{"paper_id":"de0e4b2d-9cad-45e0-b8b8-5e0ca5a3fe7d","body_text":"Endometriosis is a gynecological disorder characterized by the presence of endometrial-like\ntissue outside the uterine cavity. It affects approximately 30–50% of women with infertility\n[ 1 ]. One of the major challenges associated with\nendometriosis is the delay in both diagnosis and treatment. An estimated 6 out of 10\npatients with endometriosis are not correctly diagnosed, leading to over half of\nendometriosis patients not receiving the appropriate treatment. On average, women experience\na diagnostic delay of 7 to 11 years from the onset of symptoms to an accurate diagnosis\n[ 2 ]. These diagnostic inaccuracies and delays\nrepresent a significant global health concern, highlighting the inadequate recognition of\nclinical symptoms in affected individuals and the lack of understanding of the disease\nmechanism [ 3 ].\nThe better understanding of the pathophysiology of early-stage endometriosis is crucial for\ndeveloping new therapeutic strategies. Previous reports have shown that multiple factors\nsuch as genetic, immunological, hormonal, and environmental factors contribute to the onset\nof the disease [ 4 , 5 , 6 ]. Among these multiple factors, one of\nthe major challenges is to investigate endometriosis as chronic inflammatory disease [ 7 ]. However, monitoring cytokine levels during the early\nstages of the disease in humans is difficult, as these stages are often asymptomatic [ 8 ].\nIn endometriosis, which is a chronic inflammatory disease and a debilitating condition\naccompanied by pelvic pain, there has been increasing attention on the involvement of\npro-inflammatory and anti-inflammatory cytokines in the pathogenesis and their potential as\ndiagnostic biomarkers [ 9 ]. These cytokines have been\nreported to play important roles in the invasion, angiogenesis, survival, and growth of\nendometriotic lesions [ 9 , 10 , 11 ]. Multiple studies have\nshown that pro-inflammatory cytokines such as IL-2, IL-6, TNF-α, and IFN-γ promote the\nestablishment and progression of endometriosis. On the other hand, IL-10, classified as an\nanti-inflammatory cytokine, is known to suppress the production of pro-inflammatory\ncytokines such as IL-2, IL-6, TNF-α, and IFN-γ [ 12 ].\nTo accurately evaluate the interaction between pro-inflammatory and anti-inflammatory\ncytokines and their role in the pathogenesis of endometriosis, longitudinal profile studies\nfrom the early stages of lesions are essential.\nVarious animal models of endometriosis have been reported using mice, rats, and non-human\nprimates, providing valuable insights into endometriosis research [ 13 , 14 , 15 ]. Mouse models have been most frequently reported due to their ease of\nhusbandry, cost-effectiveness, and high potential for genetic modifications [ 13 ]. Recently, a mouse model was reported that allows\nnon-invasive observation of endometriotic lesion formation by transplanting uterine tissue\nfragments of inbred transgenic mice carrying a high-luminescent reporter gene into the\nperitoneal cavity of syngeneic mice using bioluminescence imaging [ 16 ]. Such luminescent mouse models enabling non-invasive imaging are\nconsidered useful for studying the molecular mechanisms of disease onset and progression,\nincluding cytokine dynamics associated with the growth of endometriotic lesions, as well as\nfor therapeutic research.\nIn this study, we aimed to establish a mouse model capable of measuring cytokine dynamics\nassociated with endometriotic lesion growth. Following previous reports, we used C57BL/6\nmice expressing Emerald luciferase (ELuc) which is stabler and brighter than firefly\nluciferase [ 16 ,  17 ]. As recipients, we utilized a C57BL/6 congenic strain of albino\n( c/c ) and hairless ( hr/hr ) for efficient luminescent\nimaging [ 18 ]. Induced endometriotic tissues were\nanalysed using X-ray micro-computed tomography ( μ CT) imaging to understand\ncomprehensive spatial relationship between transplanted and recipient tissues in\nthree-dimension. Furthermore, by performing simultaneous multiplex analysis of serum samples\nfrom each recipient, we were able to capture characteristics patterns of temporal changes in\nthe levels of seven inflammation-related cytokines (IL-1β, IL-2, IL-6, IL-10, IL-12p70,\nIFN-γ, and TNF-α).\n\nAll animal experiments and use of genetically modified mice were approved by the\nInstitutional Animal Care and Use Committee (T2023-EP001 and T2024-EP001) and the Genetic\nRecombination Experiments Committee (T2022-02-17) of the RIKEN Tsukuba Branch,\nrespectively. All mice were cared for and used humanely in accordance with the Committee’s\nguiding principles. A total of 77 female mice were used in this study, consisting of 36\nhemizygous C57BL/6JJmsSlc-Tg(CAG-ELuc)1Nkzom transgenic mice expressing ELuc (B6-CAG-ELuc,\nRBRC11951) [ 16 ] at 8 weeks old and 41 congenic\nB6.Cg- Tyr c-2J /Tyr c-2J -Hr hr / Hr hr \n(B6.Cg- c/c - hr/hr , RBRC05798) mice [ 19 ] at 10–12 weeks old. Female B6-CAG-ELuc mice and\nB6.Cg- c/c - hr/hr  mice were obtained from the\nExperimental Animal Division, RIKEN BRC through the National BioResource Project of the\nMEXT, Japan. Mice were acclimatized for more than seven days before the start of\nexperiments. All mice were maintained as specific pathogen-free at a temperature of\n23–25°C under a 12-h light/dark cycle. The mice were fed with standard food (CE-2; CLEA\nJapan, Inc., Tokyo, Japan) and water  ad libitum .\nThe genome scanning by SNPs markers was used to investigate the genetic background of\nB6.Cg- c/c - hr/hr  mice. PCR templates for SNP analysis\nwere prepared from tail tissues of two parental breeder pairs of adult\nB6.Cg- c/c - hr/+  females and\nB6.Cg- c/c - hr/hr  males which produced\nB6.Cg- c/c - hr/hr  recipient females. The genotyping was\nperformed using custom TaqMan SNP Genotyping Assays designed for a set of validated SNPs\nat 180 polymorphic loci across all chromosomes [ 20 ]. The samples’ genotype was compared with those of various C57BL/6 substrains\nincluding C57BL/6JJcl, C57BL/6JCrlj, C57BL/6JJmsSlc, C57BL/6NCrlCrlj, C57BL/6NCrl,\nC57BL/6NJcl, C57BL/6NSlc, C57BL/6NTac, as well as DBA/2JJcl and CBA/J strains. The\nevaluation of congenic status primarily focused on the genotype matching rate with the\ndonor strain C57BL/6JJmsSlc, while also taking into consideration the matching of\ngenotypes in the major histocompatibility complex  H2  region and minor\nhistocompatibility loci [ 21 ].\nEndometriotic lesions were induced by transplantation of the uterine tissues as\npreviously described [ 16 ] with modifications to\nminimize invasive procedures for the cytokine analysis ( Supplementary Table 1 ). The B6-CAG-ELuc female\nmice of 8 weeks old as donors and congenic\nB6.Cg- c/c - hr/hr  female mice of 10–12 weeks old as\nrecipients were used for this study. Both donor and recipient females were examined for\nvaginal appearance, and those not showing characteristics of either proestrus or estrus\n(vaginal swelling or cornification) were selected for use in this study. The donor females\nwere euthanised by cervical dislocation under inhalation anesthesia with isoflurane\n(Viatris, Inc., Tokyo, Japan) and both uterine horns, confirmed not to be edematous as\ntypically seen in estrus, were dissected out into sterile saline (Otsuka Pharmaceutical\nFactory, Inc., Tokushima, Japan). The uterine horns were cut into rounds at approximately\n1 mm interval, and minced into smaller fragments of approximately 1 mm × 1 mm on a\nparaffin wax plate (GC Corp., Tokyo, Japan) using a carbon steel razor blade of 0.1 mm\nthickness (FA-10, FEATHER Safety Razor Co., Ltd., Osaka, Japan). Small incision of\napproximately 3 mm was made in the abdominal skin and muscle layer of each recipient\nanesthetized with a mixture of medetomidine (Domitor, Nippon Zenyaku Kogyo Co., Ltd.,\nFukushima, Japan), midazolam (Dormicum, Maruishi Pharmaceutical Co., Ltd., Osaka, Japan)\nand butorphanol (Vetorphale, Meiji Seika Pharma Co., Ltd., Tokyo, Japan). Approximately 30\nuterine tissue fragments of one donor were suspended in 200  µ l of sterile\nsaline and transferred into the peritoneal cavity of each recipient. Surgical incisions in\nthe muscle layer were closed using 6-0 silk suture (Natsume Seisakusho, Tokyo, Japan), and\nincisions in skin were clipped (9 mm MikRon autoclip, Becton Dickinson and Co., Franklin\nLakes, NJ, USA). The recipient was injected subcutaneously with 0.5\n µ g/mouse of 17b estradiol (E2) (Progynon ® -Depot, Fuji Pharma\nCo., Ltd., Toyama, Japan) in corn oil (FUJIFILM Wako Pure Chemical Corp., Osaka, Japan)\nafter the operation and subsequently once a week to promote the growth of lesions and\nangiogenesis [ 16 ,  22 ]. The schema of experimental procedures is shown in  Fig. 1 Fig. 1. Generation of the endometriosis mouse. (A) An experimental work flow for the\npreparation of donor tissue fragments, surgical transplantation of the donor uterine\ntissues to the recipient, and observation of luminescent signals by IVIS. (B)\nExperimental time schedule. Recipient mouse was injected subcutaneously with 0.5\n µ g/mouse of 17β estradiol (E2) weekly. Endometriotic lesions were\ncollected on 14, 28 and 42 days post transplantation (d.p.t.). n=10 mice per group\nin each time point. .\nGeneration of the endometriosis mouse. (A) An experimental work flow for the\npreparation of donor tissue fragments, surgical transplantation of the donor uterine\ntissues to the recipient, and observation of luminescent signals by IVIS. (B)\nExperimental time schedule. Recipient mouse was injected subcutaneously with 0.5\n µ g/mouse of 17β estradiol (E2) weekly. Endometriotic lesions were\ncollected on 14, 28 and 42 days post transplantation (d.p.t.). n=10 mice per group\nin each time point.\nThe growth of endometriotic lesions in the recipient mice was observed on 14, 28 and 42\ndays post transplantation (abbreviated as d.p.t.). Each recipient mouse was\nintraperitoneally injected with 150 mg/kg D-luciferin (FUJIFILM Wako Pure Chemical Corp.,\nOsaka, Japan) under inhalation anesthesia with isoflurane. Ten minutes after D-luciferin\nadministration, bioluminescence was visualized and measured by using the IVIS Lumina XRMS\nSeries III imaging system (PerkinElmer Inc., Waltham, MA, USA) with an exposure time of 30\ns.\nThe recipient and control mice were deeply anesthetized with a mixture of medetomidine,\nmidazolam, and butorphanol for blood collection via cardiac puncture on 14, 28 and 42\nd.p.t. Age-matched untreated B6.Cg- c/c - hr/hr  mice (n=5)\nserved as controls on 0 d.p.t. The mice were euthanized after blood collection. Collected\nblood samples were incubated for 30 min at RT, and centrifuged at 700×  g \nin 4°C for 10 min. The serum was collected and stored at −80°C freezer until further\nanalysis. Tissue samples of endometriotic lesion were collected for morphological\nobservation and recording under a dissecting microscope (Stereozoom S9i, Leica\nMicrosystems, Ltd., Heerbrugg, Switzerland) and IVIS imaging system. Subsequently, the\nlesion tissues were fixed with 4% paraformaldehyde (PFA) in PBS (pH 7.4) for further\nmorphological analysis.\nThe three-dimensional (3D)  µ CT imaging [ 23 ] was conducted with a Comscan-Xmate E90S system (Comscantecno Co., Ltd.,\nKanagawa, Japan). The fixed tissues in PFA were rinsed with PBS three times, each for 5\nmin. The tissues were post-fixed in Bouin’s fixative solution for 24 h at RT and stored in\n70% ethanol (EtOH) at RT until further processing. Prior to  µ CT scanning,\nthe fixed tissues were immersed overnight in a contrast reagent consisting of 1%\nphosphotungstic acid (PTA) in 70% EtOH at RT to enhance the contrast of the image of soft\ntissues [ 24 ]. The contrast-enhanced tissues were\nscanned using a tube peak voltage of 40 kV and a tube current of 100  µ A.\nThe tissue samples were rotated 360° in 0.3° steps, generating 1,200 projection images.\nThe acquired images were reconstructed and analysed to generate three-dimensional\ntomographic visualizations using OsiriX software (Pixmeo, Geneva, Switzerland).\nThe fixed tissues after  µ CT imaging were processed by automated\ntissue-processor (Tissue-Tek ®  VIP TM , Sakura Finetek, Tokyo, Japan)\nand embedded in paraffin block. The embedded tissues were sectioned by a sliding microtome\n(Yamato Kohki, Saitama, Japan) at 4  µ m thickness on glass slides,\ndeparaffinized and stained with hematoxylin and eosin for histological observation. The\ntissue section was observed by using an upright Olympus BX51 microscope (Evident, Tokyo,\nJapan).\nLevels of serum cytokines including IL-1β, IL-2, IL-6, IL-10, IL-12p70, TNF-α, and IFN-γ\nwere quantified using multiplex magnetic immunobeads assay system [ 25 ] and relevant reagents (Mouse Bio-Plex Pro, Catalog No. L6000004C6,\nBio-Rad, Hercules, CA, USA). Each serum sample of 30  µ l was used for the\nassay according to the manufacturer’s instructions. Cytokine levels were expressed as\npg/ml.\nData were analysed using GraphPad Prism 9.0 (GraphPad Software Inc., La Jolla, CA, USA).\nWelch’s  t -test was used to analyse total luminescence data. The temporal\npatterns of cytokine levels were analysed by the two-way repeated measures ANOVA with\nfactor 1 (type of cytokine: 7 cytokines) and factor 2 (Time course: 0, 14, 28, 42 d.p.t.),\nfollowed by Tukey’s HSD test for post-hoc analysis. The correlation between each cytokine\nconcentration and luminescence value (log 10 [total flux]) at each time point was\nanalysed by Pearson’s correlation coefficient analysis. The temporal luminescence and\ncytokine level data were presented as the mean ± SEM.\n\nThe whole genome scanning using SNP markers was conducted to clarify the genetic\nbackground of the recipient B6.Cg- c/c - hr/hr  strain. The\nresults revealed that the genotype matching rate of the recipient strain with\nC57BL/6JJmsSlc was 91% (164 out of 180 loci), with all SNP markers on chromosome 17,\ncontaining the major histocompatibility complex  H2  region, being of\nB6-genotype. The remaining 9% of non-B6-genotype SNP loci were distributed across nine\nchromosomes 3, 5, 8, 10, 11, 13, 14, 16 and 18 ( Supplementary Table 2 ). Among these chromosomal\nsegments, the minor histocompatibility 8 locus ( H8 ) is known to\ncolocalize with  hr  gene on chromosome 14 [ 19 ,  21 ].\nUterine tissue fragments from B6-CAG-ELuc transgenic mice expressing Emerald luciferase\nwere transplanted into the peritoneal cavity of\nB6.Cg- c/c - hr/hr  recipients, and E2 was administered\n(E2(+)) to induce endometriotic lesions. As shown in  Fig. 2A Fig. 2. Longitudinal observation of endometriosis mouse model by non-invasive  in\nvivo  imaging. The ELuc light emission from the lesion in the abdominal\ncavity was detected by IVIS on 0, 14, 28, 42 d.p.t. in the same recipient mouse\nadministered with E2 (A) and without E2 (B). The exposure time was 30 seconds. (C)\nTemporal changes of the total luminescent flux (photon/second) with E2 (A) and\nwithout E2 (B) at four time points. E2(+): n=10 at each time point, E2(−): n=6.\nBars: mean ± SEM. Significant  P -values by Welch’s\n t -test are indicated: * P <0.05,\n** P <0.01, *** P <0.001. , we were able to detect non-invasively the location and growth progression of the\ntransplanted tissue fragments in the peritoneal cavity through bioluminescent signals\nimmediately after transplantation (day 0) and up to 14, 28, and 42 d.p.t. In\nnon-E2-administered (E2(−)) recipient mice, an increase in luminescence was observed from\nday 0 to day 28, but reached a plateau between days 28 and 42 ( Fig. 2B ).\nLongitudinal observation of endometriosis mouse model by non-invasive  in\nvivo  imaging. The ELuc light emission from the lesion in the abdominal\ncavity was detected by IVIS on 0, 14, 28, 42 d.p.t. in the same recipient mouse\nadministered with E2 (A) and without E2 (B). The exposure time was 30 seconds. (C)\nTemporal changes of the total luminescent flux (photon/second) with E2 (A) and\nwithout E2 (B) at four time points. E2(+): n=10 at each time point, E2(−): n=6.\nBars: mean ± SEM. Significant  P -values by Welch’s\n t -test are indicated: * P <0.05,\n** P <0.01, *** P <0.001.\nWhen comparing the luminescence of transplanted tissue fragments between E2(+) and E2(−)\nmice, as shown in Fig. 2C, the E2(+) mice showed significantly higher luminescence\ncompared to the E2(−) mice after 14 d.p.t., with a dramatic increase observed by 42 d.p.t.\nThese results indicate that the tissue transplants successfully engrafted and either grew\nor survived for 42 days regardless of E2 administration, with particularly notable growth\nobserved in the E2(+) mice.\nThe tissue engraftment sites were monitored by recording bioluminescent signals followed\nby autopsy examination on 14, 28, and 42 d.p.t. Engrafted lesions were observed around the\nsurgical suture sites in all recipients at all time points. In addition, consistent\nengraftment was observed around the pancreatic tissues throughout the experimental period,\nthough at a lower frequency than the suture sites, while a few lesions adhering to\ninguinal adipose tissues were observed in later stages on 28 and 42 d.p.t. ( Supplementary Fig. 1 ).\nPost-mortem endometriotic lesion tissues were observed under a dissecting microscope,\nfocusing on bioluminescent regions detected by IVIS imaging, to confirm the morphology of\nengrafted lesion tissues. Endometriotic tissue masses were confirmed to be successfully\nengrafted at all luminescent sites.\nAt 14 d.p.t., a dome-shaped tissue mass was found to be engrafted near the suture site\naround the median peritoneum. Blood capillaries branching from the recipient’s bilateral\ninferior epigastric vessels (arrowheads) were observed extending to the base where the\ndome-shaped tissue mass closely adhered to the peritoneum, reaching the dome-shaped mass\n( Fig. 3A Fig. 3. Representative images of endometriotic lesions by dissecting microscopy. (A) A\ndome-shaped tissue mass mainly consisted of two cystic structures (*) engrafted at\nthe suture site on the peritoneum on 14 d.p.t. The recipient’s epigastric vessels\nare indicated with arrowheads. White arrow: a part of black suture thread is faintly\nvisible through the tissue. (B) An engrafted tissue mass at the suture site (white\narrow) on the peritoneum on 28 d.p.t. The engrafted tissue consisted of multiple\ncysts containing pale yellow to transparent fluid (*). (C) An expanded cystic mass\n(*) found near the pancreas. The engrafted tissue was partly covered with the\nperipancreatic adipose tissue ( pat ). ). At 28 d.p.t., the engrafted tissue mass near the suture site on the peritoneum\nhad formed a cluster of multiple cysts containing pale yellow to transparent fluid.\nMultiple blood vessels had invaded the base of the engrafted tissue, and the entire\nsurface of the cystic clusters was covered by a thin membranous tissue with a network of\ncapillaries ( Fig. 3B ). At 42 d.p.t., larger\ncystic clusters were observed around the suture site. Additionally, cystic masses covered\nby a thin membrane, an adipose tissue and associated blood capillaries were observed near\nthe pancreas ( Fig. 3C ).\nRepresentative images of endometriotic lesions by dissecting microscopy. (A) A\ndome-shaped tissue mass mainly consisted of two cystic structures (*) engrafted at\nthe suture site on the peritoneum on 14 d.p.t. The recipient’s epigastric vessels\nare indicated with arrowheads. White arrow: a part of black suture thread is faintly\nvisible through the tissue. (B) An engrafted tissue mass at the suture site (white\narrow) on the peritoneum on 28 d.p.t. The engrafted tissue consisted of multiple\ncysts containing pale yellow to transparent fluid (*). (C) An expanded cystic mass\n(*) found near the pancreas. The engrafted tissue was partly covered with the\nperipancreatic adipose tissue ( pat ).\nAfter observing endometriotic lesions under a dissecting microscope, the lesion tissues\nwere fixed with PFA and treated by phosphotungstic acid for contrast staining of soft\ntissues. Using  μ CT imaging, three-dimensional images were generated to\nassess how the engrafted lesion tissue adhered to and formed lesions on either the host’s\nperitoneum or pancreatic adipose tissues ( Figs.\n4 A–C Fig. 4. Representative images of endometriotic lesions by 3D mCT imaging and conventional\nhistology. (A1) A 3D reconstructed mCT image of a dome-shape engrafted tissue on 14\nd.p.t. (see  Supplementary\nMovie 1A  for 360° view). (A2) A digital cross-section of A1 to visualize\nthe internal structure of the cyst. (B1) A cystic engrafted lesion associated with a\npancreatic tissue (see  Supplementary Movie 1B  for 360° view) and (B2) a digital cross-section of\nthe cystic engrafted lesion (*) and the surrounding capillaries on 28 d.p.t.\n pnc : pancreatic tissue. (C1) An expanded cystic lesion on the\nperitoneum with a dense capillary network extended on the surface of the cystic\nlesion on 42 d.p.t. (see  Supplementary Movie 1C  for 360° view). (C2) Two tissue cross-sections 1\nand 2 cut at different positions and angles, created from three-dimensional\n μ CT data. Expanded cysts (*) fused to form the lesion. (D)\nH&E-stained tissue section on 42 d.p.t. prepared by embedding in standard\nparaffin blocks after mCT image acquisition. An expanded cyst (*) with a\nfluid-filled luminal space was lined with single-layered epithelium and surrounded\nby uterine gland-like ( gl ) structures and interstitial stroma. ). We obtained three-dimensional voxel information for repeatative observations from\nany angle and created movies for the overall view (Figs. 4A1, B1, and C1,  Supplementary Movies 1A–C ). At 14\nd.p.t., the lesion tissue adhered tightly to the peritoneum, penetrating the muscular\nlayer to such an extent that the adhesion site could be confirmed from the outer side of\nthe muscular layer (behind the peritoneum) ( Supplementary Movie 1A ). Furthermore, observing\ntissue sections at arbitrary angles revealed that many lesions had developed into fused\nlesions consisting of multiple cysts of approximately 2–3 mm in diameter (Figs. 4A2, B2,\nand C2). The lesion tissue near the pancreas did not directly adhere to the pancreas\nitself but became engrafted in the surrounding pancreatic adipose tissue, where the lesion\ntissue was enveloped by a reticular network of capillaries extending from either the\nadipose tissue or pancreatic tissue. In the lesion tissue that had adhered to the\nperitoneum at 42 d.p.t., we could observe an intricate vascular network surrounding the\nbase of the lesion tissue (Figs. 4C1 and C2,  Supplementary Movie 1C ). After\n μ CT analysis, cyst-containing lesion tissues were embedded in paraffin,\nsectioned, and observed with H&E staining. The lesion tissue consisted of large cysts\nlined by columnar epithelium along with glandular tissues and interstitial stroma ( Fig. 4D ).\nRepresentative images of endometriotic lesions by 3D mCT imaging and conventional\nhistology. (A1) A 3D reconstructed mCT image of a dome-shape engrafted tissue on 14\nd.p.t. (see  Supplementary\nMovie 1A  for 360° view). (A2) A digital cross-section of A1 to visualize\nthe internal structure of the cyst. (B1) A cystic engrafted lesion associated with a\npancreatic tissue (see  Supplementary Movie 1B  for 360° view) and (B2) a digital cross-section of\nthe cystic engrafted lesion (*) and the surrounding capillaries on 28 d.p.t.\n pnc : pancreatic tissue. (C1) An expanded cystic lesion on the\nperitoneum with a dense capillary network extended on the surface of the cystic\nlesion on 42 d.p.t. (see  Supplementary Movie 1C  for 360° view). (C2) Two tissue cross-sections 1\nand 2 cut at different positions and angles, created from three-dimensional\n μ CT data. Expanded cysts (*) fused to form the lesion. (D)\nH&E-stained tissue section on 42 d.p.t. prepared by embedding in standard\nparaffin blocks after mCT image acquisition. An expanded cyst (*) with a\nfluid-filled luminal space was lined with single-layered epithelium and surrounded\nby uterine gland-like ( gl ) structures and interstitial stroma.\nWe measured the serum level of inflammation-related cytokines such as IL-1β, IL-2, IL-6,\nIL-10, IL-12p70, TNF-α, and IFN-γ on 14, 28 and 42 d.p.t. in the endometriosis mouse model\nusing a multiplex magnetic immunobeads assay. As shown in  Figs. 5 A–G Fig. 5. Change in serum level of cytokines overtime. Serum levels of IFN-γ (A), TNF-α (B),\nIL-12p70 (C), IL-1β (D), IL-6 (E), IL-2 (F), IL-10 (G) in the recipient mice with\nendometriotic lesions (n=10 per group) and non-treated control as 0 d.p.t. (n=5).\nBars: mean ± SEM. Significant  P -values are indicated as follows:\n* P <0.05, ** P <0.01,\n*** P <0.001, and **** P <0.0001. , examining statistically the graphs of each cytokine concentration revealed that\nthe cytokines could be classified into four groups with characteristic patterns of\ntemporal changes ( Supplementary\nTable 2 ). IFN-γ ( Fig. 5A ) and TNF-α\n( Fig. 5B ) showed significant increases at each\ntime point, demonstrating stable increases ( P <0.01). IL-12p70 ( Fig. 5C ) and IL-1β ( Fig. 5D ) showed gradual increases in the earlier stages\n( P <0.05) followed by marked elevation in the later stages\n( P <0.001). The increases in IL-6 ( Fig. 5E ) and IL-2 ( Fig. 5F ) were\nmodest between 0 and 14 d.p.t. (not significant,  P >0.05) but showed\ndramatic increases between 28 and 42 d.p.t. ( P <0.001). IL-10 ( Fig. 5G ) uniquely showed a transient increase in the\nearly stage ( P <0.01) followed by gradual decreases from 14 to 42\nd.p.t. ( P <0.01), displaying a pattern different from other cytokines.\nThese distinct patterns suggest that each cytokine plays a distinct role in inflammation\nand immune response.\nChange in serum level of cytokines overtime. Serum levels of IFN-γ (A), TNF-α (B),\nIL-12p70 (C), IL-1β (D), IL-6 (E), IL-2 (F), IL-10 (G) in the recipient mice with\nendometriotic lesions (n=10 per group) and non-treated control as 0 d.p.t. (n=5).\nBars: mean ± SEM. Significant  P -values are indicated as follows:\n* P <0.05, ** P <0.01,\n*** P <0.001, and **** P <0.0001.\nFurthermore, when the correlation between temporal changes of each cytokine level and\ntemporal changes of luminescence values indicating the lesion growth of E2(+) recipients\nwas analysed by calculating Pearson correlation coefficients ( Supplementary Table 3 ), IL-6 and IL-2 showed\nthe strongest positive correlations with luminescence/tissue growth (IL-6: r=0.967, IL-2:\nr=0.945,  P <0.001). IFN-γ, TNF-α, IL-1β, and IL-12p70 also showed\nsignificant positive correlations. IL-10 was the only cytokine showing a negative\ncorrelation with luminescence, though without statistical significance. Particularly\nduring 28–42 d.p.t., the increase in IL-6 and IL-2 showed the strongest association with\nthe luminescence increase/lesion growth.\n\nIn this study, we developed an improved endometriosis mouse model optimized for\ninflammatory cytokine quantification by minimizing experimental invasiveness ( Supplementary Table 1 ). The\nprotocol utilized B6-CAG-ELuc transgenic mice [ 16 ] as\ndonors and B6.Cg- c/c - hr/hr  mice as recipients, with\nmodifications aimed at reducing confounding factors. The use of\nB6.Cg- c/c - hr/hr  recipients enhanced luminescent singal\ndetection by eliminating interference from skin pigmentation and fur [ 18 ], while avoiding inflammation from repeated shaving [ 13 ]. For synchronizing estrus cycles, we relied solely on\nvaginal appearance observation and donors’ uterine edematous changes as indicators, avoiding\nsurgical ovariectomy and hormone administration. By limiting surgical invasion to a single\nabdominal incision, we achieved consistent lesion establishment while minimizing invasive\nprocedures that could affect cytokine measurements. Following transplantation, endometriotic\nlesions were established using E2 administration alone, which promotes both endometrial\nproliferation and angiogenesis [ 16 ,  22 ]. The successful engraftment over 40 days demonstrated\nacceptable histocompatibility between the strains, despite the recipients retaining\napproximately 9% non-B6-genotype regions potentially including the region containing the\n H8  on chromosome 14, one of the classical minor histocompatibility loci\n[ 21 ].\nA key advantage of our model is that we successfully induced endometriotic lesions with E2\nadministration while preserving recipients’ ovaries intact without ovariectomy. This feature\nmakes our model particularly suitable for research on endometriosis-related infertility\n[ 1 ,  26 ]. The\ncystic lesions were primarily composed of fluid-filled cysts, uterine gland-like epithelium,\nand the interstitial stroma as reported in previous mouse models [ 13 ,  16 ,  18 ]. These morphological features were similar to the pathological\nfindings observed in human endometriosis [ 27 ].\nHowever, it has been reported that in experimental mouse models, morphological\ncharacteristics of lesion tissues can vary depending on genetically different strains and\nestrous cycles of donors and recipients [ 28 ].\nTherefore, careful attention must be paid to these variables when designing and interpreting\nexperimental studies.\nTwo prominent sites were observed for high-frequency and stable engraftment of uterine\ntissue transplants: the post-surgical suture sites and the pancreatic region ( Supplementary Fig. 1 ). The similar\nadhesion sites reported in other mouse experiments [ 16 ], which aligns with clinical observations where endometriosis can develop in\ncesarean section scars [ 29 ]. By our morphological\nobservations, sites undergoing angiogenesis and tissue repair processes are thought to\nprovide favorable conditions for endometrial tissue engraftment. The preferential adhesion\nof the endometriotic lesions to the pancreatic region may be attributed to the presence of\nperipancreatic adipose tissue (PAT), a metabolically active tissue containing small\nadipocytes and high amounts of stromal vascular cells [ 30 ]. Unlike other adipose tissues, PAT’s unique properties in promoting\ninflammatory responses [ 30 ] suggest PAT provides more\nfavorable conditions for endometriotic lesion engraftment. The significant vascularization\nobserved around the engrafted lesion tissues in our 3D images further supports this\nhypothesis. Additionally, since E2 is known to promote vascular endothelial growth factor\n(VEGF) expression in blood vessels [ 22 ], E2\nadministration may have also contributed to the rapid growth of lesion tissue via enhanced\nangiogenesis in these preferential sites.\nOur longitudinal analysis of cytokine profiles in the mouse model revealed dynamic changes\nthat closely parallel observations reported in endometriosis patients [ 31 , 32 , 33 ]. We observed significantly elevated levels of pro-inflammatory\ncytokines (IL-1β, IL-2, IL-6, IL-12p70, TNF-α, and IFN-γ) that increased in a time-dependent\nmanner, peaking on 42 d.p.t., coinciding with the period of rapid lesion growth. Notably,\nIFN-γ and TNF-α showed consistent increases throughout the observation period, suggesting\ntheir crucial role in disease progression, which aligns with clinical findings of elevated\nIFN-γ and TNF-α levels in the peritoneal fluid of patients [ 34 ,  35 ]. Interestingly, anti-inflammatory\ncytokine IL-10 showed a contrasting pattern, with initial elevation followed by decline,\nreflecting the inflammatory imbalance characterized by the predominance of pro-inflammatory\ncytokines [ 12 ]. The sharp increase in IL-6 and IL-2\nlevels on 42 d.p.t. further supports the establishment of an activated inflammatory state,\nconsistent with clinical observations [ 36 ,  37 ]. These temporal changes in our model provide valuable\ninsights into the early inflammatory dynamics of endometriosis, offering an experimental\nfoundation for understanding disease progression and potential therapeutic\ninterventions.\nRahmawati  et al.  (2023) reported elevated serum levels of IL-1β, IL-6,\nIL-8, and IL-12p70 in a study examining women with endometriosis and infertility,\nparticularly noting that IL-12p70 correlated with pain assessment scores in endometriosis\n[ 33 ]. Since a significant increase in IL-12p70 was\nalso confirmed in the later stage of lesion growth in this mouse model ( Fig. 5C ), combining the model with appropriate methods to evaluate\npain, such as measuring changes in spontaneous behavior [ 38 ], may be useful in developing treatments for pain associated with\nendometriosis.\nIn conclusion, our study has yielded novel insights that contribute to the understanding of\nendometriosis through the development of an improved mouse model that effectively reflects\nhuman disease characteristics. In addition to the successful utilization of\nB6.Cg- c/c - hr/hr  mice as recipients, the combination of\nadvanced imaging techniques and comprehensive cytokine profiling has enabled us to\ndemonstrate new resources and fundamental technologies that promote molecular and cellular\nlevel research into the pathophysiology of endometriosis.","source_license":"CC0","license_restricted":false}