two-tailed unpaired t-tests. *P £ 0.05, **P £ 0.01, ***P £ 0.001, ****P £ \n0.0001. \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.21.671604doi: bioRxiv preprint \n\n \nFig. 2 | The vaginal microbiome of endometriosis mice is a target for treating pain-like behaviors. a, Schematic depicting the \ntimeline and details of antibiotic treatment of endometriosis mice. b, Mouse grimace scores of vehicle- and antibiotic-treated \nendometriosis animals throughout treatment paradigm (N=7-8). c, Abdominal mechanical allodynia of vehicle- and antibiotic-treated \nendometriosis mice displayed as a percent of trials with response to a 0.16g von Frey filament from baseline to 7 days post-treatment. \nd, Area under the curve of the graph in (c) along with area under the curve of von Frey filaments 0.64g and 2.56g. e, Schematic of \nVMT from endometriosis to wildtype mice. f, Mouse grimace scores of sham and endometriosis VMT wildtype recipients from \nbaseline to 7 days post-treatment (N=10). g, Abdominal mechanical allodynia of sham and endometriosis VMT recipients displayed as \na percent of trials with response to a 0.16g von Frey filament throughout the treatment paradigm. h, Area under the curve of the graph \nin (g) along with area under the curve of von Frey filaments 0.64g and 2.56g. i, Schematic of sham VMT to endometriosis mice. j, \nMouse grimace scores of sham VMT endometriosis mice from baseline to 7 days post-VMT (N=7-8). k, Abdominal mechanical \nallodynia of sham VMT endometriosis mice displayed as a percent of trials with response to a 0.16g von Frey filament throughout the \ntreatment paradigm. l, Area under the curve of the graph in (k) along with area under the curve of von Frey filaments 0.64g and 2.56g. \nPercent of trials with response and mouse grimace scores were compared using 2-way repeated measures ANOV A with post hoc \nBonferroni correction. Area under the curve for von Frey stimulation response was compared using a 2-way mixed effects ANOV A \nwith post hoc Bonferroni correction. *P £ 0.05, **P £ 0.01, ***P £ 0.001, ****P £ 0.0001. Dotted lines indicate the mean percent \ntrials with response and mouse grimace score for sham animals. \n \n \n \n \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.21.671604doi: bioRxiv preprint \n\nMethods  \nAnimals  \nFemale C57BL/6 (Jackson Laboratory) mice were bred in house and maintained on a 12-hour light/12-hour \ndark schedule with ad libitum access to rodent chow and water. Animals were 7- to 8-weeks-old at induction of \nendometriosis; similar-aged mice were used as uterine tissue donors and endometriosis VMT recipients. All \nprotocols were in accordance with the National Institutes of Health guidelines and were approved by the \nInstitutional Animal Care and Use Committee at the University of Texas at Dallas (Richardson, TX; 2022-\n0088).  \n \nNon-surgical mouse model of endometriosis  \nEndometriosis was induced using procedures identical to those described by Fattori and colleagues9. Briefly, \nuterine horn donors received a single injection of 3 µg/100 µL b-estradiol 3-benzoate (E2) 7 days prior to tissue \ncollection. E2 was prepared by dissolving 1 mg of E2 (Sigma Aldrich) in 1 mL of 100% molecular grade \nethanol (Sigma Aldrich) to a final stock concentration of 1 mg/mL. To create a working solution, 3.3 µL of \nstock solution was added to 106.7 µL of sesame oil (Sigma Aldrich) such that the final concentration was 3 \nµg/100 µL. Using a 1cc syringe (Medline), 100 µL of the working solution was injected subcutaneously in each \ndonor mouse. 7 days post-injection, donor mice were sacrificed, and their uterine horns were removed. Uterine \nhorns were cut longitudinally with a pair of dissecting scissors and then minced using scissors and a blade until \nfragments were < 1 mm in length. Further homogenization of tissue fragments was achieved using a dounce \ntissue homogenizer (Grainger). Homogenized tissue from all donors was combined into one solution prior to \ntransplant to decrease potential inter-individual variability effects; tissue from 1 donor animal was needed for \nevery 2 recipients. Recipient mice were maintained on inhalable isoflurane (1.8-2.2%; Covetrus) for the \nduration of induction. Using a 23G needle (PrecisionGlide™), 500 µL of uterine horn solution was injected \nintraperitoneally into each recipient mouse. Sham mice were given an intraperitoneal injection of 500 µL of \nHank’s balanced salt solution. Mice were immediately returned to their home cage following induction.   \n \nVaginal lavage  \nVaginal lavage was obtained as previously described22. Briefly, mice were scruffed then, using a p200 pipette, \n50 µL of sterile saline (Medline) was flushed in and out of the vagina 5 times. Samples were collected in sterile \ntubes and either frozen at -80°C or placed on ice for immediate use.   \n \nBehavioral tests  \nPrior to the start of all behavioral tests, mice were habituated to the testing apparatus for 2 hours and \nexperimenter presence for 30 minutes. The experimenter was blinded to injury and drug treatment where \napplicable. Mice were randomly assigned to all treatment groups.  \n \nSpontaneous behaviors  \nSpontaneous behavior was hand scored from 10-minute bottom-up videos. Videos were taken prior to other \nbehavior testing at each timepoint. Scored behaviors included rearing and abdominal or non-abdominal \nlicking/grooming behaviors. Rearing behavior was classified as the lifting of both front paws from the ground \nand standing in an upright position on one or both hindlimbs. Supported rearing (against the walls of the \nbehavior chamber) and unsupported rearing were considered equal behaviors. For spontaneous abdominal pain \nmeasurements, licking of the abdomen was considered and analyzed as previously reported9. Non-abdominal \nlicking was also analyzed. For each spontaneous behavior measured, the number of bouts per activity was \nreported.   \n \nMouse grimace score  \nFacial grimace was scored manually by a trained experimenter as previously described23. This was measured \nfollowing the completion of bottom-up video recording and prior to further behavior testing. Mice were given a \nscore of 0-2 for 5 different facial features: orbital tightening, nose bulge, cheek bulge, ear position, and whisker \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.21.671604doi: bioRxiv preprint \n\nchange. Each animal was scored twice with approximately 5 min between trials. The scores for all features were \naveraged to generate the overall mouse grimace score.  \n \nvon Frey mechanical allodynia testing  \nAbdominal sensitivity to punctate mechanical stimulation was assessed via calibrated von Frey filaments \n(0.16g; 0.64g; 2.56g)24. These filaments were selected based on preliminary ‘up/down’ abdominal von Frey \ntesting completed in sham animals; the average 50% withdrawal threshold in these mice was 0.64g. Thus, we \nincluded the 0.64g filament and 2 filaments ‘up’ and ‘down’ from that average: 0.16g and 2.56g. The decision \nto include 3 filaments was made to better capture a mechanical allodynia phenotype and/or a change in that \nphenotype. Animals’ abdomens were stimulated 5 times with each filament with at least 1 minute between \nstimulations. Response frequency was recorded for each filament. Care was taken to avoid stimulating the same \nspot on the abdomen twice. A jump, paw flinch, or kicking of hindpaws was counted as a withdrawal \nresponse.    \n \nVaginal treatments  \nVolumes for all vaginal treatments (25 µL) was determined through pilot studies wherein various amounts of \ncrystal violet (1%) was administered into the vaginal cavity of mice. N=3 mice were used for each volume \ntested (10 µL; 25 µL; 50 µL). Mice were placed supine and maintained on isoflurane (1.8-2.2%) during the \ntreatment. Following administration, 5 minutes were allowed for the crystal violet solution to sit in the vaginal \ncavity prior to removing the mouse from isoflurane and immediately euthanizing. This 5-minute period was \nprovided for all treatments to avoid immediate leakage of the solution out of the vagina. In crystal violet pilot \nstudies, the vaginal tissue was dissected from each mouse and cut open longitudinally. Images were taken and \nanalyzed using ImageJ for percent coverage (Extended Figure 2).   \n \nAntibiotic treatment  \nAntibiotic cocktail was made fresh every other day of treatment. This cocktail is commonly used to create a \npseudo germ-free intestine in rodents and contains 4 antibiotics: vancomycin (0.5 g/L), ampicillin (1 g/L), \nneomycin (1 g/L), and metronidazole (1 g/L)25. To make the solution, antibiotics were resuspended in sterile \nsaline at the appropriate concentrations. Mice were placed supine and maintained on inhalable isoflurane (1.8-\n2.2%) during treatment. 25 µL of the antibiotic cocktail was intravaginally administered using sterile 200 µL \npipet tips. Vehicle animals received 25 µL of sterile saline. Following administration, mice remained under \nisoflurane for 5 minutes. Animals were immediately returned to their home cage following the 5-minute \nabsorption period and closely observed until normal behavior resumed. Treatment was repeated daily for 5 \nconsecutive days.  \n \nVaginal microbiome transplant  \nVaginal lavage contents were obtained from donors using the lavage method described above. To avoid \npotential animal and/or cage-specific effects, vaginal lavage donors were rotated in groups such that no donor \nwas used on two consecutive days. This rotation scheme was also used to reduce potential lavage-related \ndisruptions in donor vaginal microbiomes. 50 µL of lavage contents were collected from each donor mouse and \ncombined to create a mixed donor solution. This solution was stored on ice throughout lavage collection and \nvaginal microbiome transplant (VMT). VMT was performed immediately following collection. Recipient mice \nwere placed supine and maintained on inhalable isoflurane (1.8-2.2%) during treatment. 25 µL of the pooled \nvaginal lavage contents were intravaginally administered using sterile 200 µL pipet tips. Following \nadministration, mice remained under isoflurane for 5 minutes before being returned to their home cage. Mice \nwere observed closely until normal behavior resumed. VMTs were performed daily for 5 consecutive days.   \n \nExtra-uterine lesion detection  \nAt the end of each animal’s treatment paradigm, mice were humanely sacrificed using isoflurane overdose \nfollowed immediately by cervical dislocation. The abdominal region of mice with endometriosis mice was \ncarefully examined for ectopic lesions. The entire length of the abdominal wall, uterine horns, ovaries, and all \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.21.671604doi: bioRxiv preprint \n\nother tissue located within the peritoneal cavity were searched for endometrial adhesions. Location and number \nof lesions in each endometriosis mouse was recorded to confirm success of the disease model.   \n \nStatistical analysis  \nIndividual data points were presented wherever possible in addition to group means ± SEM. Data were analyzed \nusing GraphPad Prism 10; results were considered statistically significant when   P < 0.05.   \n \n \nReferences \n1. Koninckx, P. R., Fernandes, R., Ussia, A., Schindler, L., Wattiez, A., Al-Suwaidi, S., Amro, B., Al-\nMaamari, B., Hakim, Z., & Tahlak, M. (2021). Pathogenesis Based Diagnosis and Treatment of \nEndometriosis. Frontiers in Endocrinology, 12(12), 745548. https://doi.org/10.3389/fendo.2021.745548 \n2. Vercellini, P., Fedele, L., Aimi, G., Pietropaolo, G., Consonni, D., & Crosignani, P. G. (2006). \nAssociation between endometriosis stage, lesion type, patient characteristics and severity of pelvic pain \nsymptoms: a multivariate analysis of over 1000 patients. 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Cell, 118(2), \n229–241. https://doi.org/10.1016/j.cell.2004.07.002 \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.21.671604doi: bioRxiv preprint \n\n \n \nExtended Data Fig. 1 | Additional abdominal mechanical hypersensitivity tests. Percent of trials with response to the 0.64g and \n2.56g von Frey filament for sham versus endometriosis mice (a, b; N=8-10), vehicle versus antibiotic-treated endometriosis mice (c, \nd; N=7-8), sham versus endometriosis VMT-treated wildtype (WT) recipients (e, f; N=10), and sham versus endometriosis VMT-\ntreated endometriosis mice (g, h; N=7-8). All comparisons were performed using a 2-way repeated measures ANOV A with post hoc \nBonferroni correction. *P £ 0.05, **P £ 0.01, ***P £ 0.001, ****P £ 0.0001. Dotted lines indicate the mean percent trials with \nresponse for sham animals. \n \n \n \n \n \nExtended Data Fig. 2 | Pilot study for volume of vaginal treatments. a, Plot showing percent coverage by 3 different volumes of \ncrystal violet (1%) administered to the vagina of mice (N=3 per volume group). b, Representative images of 3 reproductive tracts \nfollowing intravaginal administration of 10, 25, or 50 µL of crystal violet (1%). Tissue was cut longitudinally to expose the vaginal \ncavity.  \n \n \n \n \n(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. \nThe copyright holder for this preprintthis version posted August 21, 2025. ; https://doi.org/10.1101/2025.08.21.671604doi: bioRxiv preprint","source_license":"CC0","license_restricted":false}