{"paper_id":"bfaea40a-d3c1-4197-a097-e6e026d8675e","body_text":"Article Title: Spatiotemporal mapping of  ex vivo  myometrial \ncontractions in adenomyosis \nRunning title: Oxytocin control of contractile waves  \nEmilie Huon a*, Léna Zig a, Olympe Séguard a, Marine Planckea, Carole Abo  Md PhDb, Marie -\nCharlotte Lamau Mdb, Gauthier Dietrich Mdb, Ylann Abrahami Mdb, Jordan Kouby Mdb, Julien Adam \nMd PhDc and Nicolas R. Chevalier PhDa*,  \naLaboratoire Matières et Systèmes Complexes, Université Paris Cité, CNRS, Paris, France. \nbService de  Chirurgie Gynécologique, Hôpitaux Paris Saint -Joseph Marie Lannelongue,  Paris, \nFrance, \ncService d’Anatomie pathologique, Hôpitaux Paris Saint-Joseph Marie-Lannelongue, Paris, France \n*Corresponding Authors: \nDr Nicolas R Chevalier \nCR, CNRS \nnicolas.chevalier@u-pariscite.fr  \nPhone: +33157276259 \n \nEmilie Huon \nPhD Candidate, Université Paris Cité \nemilie.huon@u-pariscite.fr \nPhone: +33631732739 \n \nArticle type:  Case-Control study \nFunding Statement:  This study was funded by the Fondation pour la Recherche sur \nl’Endométriose, project HYPERU, under the auspices of the Fondation pour la Recherche \nMédicale, project FRE202212016469. \nDisclosure Statement: All authors declare that they have no competing interests. \nAttestation Statement: Data regarding any of the subjects in the study have not been previously \npublished unless specified. Data will be made available to the editors of the journal for review or \nquery upon request. \nData Sharing Statement: Essential data generated or analysed during this study are included in \nthis report and its supplementary information files. Further data are available from the \ncorresponding author upon reasonable request. \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \nNOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.\n\nWord Count: 326 words for abstract, 26 words for capsule and 3405 words for text. \nTrial registration: Approval for this study was granted on the 27th of January 2025 under IRB \nnumber 00012157 from the Groupe Ethique Recherche Médicale (GERM) of Hôpital Paris Saint \nJoseph. The first patient was included on the 26th of February 2025.  \nCapsule: The whole myometrium contracts ex vivo , not just the junctional zone. Myogenic \ncontractility of adenomyosis uteri was normal, in spite of oxytocin receptor overexpression . The \npropagation direction of contractile waves can be controlled by oxytocin. \nAbstract \nObjective: Uterine dyscontractility  plays a major role in pain sensation , infertility , and \nadenomyosis pathogenesis. In vivo, contractility occurs in the inner myometrium and junctional \nzone, which is thickened in adenomyosis. We examined whether the junctional zone presents \nparticular contractile features ex vivo by measuring contractility and oxytocin response in full-\nthickness serosa-to-endometrium myometr ial slices retrieved from control and adenomyosis \npatients. \nDesign: Prospective ex vivo case-control study. \nSubjects: Premenopausal women following hysterectomy: adenomyosis (n = 16) and controls (n \n= 12). Contractility: 57 strips (11 vs 8 patients), immunofluorescence: 10 vs 9 patients. \nMain Outcome Measures:  We resorted to spatiotemporal mapping , time -lapse video -based \ntechniques used in the field of gastroenterology. We measured contraction frequency, amplitude, \nduration, velocity, site of origin, and propagation direction along the serosa-to-endometrium axis, \nbefore and after oxytocin. We measured o xytocin receptor density along the serosa-to-\nendometrium axis by immunofluorescence and machine learning-assisted segmentation. \nResults: Ex vivo contractile waves propagated throughout the outer and inner myometrium, with \nsimilar amplitude and frequency. Contraction frequency, amplitude, duration, and velocity did not \ndiffer between control and adenomyosis uteri . Oxytocin inc reased contraction frequency and  \nvelocity and systematically caused contractile waves to originate at the inner myometrium in both \ngroups. In both groups, oxytocin receptor density was highest in the  inner myometrium . \nAdenomyosis patients had a 1. 8-fold higher oxytocin receptor density, but a smaller oxytocin -\ninduced increase in contraction frequency. \n \nConclusion: We provide the first e x vivo  spatiotemporal time-lapse analysis of  myometrial \ncontractions. The whole myometrium contracts ex vivo, not just the inner part (junctiona l zone). \nThis uniform myogenic motility is presumably inhibited neurally in vivo in the outer myometrium. \nEx vivo adenomyosis basal contractility was similar to that of controls. Adenomyotic myometrium \noverexpressed oxytocin receptors compared to controls, but this did not translate into a stronger \ncontractile response to oxytocin. In both groups, oxytocin caused contractile waves to originate \nat and propagate from the sub-endometrial myometrium, where receptor density is highe r. This \ndemonstrates that contractile wave direction can be controlled hormonally. \nKeywords \nEx vivo; human myometrium; endometriosis; oxytocin receptors; junctional zone. \n \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nIntroduction \nUterine contractions depend on hormonal, mechanical, and inflammatory signals (1–3). In severe \ndysmenorrhea, endometriosis, and adenomyosis, this contractility becomes disrupted, leading \nto hypercontractility and dysperistalsis (4–8). In healthy eumenorrheic women, uterine \ncontractions (peristaltic waves) concentrate in the inner myometrium (9–11), originating in the \njunctional zone (JZ) (12–14). The characteristic low T2 of the JZ remains visible on MRI of excised \nuteri, although less sharply than in vivo (15,16). Uterine peristaltic contractions vary in direction, \nfrequency, amplitude, and velocity depending on cycle phase to facilitate sperm transport (cervix-\nto-fundus direction during ovulation) or menstrual fluid evacuation (fundus-to-cervix direction \nduring menses) (6,17,18). \nEndometriosis is associated with a significant ly increased peristaltic activity  (5,19,20). This \nexcessive peristalsis generates elevated uterine pressures that can promote retrograde transport \nof endometrial fragments through the fallopian tubes into the peritoneal cavity  (21). This \nmechanism is considered a crucial early step in disease pathogenesis (6,21). Adenomyosis is \ncharacterized by the presence of endometrial glands within the myometrium, and by a thickened \n(>12 mm) JZ (12,22,23). Contractions in adenomyosis are uncoordinated, which may disrupt \nsperm transport and contribute to infertility (24–26). A study on isolated myometrial strips found \nthat adenomyotic tissue exhibits markedly higher spontaneous contraction amplitude compared \nto healthy tissue  (27). Chronic dyscontractility could induce JZ microtrauma that favours \nendometrial invasion of the myometrium, a hypothesis known as “ tissue injury and repair ” (28). \nThe underlying causes of contractile dysfunction in adenomyosis remain poorly understood. \nIn vivo cine MRI / TVUS captures contraction direction, velocity, and coordination, but do es not \nhave access to force or pressure. Conversely, ex vivo strip studies report force but obliterate wave-\nlike aspects of contraction propagation. In vivo, uterine contractility is found to be restricted to \nthe inner myometrium, which encompasses the JZ (5,9–11). It is not known whether the JZ or the \ninner myometrium exhibit distinct contractile features ex vivo, in an organ bath. To address these \nquestions and methodological gaps, we developed here an optical, ex vivo approach to measure \ncontractile wave origin, amplitude, velocity, direction, and frequency across the entire myometrial \nthickness, from serosa to endometrium. Our method draws on tissue displacement mapping \ntechniques previously applied to intestinal smooth muscle (29,30), and on speckle -tracking \nmethods used in in vivo uterine studies (31). Because oxytocin receptors (OTR) have previously \nbeen found to be overexpressed in  adenomyosis myometrium (27,32), we also tested the \ncontractile response to the uterotonic hormone oxytocin (33,34), and histologically characterized \nthe OTR distribution across the myometrium of adenomyosis and control uteri. \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nMaterials and Methods  \nPatients and tissue collection \nMyometrial biopsies were obtained after hysterectomy from 28 premenopausal women at Hôpital \nParis Saint Joseph. Patient characteristics are detailed in Table 1. Ethical approval for the study \nwas granted by the Groupe Ethique Recherche Médicale (GERM) under approval number IRB \n00012157. All patients were provided with written information about the study and consented to \nparticipate.  \nTwo adjacent full-thickness biopsies (~30 × 10 × 10 mm, serosa to endometrium) were cut out \nfrom the anterior wall, midway between cervix and fundus, avoiding any visible myomas. The first \nbiopsy was immediately fixed in 4% PFA overnight for immunofluorescence (n = 10 adenomyosis, \nn = 9 controls). The second biopsy was transported in ice-cold medium (DMEM:F-12 + GlutaMAX, \nGibco, Thermo Fisher) pre -oxygenated with carbogen (95% O2, 5% CO2). Ex vivo  contractility \nassays were performed on this second biopsy, within 6 hours of retrieval (n = 11 adenomyosis, n \n= 8 controls). Ten patients (5 per group) contributed to both ex vivo  contractility and \nimmunofluorescence analysis. \n \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nEx vivo contractility \nEach fresh biopsy was further sliced into three ~1 mm thick strips (final size ~30 × 10 × 1  mm), \nwith circumferential fiber s running along the strip width. Carbon black microparticles were \nadhered to each slice to serve as fiducial markers, and the 3 strips were pinned vertically (serosa \nat the top, endometrium at the bottom), next to each other, in a Sylgard -lined container at 37°C, \ncontinuously bubbled with carbogen  (Figure 1A,C, Video S1) . Once spontaneous phasic \ncontractions appeared (after 1-3 h), they were recorded at 1 frame per second (1Hz) with a Basler \nacA3800 camera mounted with a 25mm lens. Recording times were 20 min for baseline and  30 \nmin after 10 µM oxytocin (Sigma O4 375) administration. For each patient, c ontractile behaviour \nwas averaged over all contractions in the recording intervals and over the three strips , thus \nreducing intra-patient technical variability. \nVideo analysis \nWe tracked displacement of carbon black particles adhered to the myometrium with the TrackV2 \nImageJ plugin. Three pairs of particles were selected for each strip , at the sub -serosal, mid -\nmyometrial, and sub-endometrial levels (Figure 1C). For each pair, we computed the deformation \nrelative to the relaxed state: 𝐷𝑒𝑓𝑜𝑟𝑚𝑎𝑡𝑖𝑜𝑛 (𝑡)(%) =   100 \n𝐷(𝑡)−𝐷𝑟𝑒𝑙𝑎𝑥\n𝐷𝑟𝑒𝑙𝑎𝑥\n, where D(t) is the inter-point \ndistance (along x, i.e. perpendicular to the serosa -endometrium axis), and 𝐷𝑟𝑒𝑙𝑎𝑥 the maximum \ninterpoint-distance reached during the recording. Traces were smoothed with a Gaussian filter (σ \n= 3 s) , and contractions were detected using custom MATLAB peak-detection algorithms \n(https://github.com/emiliehuon/uterus_analysis_contractility), adjusting the minimum peak \nprominence for each recording (0.1 – 1 %) to exclude noise. We computed the frequency (peaks \nper 10 min), amplitud e (peak -to-preceding valley, %) , and duration (width at half -prominence, \nmin). \nFor each propagating wave, its site of origin was measured directly from the video and normalised \nto strip length. Velocity was measured as the distance travelled by the contractile wave divided by \nthe travel time. Sites of origin and velocity were averaged over all visible waves for each strip, and \nthen over the three strips for each patient. Other contraction parameters were averaged over the \nthree pairs of points, and over the three strips (i.e., 9 measurements per patient). \nImmunofluorescence \nThe fixed biopsy was cryoprotected in 30% sucrose, embedded in PolyFreeze (Sigma), and cut in \n18 µm sections deposited on Superfrost glass slides. Slides were permeabilized and bl ocked in \nPBS, 1% BSA, and 0.1% Triton for 1 h, incubated overnight with Oxytocin receptor antibody (OTR, \nLS-A246, LSBio, 1:100), thoroughly washed, and further incubated in Alexa Fluor 647 anti -rabbit \nIgG (Thermo Fisher, 1:400, 3 h). Slides were kept under gentle agitation throughout to optim ize \nstaining homogeneity. They were fina lly washed, cover-slipped, and imaged at 10x on a Zeiss \nwide-field epifluorescence microscope (ImagoSeine platform), in mosaic mode to capture the full \nfluorescence distribution from serosa to endometrium. Mosaic tiles (~100-200 per sample) were \nstitched with ZEN software (Zeiss). \nTable 1: Patient cohort description.  Clinical characteristics of patients who underwent  hysterectomy in the ex vivo \ncontractility cohort (n = 19: 11 adenomyosis, 8 controls) and the immunofluorescence cohort (n = 19: 10 adenomyosis, \n9 controls). Quantitative variables are expressed as median [interquartile range] and qualitative variables as  % (N). \nSecondary pathology refers to a condition diagnosed alongside the primary indication for hysterectomy. Groups were \ncompared with Welch’s t-test (age) and Fisher’s exact test ( parity, hormonal treatment). Cycle phase and pathologies \nare reported descriptively. \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nImage Analysis \nTen ROIs (each 780 µm x 780 µm) were placed along the serosa-endometrium axis, avoiding large \nvessels, holes, and folds, using a custom Fiji macro (GitHub, as above). OTR-positive pixels were \nsegmented with Trainable Weka Segmentation (35). OTR density was expressed as the OTR-\npositive area divided by the ROI area. ROI positions along the serosa -to-endometrium axis were \nnormalised, and a smooth expression profile fitted through the 10 points. The average OTR density \nwas computed as the area under the smoothed expression profile. \nStatistical analysis \nStatistical analyses were performed under GraphPad Prism 11. Tests are specified in figure and \ntable legends. Data are reported as median [IQR], or % (N); p < 0.05 was considered significant. \nResults \nCharacteristics of ex vivo spontaneous myometrial contractile waves  \nWe characterized the contractile behaviour of full-thickness – from serosa to endometrium - \nhuman myometrial  strips (Figure 1) . Contractile activity occurred mostly as propagating \ncontractile waves that travelled along the slice length, from the serosa to the endometrium, or in \nthe opposite direction  (Figure 1B-D, Video S1) . The cross -sample average (n = 19) contraction \nfrequency was 2.3 cycles per 10 minutes (cp10m), amplitude 13 %, duration 1.0 min, and velocity \n1.4 cm/min. Importantly, contraction frequency and amplitude were homogeneous along the \nslice length (Figure 1D, E): the outer myometrium contracted as much as the inner myometrium \nin our ex vivo organ bath.  \nIndividual contractile waves originated at various positions along the slice le ngth. Waves that \noriginated at the serosa travelled predominantly towards the endometrium, and conversely those \nnucleating at the endometrium propagated to the serosa; waves originating in the middle of the \npreparation travelled in both directions. This behavio ur was similar to that of myogenic \ncontraction waves in the intestine  (36). The cross -sample average normalized origin of \ncontraction was 0.52 (0 = endometrium | 1= serosa; n = 19), i.e., close to the mid-myometrium. \nSpontaneous contraction f requency, amplitude, duration, and vel ocity did not differ between \ncontrol and adenomyosis uteri (Figure 2A-D). Contractile waves tended to originate closer to the \nendometrium in adenomyosis than in controls (Figure 2F), but the difference was not statistically \nsignificant.  \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\n \n \n \n \nFigure 1. Uterine contractile waves propagate with uniform amplitude across the whole myometrial thickness. (A) \nExperimental setup. Three full-thickness strips per patient are pinned vertically in an organ bath (1), thermoregulated at \n37°C (2), continuously bubbled with carbogen (3), and recorded with a video camera (4) at 1 Hz for up to an hour. (B) \nRepresentative 20-min kymograph showing three serosa-to-endometrium waves (arrows). (C) A strip covered with carbon \nblack particles for displacement tracking  (still shot from Video S1) . Shown in red are the successive positions of three \npairs of points (at the sub -serosa, mid-myometrium, and sub -endometrium), tracked over 20 min. (D) Representative \ndeformation traces of the three pairs of points at the sub-serosa (blue), mid-myometrium (orange), and sub-endometrium \n(yellow). Frequency was derived from detected peaks (○), amplitude as peak -to-preceding-valley (▽) difference, and \nduration as width at half -prominence (-). (E) Contraction amplitude at different positions along the myometrium, for all \npatients (n = 19: 11 adenomyosis, 8 controls). Each point is the mean of three slices per patient (~5 contractions per slice \nper recording). Repeated-measures one-way ANOVA with Tukey’s post-hoc test; **p = 0.0048. \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\n \nFigure 2. Effects of oxytocin on the contractility of control and adenomyosis myometrium . (A -F) Contractile \nparameters before and 7-30 min after oxytocin, in controls (blue, n = 8) and adenomyosis (red, n = 11); open boxes before \noxytocin, filled boxes after oxytocin. Each point represents one patient (mean of 5.0 ±1.8 contractions per strip, and for \nthree strips per patient). Two-way ANOVA (group x period, repeated measures on period), mixed-effects model, followed \nby Sidak’s post-hoc test. Comparisons not in dicated were not significant. (E, F ) The sites of origin of the contractile \nwaves are shown for all patients, with adenomyosis patients separated from controls. (G) Representative kymograph of \na myometrial strip along the serosa-to-endometrium axis, showing contraction frequency and direction change after \nadministration of 10 µM oxytocin. \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nOxytocin-induced contractility in adenomyosis and control patients \nBecause oxytocin receptors have been previously reported to be overexpressed in adenomyosis \nmyometrium (27,32), w e next examined the contractile response of control and adenomyosis \ntissue to 10 µM oxytocin. This concentration was chosen because it elicited pronounced changes \nin motility, which  were difficult to detect at lower concentrations (≤1 µM);  non-pregnant \nhysterectomy myometrium is known to be much less oxytocin-sensitive than pregnant cesarean \nsection myometrium (33,37,38). Oxytocin induced time-dependent alterations. Right after \nadministration, it caused a transient (0-7 min) state of “tremor” , characterized by short-duration, \nlow-amplitude, high-frequency contractions (Figure 2G, Video S1, Figure S1). At longer times after \nadministration (7-30 min), the slice reached a stationary state where contractions were more \nfrequent than before administration (Figure 2A), but less frequent than in the transient “tremor” \nepisode (Figure S1 ). While oxytocin did not affect wave amplitude (Figure 2B, Figure S1), it \nincreased wave velocity (Figure 2D, Figure S1) and concomitantly decreased contraction duration \n(Figure 2C, Figure S1). Changes induced by oxytocin  were similar in control and adenomyosis \npatients (Figures 2B-E) except for frequ ency, where control uteri exhibited  a stronger response \nthan adenomyosis (Figure 2A). \nA striking effect of oxytocin was that it consistently shifted the site of origin of contractile waves \ntowards the endometrium (Figure 2E). This shift was present in both patient groups, but was more \npronounced in controls  (Figure 2F). This resulted in a majority of waves traveling from the \nendometrium towards the serosa after oxytocin administration (Figure 2G, Video S1).  \nTo understand the molecular origin of this change, we mapped the density of OTR receptors along \nthe serosa -to-endometrium axis (Fig ure 3A-D).  We found that OTR e xpression continuously \nincreased from the serosa to the endometrium (Figure 3A,E): OTR expression was highest in the \nsub-endometrial myometrium in both patient groups. Adenomyosis patien ts showed a \nsignificantly higher average OTR density, 1.8-fold, than controls (Figure 3F). \n \n \n \n \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\n \nFigure 3: Oxytocin receptor density follows an increasing serosa -to-endometrium gradient and is higher in \nadenomyosis patients. (A) Full-thickness myometrium section immunostained for OTR, mosaic of ~200 stitched tiles. \n(B) Magnified view of one ROI used for analysis (780 x 780 µm; black square in A). (C) Magnified view of the raw signal \n(blue square in B). (D) Binary obtained by thresholding the Trainable Weka Segmentation probability map in Fiji. (E) OTR \ndensity profiles ± SD along the serosa-to-endometrium axis (0 = serosa | 1 = endometrium). (F) Integrated OTR density \n(area under each profile) for adenomyosis (n = 10) and controls (n = 9). Violin plots with median and quartiles; Welch’s \nt-test, **p = 0.0015. \n \nDiscussion \nMost uterine contractility organ bath studies focus on the force signal, without looking at the strip \nitself. In this study, we harnessed optical tracking techniques commonly used in the assessment \nof intestinal motility to monitor the contractility of full-thickness myometrium strips. This revealed \nkey spatiotemporal - as opposed to just temporal - information about myometrial contractility. \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nThis approach allowed us to compare outer vs inner myometrium and to derive data pertaining to \ncontractile wave initiation and propagation. The baseline ex vivo frequencies we find (~2 cycles / \n10 min) are similar to ex vivo values reported with force tran sducers (33,39), but are ~5-10 times \nlower than in vivo values (10,17,18). \nThe whole myometrium contracts ex vivo, not just the JZ \nMRI (4) and ultrasound (9–11,40) investigations have consistently shown that , in vivo, only the \ninner myometrium contracts (is strained, deformed). In contrast, in our ex vivo study, we found \nthat outer and inner myometrium displayed similar contractile amplitude and frequency (Fig ure \n1): contractile waves propagated seamlessly from one region to the other, and could alternatively \noriginate in the outer or inner myometrium (Fig ure 2 , Video S 1). How do we reconcile these \napparently contradictory in vivo and ex vivo observations? We have previously shown that ex vivo \nuterine contractility is insensitive to tetrodotoxin (41), i.e., it is myogenic. Although nerve fibers \nare still present in the tissue we monitor, they have been severed from their nerve cell bodies in \nthe hypogastric plexus (42) during the hysterectomy operation and are de facto no longer \nfunctional. In a forthcoming publication, we show that adrenergic inhibitory innervation is mostly \nconcentrated in the outer myometrium: it is reasonable to conclude that this innervation actively \nquenches, in vivo, the potential myogenic contractility of the outer myometrium. \nOxytocin receptor distribution can control contractile wave direction \nAt baseline, contractile waves originated at various sites along the myometrium thickness, either \nin the outer or the inner myometrium. After oxytocin administration , contractile waves \nsystematically originated in the inner myometrium (close to the endometrium) in both  patient \ngroups (Figure 2), and thereafter propagated to the outer myometrium. We further found that this \ninner myometrium region corresponds to the one where OTR expression is highest (Figure 3). This \nsuggests that the spatial distribution of OTR can determine the preferential site o f origin of \ncontractions and their ensuing direction . It is known that oxytocin injection modulates the \nfrequency and direction of uterine peristalsis in vivo (6,18), and that OTR expression fluctuates \nacross the menstrual cycle (43). A differential expression of OTR at the fundus vs cervix at different \nphases of the menstrual cycle could serve as a guiding cue for cervico -fundal vs fundo-cervical \nwaves (32,44).  \nAdenomyosis vs control: more oxytocin receptors but similar myogenic \ncontractility  \nWe found that spontaneous contraction frequency, amplitude, duration, and velocity did not differ \nbetween adenomyosis patients and controls. This is consistent with a recent in vivo ultrasound \nstudy of adenomyotic patients (24) that found only minor alterations to frequency, amplitude, \nduration, and velocity, but a significant alteration of contraction coordination. In contrast, Guo et \nal. (27) reported higher spontaneous amplitude in ex vivo strips from adenomyosis patients. Their \nmeasurements were restricted to the outer myometrium of patients with highly symptomatic \nadenomyosis, whereas we averaged contractility over the whole myometrial thickness in a cohort \nthat was not selected based on symptom severity. Our results indicate that the spontaneous \nmyogenic contractility of adenomyosis myometrium does not significantly differ from that of \ncontrols: our experiment does not capture alterations o f the neurogenic component, which are, \nas we will show in a forthcoming publication, substantial. \nWe found that  OTR density was ~2 times higher in adenomyosis than in controls (Fig ure 3), in \nagreement with previous reports of OTR overexpression in adenomyotic myometrium (27,32). \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nAlthough we did not assess OTR distribution along the cervico -fundal axis, it is possible that \nfurther alterations in the spatial distribution of these receptors could lead to a loss of contractile \nwave coordination and direction (24,32,45). \nAdministration of 10 µM oxytocin increased contraction frequency in both patient grou ps. The \nfrequency increase was , however, not correlated with OTR e xpression: it was  1.6-fold lower in \nadenomyosis uteri than in controls, whereas OTR density was 1.8 times-fold higher in \nadenomyosis patients than in controls. It is likely that the concentration we used (10 µM) is \nsaturating, i.e., it yielded a response independent of OTR expression. Dose-response experiments \ncould reveal a sensitivity to lower oxytocin concentrations in adenomyosis uteri. It is also possible \nthat receptor overexpression does not translate to a stronger response if receptors are \ndesensitised or internalised, or if downstream signalling is altered (33,34). \nStudy limitations \nSeveral limitations of this study should be acknowledged. First, our sample size was modest (n = \n8-11 per group), which limited statistical power: smaller between -group differences in oxytocin-\ninduced contractility may have gone undetected. Many novel results were, however, common to \nboth groups and therefore based on a more substantial number of patients (n = 19 total). Second, \nwe extracted contractile signals from operator -defined pairs of points : a more sophisticated \napproach would consider the full defo rmation field of the slice (31). Third, we used a single \noxytocin concentration and did not obtain full dose-response curves for adenomyosis vs control \ntissue. These data will be provided in a future report. The high concentration used (10 µM)  \nappeared necessary to elicit changes in contractility in the non -pregnant, hysterectomy \nmyometrium we examined. The literature confirms that non-pregnant myometrium is much less \nsensitive to oxytocin than pregnant caesarean-section tissue  (33,37,38). Fourth, most of the \nadenomyosis tissue we collected did not present with adenomyosis lesions in the particular \nbiopsy we retrieved; our data therefore pertains to potential systemic (myometrium -wide) \nalterations to myogenic contractility, rather than local alterations induced specifically by lesions.  \nConclusion \nWe provided spatially resolved measurements of contractility across the whole thickness of the \nnon-pregnant human myometrium, from serosa  to endometrium. Ex vivo contractility is purely \nmyogenic, and myogenic motility  was similar in the outer, middle, and inner myometrium . This \nsharply contrasts with the in vivo  (MRI, TVUS) observation that only the inner myometrium is \ninvolved in contractile activity. We suggest that the difference resides in the neuro-myogenic \nactivity of the uterus in vivo, i.e., active quenching of contractions by inhibitory nerve activity. \nOxytocin (10  µM) caused contractile waves to originate in the inner myometrium close to the \nendometrium, in a region where OTR de nsity was highest. This indicates  that OTR spatial \nheterogeneity in the uterus can control the direction of uterine contractile waves during the \nmenstrual cycle. Although adenomyosis uteri exhibited higher OTR expression, their spontaneous \nand oxytocin -induced myogenic contractile behavio ur did not differ substantially from t hat of \ncontrol samples. \nAcknowledgement  \nWe thank Olivier Cardoso (MSC laboratory) for developing and sharing the TrackV2 plugin. We \nthank Thomas Rios, Marine Panza and Guillaume Quibeuf  for their help with oxytocin receptor \nimaging and acknowledge the ImagoSeine core facility of the Institut Jacques Monod, member of \nthe France BioImaging infrastructure (https://ror.org/01y7vt929) supported by the French National \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nResearch Agency (ANR -24-INBS-0005 FBI BIOGEN) and GIS -IBiSA. We thank the \nanatomopathology staff of Hôpital Paris Saint -Joseph: A. Gérard, G. Toutain, M. Peronnet and \nA.Bibollet, Olivia and S. Dequeant.  \n  \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\nReferences \n1.  Myers KM, Elad D. Biomechanics of the human uterus. Wiley Interdiscip. Rev. Syst. Biol. \nMed. 2017;9(5).  \n2.  Núñez-Troconis J, Carvallo D, Martínez-Núñez E. Primary Dysmenorrhea: \npathophysiology. Investigacion Clinica (Venezuela) 2021;62(4):378–406.  \n3.  Woodbury RA, Torpin R, Child GP , Watson H, Jarboe M. Myometrial physiology and its \nrelation to pelvic pain. J Am Med Assoc 1947;134(13):1081–5.  \n4.  Kido A, Togashi K, Nishino M, Miyake K, Koyama T, Fujimoto R, et al. Cine MR imaging of \nuterine peristalsis in patients with endometriosis. Eur Radiol 2007;17(7):1813–9.  \n5.  Leyendecker G, Kunz G, Wildt L, Beil D, Deininger H. Uterine hyperperistalsis and \ndysperistalsis as dysfunctions of the mechanism of rapid sperm transport in patients with \nendometriosis and infertility. Human Reproduction 1996;11(7):1542–51.  \n6.  Leyendecker G, Kunz G, Herbertz M, Beil D, Huppert P , Mall G, et al. Uterine peristaltic \nactivity and the development of endometriosis. Ann N Y Acad Sci 2004;1034:338–55.  \n7.  Salmeri N, Di Stefano G, Viganò P , Stratton P , Somigliana E, Vercellini P . Functional \ndeterminants of uterine contractility in endometriosis and adenomyosis: a systematic \nreview and meta-analysis. Fertil Steril 2024;122(6):1063–78.  \n8.  Pinto V , Matteo M, Tinelli R, Mitola PC, De Ziegler D, Cicinelli E. Altered uterine \ncontractility in women with chronic endometritis. Fertil Steril 2015;103(4):1049–52.  \n9.  de Vries K, Lyons EA, Ballard G, Levi CS, Lindsay DJ. Contractions of the inner third of the \nmyometrium. Am J Obstet Gynecol 1990;162(3):679–82.  \n10.  Lyons EA, Taylor PJ, Xin Hua Zheng, Ballard G, Levi CS, Kredentser J V . Characterization of \nsubendometrial myometrial contractions throughout the menstrual cycle in normal fertile \nwomen. Fertil Steril 1991;55(4):771–4.  \n11.  Birnholz JC. Ultrasonic visualization of endometrial movements. Fertil Steril \n1984;41(1):157–8.  \n12.  Levy G, Dehaene A, Laurent N, Lernout M, Collinet P , Lucot JP , et al. An update on \nadenomyosis. Diagn Interv Imaging 2013;94(1):3–25.  \n13.  Noe M, Kunz G, Herbertz M, Mall G, Leyendecker G. The cyclic pattern of the \nimmunocytochemical expression of oestrogen and progesterone receptors in human \nmyometrial and endometrial layers: characterization of the endometrial–subendometrial \nunit. Human Reproduction 1999;14(1):190–7.  \n14.  Brosens JJ, de Souza NM, Barker FG. Uterine junctional zone: function and disease. The \nLancet 1995;346(8974):558–60.  \n15.  Brown HK, Stoll BS, Nicosia S V ., Fiorica J V ., Hambley PS, Clarke LP , et al. Uterine \njunctional zone: correlation between histologic findings and MR imaging. Radiology \n1991;179(2):409–13.  \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\n16.  McCarthy S, Scott G, Majumdar S, Shapiro B, Thompson S, Lange R, et al. Uterine \njunctional zone: MR study of water content and relaxation properties. Radiology \n1989;171(1):241–3.  \n17.  Rees CO, de Boer A, Huang Y , Wessels B, Blank C, Kuijsters N, et al. Uterine contractile \nactivity in healthy women throughout the menstrual cycle measured using a novel \nquantitative two-dimensional transvaginal ultrasound speckle tracking method. Reprod \nBiomed Online 2023;46:115–22.  \n18.  Kunz G, Noe M, Herbertz M, Leyendecker G. Uterine peristalsis during the follicular phase \nof the menstrual cycle: Effects of oestrogen, antioestrogen and oxytocin. Hum Reprod \nUpdate 1998;4(5):647–54.  \n19.  Bulletti C, Ziegler D De, Polli V , Del Ferro E, Palini S, Flamigni C. Characteristics of uterine \ncontractility during menses in women with mild to moderate endometriosis. Fertil Steril \n2002;77(6):1156–61.  \n20.  de Ziegler D, Bulletti C, Fanchin R, Epiney M, Brioschi PA. Contractility of the nonpregnant \nuterus: The follicular phase. Ann N Y Acad Sci 2001;943:172–84.  \n21.  Leyendecker G, Wildt L, Mall G. The pathophysiology of endometriosis and adenomyosis: \nTissue injury and repair. Arch Gynecol Obstet 2009;280(4):529–38.  \n22.  Gordts S, Grimbizis G, Tanos V , Koninckx P , Campo R. Junctional zone thickening: an \nendo-myometrial unit disorder. Facts Views Vis Obgyn 2023;15(4):309.  \n23.  Harmsen MJ, Trommelen LM, de Leeuw RA, Tellum T, Juffermans LJM, Griffioen AW, et al. \nUterine junctional zone and adenomyosis: comparison of MRI, transvaginal ultrasound \nand histology. Ultrasound in Obstetrics and Gynecology. 2023;62(1):42–60.  \n24.  Rees CO, Thomas S, de Boer A, Huang Y , Zizolfi B, Foreste V , et al. Quantitative ultrasound \nmeasurement of uterine contractility in adenomyotic vs. normal uteri: a multicenter \nprospective study. Fertil Steril 2024;121(5):864–72.  \n25.  Arena A, Zanello M, Orsini B, Degli Esposti E, Iodice R, Altieri M, et al. Uterine peristalsis in \nwomen affected by adenomyosis: A step towards functional assessment. International \nJournal of Gynecology and Obstetrics 2024;165(2):666–71.  \n26.  Kissler S, Zangos S, Wiegratz I, Kohl J, Rody A, Gaetje R, et al. Utero-tubal sperm transport \nand its impairment in endometriosis and adenomyosis. Ann N Y Acad Sci \n2007;1101(1):38–48.  \n27.  Guo SW, Mao X, Ma Q, Liu X. Dysmenorrhea and its severity are associated with increased \nuterine contractility and overexpression of oxytocin receptor (OTR) in women with \nsymptomatic adenomyosis. Fertil Steril 2013;99(1):231–40.  \n28.  Leyendecker G, Bilgicyildirim A, Inacker M, Stalf T, Huppert P , Mall G, et al. Adenomyosis \nand endometriosis. Re-visiting their association and further insights into the mechanisms \nof auto-traumatisation. An MRI study. Arch Gynecol Obstet 2015;291(4):917–32.  \n29.  Janssen PWM, Lentle RG. Spatiotemporal mapping techniques for quantifying gut \nmotility. Lecture Notes in Computational Vision and Biomechanics 2013;10:219–41.  \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\n30.  Lentle RG, Hulls CM. Quantifying patterns of smooth muscle motility in the gut and other \norgans with new techniques of video spatiotemporal mapping. Front Physiol 2018;9(338).  \n31.  Huang Y , Rees C, Sammali F , Blank C, Schoot D, Mischi M. Characterization of Uterine \nPeristaltic Waves by Ultrasound Strain Analysis. IEEE Trans Ultrason Ferroelectr Freq \nControl 2022;69(6):2050–60.  \n32.  Zhang Y , Yu P , Sun F , Li TC, Cheng JM, Duan H. Expression of oxytocin receptors in the \nuterine junctional zone in women with adenomyosis. Acta Obstet Gynecol Scand \n2015;94(4):412–8.  \n33.  Shmygol A, Gullam J, Blanks A, Thornton S. Multiple mechanisms involved in oxytocin-\ninduced modulation of myometrial contractility. Acta Pharmacol. Sin. 2006;27(7):827–32.  \n34.  Arrowsmith S, Wray S. Oxytocin: Its mechanism of action and receptor signalling in the \nmyometrium. J. Neuroendocrinol. 2014;26(6):356–69.  \n35.  Arganda-Carreras I, Kaynig V , Rueden C, Eliceiri KW, Schindelin J, Cardona A, et al. \nTrainable Weka Segmentation: a machine learning tool for microscopy pixel \nclassification. Bioinformatics 2017;33(15):2424–6.  \n36.  Chevalier NR. The first digestive movements in the embryo are mediated by \nmechanosensitive smooth muscle calcium waves. Philos Trans R Soc Lond B Biol Sci \n2018;373(1759).  \n37.  Arrowsmith S, Quenby S, Weeks A, Burdyga T, Wray S. Poor spontaneous and oxytocin-\nstimulated contractility in human myometrium from postdates pregnancies. PLoS One \n2012;7(5).  \n38.  Balki M, Erik-Soussi M, Kingdom J, Carvalho JCA. Oxytocin pretreatment attenuates \noxytocin-induced contractions in human myometrium in vitro. Anesthesiology \n2013;119(3):552–61.  \n39.  Arrowsmith S, Robinson H, Noble K, Wray S. What do we know about what happens to \nmyometrial function as women age? J Muscle Res Cell Motil 2012;33(3–4):209–17.  \n40.  Kunz G, Beil D, Deininger H, Wildt L, Leyendecker G. The dynamics of rapid sperm \ntransport through the female genital tract: evidence from vaginal sonography of uterine \nperistalsis and hysterosalpingoscintigraphy. Hum Reprod [Internet] 1996 [cited 2026 Nov \n10];11(3):627–32. Available from: https://pubmed.ncbi.nlm.nih.gov/8671281/ \n41.  Huon E, Rayssac J, Lamau M-C, Abo C, Dietrich G, Abrahami Y , et al. A high-throughput \n96-well plate method to measure human uterine contractility. Front Pharmacol \n2026;17:1953934.  \n42.  Astruc A, Roux L, Robin F , Sall NR, Dion L, Lavoué V , et al. Advanced Insights into Human \nUterine Innervation: Implications for Endometriosis and Pelvic Pain. J Clin Med \n2024;13(5):1433.  \n43.  Fuchs AR, Behrens O, Maschek H, Kupsch E, Einspanier A. Oxytocin and vasopressin \nreceptors in human and uterine myomas during menstrual cycle and early pregnancy. \nHum Reprod Update 1998;4(5):594–604.  \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint \n\n44.  Huang M, Li X, Guo P , Yu Z, Xu Y , Wei Z. The abnormal expression of oxytocin receptors in \nthe uterine junctional zone in women with endometriosis. Reproductive Biology and \nEndocrinology 2017;15(1).  \n45.  Vidal A, Tepasse P , Vinayahalingam V , Cottagnoud S, Gulz M, Karrer T, et al. Uterine \nContractility Changes in Adenomyosis: Evidence from a Systematic Review and Meta-\nAnalysis. Biomedicines. 2025;13(11).  \n  \n . CC-BY-NC 4.0 International licenseIt is made available under a \nperpetuity. \n is the author/funder, who has granted medRxiv a license to display the preprint in(which was not certified by peer review)preprint \nThe copyright holder for thisthis version posted November 11, 2026. ; https://doi.org/10.64898/2026.11.10.26367720doi: medRxiv preprint","source_license":"CC0","license_restricted":false}