RO8191, a new compound for initiating embryo implantation in mice

RO8191, a new compound for initiating embryo implantation in mice | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article RO8191, a new compound for initiating embryo implantation in mice Junlan Shu, Jumpei Terakawa, Satoko Osuka, Ayako Muraoka, Jiali Ruan, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5350329/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract During early pregnancy in mice, leukemia inhibitory factor (LIF) regulates embryo implantation by activating the JAK/STAT3 signaling pathway. The STAT3 pathway has been recognized to play a critical role in embryo implantation. However, it is not clear whether STAT3 activation itself can cause induction of embryo implantation. In this study, the effects of RO8191, a potential STAT3 activator, on embryo implantation were investigated through a series of studies with different mouse models. We found that RO8191 can induce embryo implantation by activating the STAT3 pathway in delayed implantation mice. Furthermore, RO8191 can initiate decidualization, which is essential for embryo implantation, even in uterine epithelial-specific Stat3 , Gp130 , or Lifr conditional knockout (cKO) mice that exihbits infertility due to embryo implantation failure. Histomorphological observations revealed successful embryo implantation and embryonic development in Lifr cKO mice. Increased epithelial detachment and vascularization were observed in Stat3 cKO mice, and excessive inflammatory response and embryo death were observed in Gp130 cKO mice. These results suggest that STAT3, Gp130 and LIFR each play a distinct role in embryo implantation and development. Although the specific mechanisms of RO8191 are not fully understood, this study providedinsights to support the application of RO8191 in treating recurent implantation failure. Biological sciences/Physiology/Reproductive biology/Reproductive disorders Biological sciences/Physiology/Reproductive biology RO8191 STAT3 Implantation Uterus Mice Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Embryo implantation is a crucial stage in early mammalian gestation. The blastocyst hatches, attaches, adheres, and invades the receptive uterus in humans and mice [ 1 ] . Recovery of embryo implantation is a significant challenge in the field of infertility treatment. Previous studies have shown that the success rate of implantation per embryo hovers around 25% in human populations with normal fertility, according to both natural cycles and assisted reproductive technology indications [ 1 ] . Although the exact molecular mechanisms of blastocyst implantation are not fully understood, existing research has revealed that successful implantation depends on the synchronization of endometrial receptivity with blastocyst activation [ 2 ] . For example, insufficient endometrial receptivity can lead to recurrent implantation failure (RIF) in clinical pregnancies [ 3 ],[ 4 ] . Endometrial receptivity is controlled by the hormones 17β-estradiol (E2) and progesterone (P4) [ 5 ] , which are primarily synthesized by the ovaries. In mice, embryo implantation occurs on the fourth day of pregnancy (D4) (D1=the day the vaginal plug is found). This process is triggered by the transient elevation of E2 [ 6 ],[ 7 ] . E2 can stimulate the release of leukemia inhibitory factor (LIF), a cytokine that belongs to the interleukin (IL)-6 family cytokines (including oncostatin M, IL-11, IL-27, ciliary neurotrophic factor [CNTF], and cardiotrophin-1 [CT-1]) [ 8 ] . It is widely accepted that LIF is expressed in the uterine glandular epithelium (GE) during the pre-implantation phase [ 9 ] and is considered crucial for implantation [ 10 ] . The LIF interacts with the LIF receptor (LIFR) on the luminal epithelium and forms a heterodimer with the glycoprotein 130 (Gp130) [ 11 ] . This complex heterodimer activates the JAK/STAT3 signaling pathway, leading to the phosphorylation of Janus kinase and subsequent phosphorylation of STAT3 [ 12 ],[ 13 ] . Following phosphorylation, p-STAT3 translocates to the nucleus, where it mediates the expression of a variety of genes in the cell [ 11 ] . Infertility due to failure of embryo adhesion has been observed in Lif -deficient mice [ 14 ] . The importance of LIF for embryo implantation has been further demonstrated by the finding that intraperitoneal administration of a LIF antagonist [ 15 ] or anti-LIF antibody [ 16 ] in the C57BL/6J (B6) mice prevented embryo implantation, leading to infertility. Intriguingly, injection of recombinant LIF or recombinant CT-1 can induce embryo implantation by activating STAT3 signaling in the uterine epithelium in delayed implantation (DI) mice (ICR or B6 strain) [ 17 ] . In contrast, CNTF, which belongs to the same IL-6 cytokine family as LIF and CT-1, neither induced embryo implantation nor activated STAT3 phosphorylation in DI mice. Mice with a conditional knockout of Stat3 in the uterine epithelium exhibit infertility due to implantation failure [ 18 ] . Similarly, mice with a conditional knockout of either Lifr [ 19 ] or Gp130 [ 20 ] in the uterine epithelium are also infertile due to failure of blastocyst implantation. These findings suggest that the epithelial-originated LIFR/Gp130-JAK/STAT3 signaling pathway is essential for successful embryo implantation in mice. Successful embryo implantation can be achieved through activation of STAT3, but to date, no pharmacological agents have been used to activate the STAT3 pathway and induce implantation in mice. The purpose of the present study was to determine whether the pharmacological agents could induce embryo implantation by activating the JAK/STAT3 signaling pathway. We found that RO8191, an interferon-α receptor 2 agonist [ 21 ] , is a useful STAT3 activator that can induce embryo implantation. Materials and methods Animals This study was approved by the Ethics Review Board for Animal Experiments at Nagoya University (approval number: A240034-002) and the Ethical Committee for Vertebrate Experiments at Azabu University (approval number: 230327-9). All experiments were conducted in accord with the relevant guidelines and regulations, including the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines. ICR (Japan SLC, Shizuoka, Japan), C57BL/6J (Jackson Laboratory Japan, Kanagawa, Japan), and the following mouse strains, all aged over seven weeks, were used in the experiments: Ltf iCre/+ mouse [ 22 ] , Stat3 flox/flox ( Stat3 f/f ) [ 23 ] , Gp130 flox/flox ( Gp130 f/f ) mouse [ 24 ] , and Lifr flox/flox ( Lifr f/f ) . To obtain the Lifr flox/flox mice (tm1c), Lifr tm1a(EUCOMM)Hmgu purchased from the European Mouse Mutant Archive (EMMA) (strain ID: EM:06941), harboring a knockout first allele, were crossed with FLPe transgenic mice (RBRC01834) [ 25 ] and FRT-LacZ-neo-FRT cassette was removed and loxP-flanked exon 4 was left. Stat3 f/f , Gp130 f/f and Lifr f/f mice were crossed with Ltf iCre/+ mouse to generate uterine epithelial-specific gene deficient (conditional knockout, cKO) ( Stat3 cKO, Gp130 cKO or Lifr cKO) mice as previously described [ 20 ] , respectively. The primers used for genotyping were; 5′-GTTTCCTCCTTCTGGGCTCC-3′, 5′-TTTAGTGCCCAGCTTCCCAG-3′ and 5′-CCTGTTGTTCAGCTTGCACC-3′ for Ltf iCre ; 5′-CCTGAAGACCAAGTTCATCTGTGTGAC-3′, 5′-CACACAAGCCATCAAACTCTGGTCTCC-3′ and 5′-GATTTGAGTCAGGGATCCTTATCTTCG-3′ for Stat3 flox ; 5′-GGCTTTTCCTCTGGTTCTTG-3′ and 5′-CAGGAACATTAGGCCAGATG-3′ for Gp130 flox ; 5′-TGAGAGCACGGAAGCTCTTT-3′ and 5′-ACTGCCCGACAAGGTTTTTA-3′ for Lifr flox . All the mouse strains, except ICR, were maintained in a C57BL/6 strain background and housed in the barrier facility at Azabu University. ICR mice were housed in the barrier facility at Nagoya University. The mice were fed ad libitum under controlled light-dark cycles (12 hours of light followed by 12 hours of dark) at 22 ± 3°C. The first day of pregnancy (D1) was determined as the morning when a vaginal plug was observed in the female that had been mated with fertile wildtype males on the previous evening. RO8191 treatment in DI mice The experimental procedure is shown in Fig. 1 A. To induce delayed implantation (DI) model mice, plug-positive ICR females were ovariectomized between 1300 and 1530 h on D3 under sevoflurane (193-17791, FujiFilm Wako Pure Chemicals, Osaka, Japan) anesthesia, and medroxyprogesterone acetate (100 µl/head; Pfizer Inc, New York, NY, USA) was injected subcutaneously in the ventral region. The RO8191 (T22142, TargetMol, Boston, MA, USA) was dissolved in sesame oil (196-15385, FujiFilm Wako Pure Chemicals), and a single intraperitoneal (i.p.) injection of RO8191 (400 µg/head; dissolved in sesame oil) or sesame oil was performed at 1300 h on D7. These female mice were euthanized in a carbon dioxide chamber, followed by dissection at 1300 h on D10 to count the number of implantation sites. The saline solution was used to flush the uterine horns to check for the presence of embryos if the uteri did not show the implantation sites. DI mice were excluded from the statistical analysis if they had no implantation sites and no blastocyst was recovered. ICR females injected with sesame oil were used as a control. The implantation rates of two groups (oil and RO8191), which were defined as the ratio of the number of mice with implantation sites to the total number of mice after being injected with oil or RO8191. RO8191 treatment in conditional knockout mice The experimental procedure is shown in Fig. 3 A. Plug-positive females of Stat3 , Gp130 or Lifr cKO were administered with a single i.p. injection of RO8191 (400 µg/head; dissolved in sesame oil) or sesame oil at between 1330 and 1700 h on D4. Plug-positive females of Stat3 f/f , Gp130 f/f or Lifr f/f were injected with sesame oil as a pregnant control group. The number of implantation sites was counted macroscopically on D7. If there was no implantation site, the uterine horns were flushed with saline solution to retrieve the embryos and determine the success of mating. Mice were excluded from the statistical analysis, if they had no implantation sites and no blastocyst was recovered. Images were saved in PNG format at a resolution of 806x1441 pixels. ImageJ software (version 1.54f; National Institutes of Health, Bethesda, MD, USA) was utilized to measure the areas of uterine bulges. The scale was set using the 'Set Scale' function based on a 1 cm scale bar. The bulges were selected using 'Oval selection'. The measurements were recorded in square centimeters (cm²). The size of each bulge was measured in the control and cKOs ( Stat3 , Gp130 , and Lifr ) uteri on D7. Statistical analysis was conducted using GraphPad Prism (version 10.0; GraphPad Software), with results expressed as mean ± standard error of the mean (SEM). One-way ANOVA followed by Tukey's multiple comparison test was used to assess group differences, with significance set at p < 0.05. In this study, we defined the bulges in the cKOs uterus as the putative implantation site. Isolation of LE After the injection of RO8191 or sesame oil for 24 hours, DI mice were sacrificed by inhalation of carbon dioxide, and the uterine horns were collected. For the sesame oil injection group, each uterine horn was rinsed with 0.5 mL saline solution to recover blastocysts, while the uteri without embryos were excluded from sampling and discarded. The uteri were washed three times briefly with saline solution on ice. All uterine horns were opened longitudinally and cut into 2 mm pieces. The uterine pieces were collected in a tube and kept at -80℃ overnight and incubated with RNA stabilization solution (AM7020, Thermo Fisher Scientific, Waltham, MA, USA) at -80℃ for 1 hour. The luminal epithelium (LE) was isolated from the uterine pieces using forceps on the inner surface of the endometrium and collected by gravity sedimentation, washed in RNA stabilization solution, and stored at -80°C prior to protein extraction. Protein extraction and Western blot analysis Protein extraction and Western blot analysis The RIPA lysis buffer containing a 1% protease inhibitor cocktail (25955-24; Nacalai tesque, Kyoto, Japan) was used for total protein isolation. Liver tissue was obtained from nonpregnant ICR mice. The LE or liver was lysed for 5 minutes on ice and centrifuged at 15,000 rpm for 30 minutes at 4°C for protein extraction. Total protein concentrations were determined using the Qubit™ protein assay kit (2411539, Thermo Fisher Scientific). LE protein (25 µg) and liver protein (10 µg) were separated on an 8% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membranes, which were followed by the blocking with 5% nonfat dry milk (Megmilk Snow Brand, Tokyo, Japan)/TTBS (Tris buffered saline with 0.01%-detergent) for 30 minutes at room temperature (RT). The membranes were then incubated with a primary antibody dilution buffer, consisting of TTBS diluted with 5% bovine serum albumin (010-25783, FujiFilm Wako Pure Chemicals) and primary antibodies, either STAT3 (1:2,000, 79D7, Cell Signaling Technology, Danvers, MA, USA) or phospho-STAT3 (p-STAT3 (Tyr705), 1:2,000, Cell Signaling Technology) overnight at 4°C. After six 5-minte washes in TTBS solution, the membranes were incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit antibody (PI-1000-1, Vector Labs, Newark, CA, USA) for 1 hour at RT, followed by six 5-minte washes in. The Western BLoT Ultra Sensitive HRP Substrate (#T7104A, Takara, Japan) immersed nitrocellulose membrane was placed in the Lumicube (Liponics, Tokyo, Japan), the aperture was adjusted to f/2.8, and the ISO was set to 12,800 with an exposure time of 30 seconds for photography. ImageJ software (version 1.54f; National Institutes of Health, Bethesda, MD, USA) was utilized to measure the molecular weight. Histological methods Uterine tissues from the mice were fixed in 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) (pH 7.4) using standard histological techniques (Presnell and Schreibman, 2013; Suvarna et al., 2013). These tissues were dehydrated through increasing concentrations of ethanol solutions, cleared with xylene, and embedded in paraffin blocks. A plane of transverse sections was cut at 5 µm thickness and stained with the routine hematoxylin and eosin (H&E). Micrographs were captured by BZ-X700 microscopy (Keyence, Osaka, Japan). Statistical analysis The parameter data including the implantation sites and implantation rate in the DI and cKOs mice were expressed as mean ± SEM or mean. Statistical analysis was performed by using independent Student’s t test at the GraphPad Prism (version 10.0; GraphPad Software). P values less than 0.05 were considered statistically significant. Results RO8191 promotes blastocysts implantation in DI mice RO8191 has been reported to bind to the interferon-α receptor 2 and promote phosphorylation of STAT3 [ 21 ] . To validate the promotion activity of RO8191 for implantation in mice, RO8191 was injected into the DI mice (Fig. 1 A). No implantation site was detected in the control mice, and nonadherent blastocysts were recovered after washing the uterus lumen (n = 5) (Fig. 1 B). On the other hand, distinct implantation sites were observed in approximately 80% (15/19) of the RO8191-injected DI mice (Fig. 1 B-D). In the negative samples after RO8191 injection (4/19), no implantation site was found, and the blastocysts were recovered. We confirmed the growing embryo in the implantation sites in RO8191-treated mice by the histological observation (Fig. 1 E, upper panels), although some part of implantation sites showed abnormal embryo development and decidual reaction (Fig. 1 E, lower panels). The implantation rate was significantly different between the two groups (Fig. 1 C). The number of average implantation sites was 6.7 ± 1.0 in RO8191 group (Fig. 1 D). Phosphorylation of STAT3 by RO8191 To evaluate whether RO8191 initiates implantation by regulating the JAK/STAT3 signaling pathway, the LE cells was collected from DI mice 24 hours after RO8191 injection. STAT3 expression was comparable between the control and RO8191-injected mice by Western blot analysis (Fig. 2 ). Phosphorylated-STAT3 (p-STAT3) was detectable in the LE after the intraperitoneal injection of RO8191 in DI mice compared to the control. High expression levels of both STAT3 and p-STAT3 were observed in the liver tissue from nonpregnant ICR mice as a control. The effects on RO8191 for cKOs mice Since the epithelial-derived LIF-LIFR/Gp130-JAK/STAT3 signaling pathway is critical for establishing embryo implantation in mice, RO8191 was administered to Stat3 cKO [ 18 ] , Gp130 cKO [ 20 ] and Lifr cKO [ 19 ],[ 26 ] mice to examine whether their implantation failure phenotype can be restored (Fig. 3 A). As a pregnant control, Stat3 f/f , Gp130 f/f and Lifr f/f mice were injected with the same dose of sesame oil (Fig. 3 A and Table 1 ). Table 1 The percentage of implantation rate and the number of implantation sites of the flox mice. Strain Injection Implantation/n The number of implantation sites (mean ± SEM) Stat3 f/f 5/5 5.6 ± 0.8 Gp130 f/f Oil 3/3 9.7 ± 0.9 Lifr f/f 4/4 8.3 ± 0.5 In the Stat3 f/f (n = 5), Gp130 f/f (n = 3), and Lifr f/f (n = 4) mice after oil injection on D4, implantation sites were lined along with the uteri on D7 and no defect of embryo implantation was found in each mouse strain (Fig. 3 A-D and Table 1 ). The average number of implantation sites was 5.6, 9.7, and 8.3 in Stat3 f/f , Gp130 f/f and Lifr f/f mice, respectively (Table 1 ). Implantation sites, including small uterine bulges, were observed in Stat3 cKO (2/4), Gp130 cKO (4/5), and Lifr cKO (3/3) cKO mice injected with RO8191 (Fig. 3 B-F). The average number of implantation sites was 2.5, 4.4, and 8.7 in Stat3 , Gp130 , and Lifr cKO mice, respectively (Fig. 3 F). Administration of sesame oil in cKOs mice did not promote implantation (Fig. 3 B-F). The size of the implantation bulges varied among the different cKOs mouse strains. The average area of implantation bulges was 0.077 cm², 0.061 cm², 0.039 cm², and 0.121 cm² in control, Stat3 , Gp130 , and Lifr cKO mice strains, respectively (Fig. 3 G). The areas of the bulges in Stat3 cKO and Gp130 cKO were notably smaller compared to the control, whereas those in Lifr cKO was larger (p < 0.05). The bulges areas in Stat3 cKO mice were markedly larger than those in Gp130 cKO mice (Fig. 3 G; p < 0.05). Histological analysis revealed that uterine sections of all cKOs mouse exhibited the decidualized stromal cells (Fig. 4 C-H). In both the control and Lifr cKO uterus, blastocysts were located at the anti-mesometrial side of the crypt (Fig. 4 A, B, G, H). No blastocyst was observed in the uterus of Stat3 and Gp130 cKO mice (Fig. 4 C, D, E, F). In the Stat3 cKO uterus, LE was broken and shed, and the development of blood vessels was observed at implantation sites (Fig. 4 D). Histological analysis of Gp130 cKO mice uterus showed that the leukocyte infiltration and embryo death had occurred (Fig. 4 F). Discussion It is widely accepted that a transient elevation of E2 in the uterus can induce LIF secretion from the glandular epithelium at D4 of pregnancy in mice. LIF activates the JAK/STAT3 signaling pathway through LIFR/Gp130 and phosphorylation of STAT3 in the uterine epithelium, which is critical for embryo implantation [ 14 ],[ 27 ] . Therefore, uterine epithelial cKO mice lacking Stat3 , Gp130 , or Lifr exhibit defects in STAT3 signaling, resulting in implantation failure [ 18 ]–[ 20 ] . Although the detailed mechanisms of downstream STAT3 signaling remain elusive, rescuing implantation failure through STAT3 activation offers considerable potential for future applications. The purpose of this study was to test whether activation of STAT3 signaling by RO8191 can initiate embryo implantation. RO8191 functions similarly to type I interferons (IFNs) as a ligand: it binds to the IFN-α receptor 2 (IFNAR2) as a homodimer, phosphorylates and activates the STAT protein family, including STAT1, STAT2, STAT3, STAT5, and STAT6, and induces IFN-inducible gene expression [ 21 ] , while type I IFNs phosphorylate STAT proteins by inducing heterodimerization of IFNAR1 and IFNAR2. In the present study, DI mice injected with RO8191 showed high implantation rates, a substantial number of implantation sites, succeed embryo development and decidualization (Fig. 1 ), indicating its potential as a STAT3 activator. On the other hand, individual physiological differences among the DI mice may have led to abnormal embryo development and decidualization at some implantation sites. RO8191 may bind the IFNAR2, which is known to be expressed in the luminal epithelium [ 28 ] , and/or an unknown receptor, to activate STAT3 and regulate gene expression, and eventually rescue embryo implantation in DI mice. Previous research has shown that mouse LE cells treated with LIF in vitro exhibited a clear single band of p-STAT3 by Western blot analysis [ 27 ] and DI mice injected with LIF also showed a high expression level of p-STAT3 [ 17 ] . Consistent with these findings, p-STAT3 was found in the uterine LE of DI mice 24 hours after RO8191 treatment (Fig. 2 ), while it was absent in the control, suggesting that RO8191 can directly activate the STAT3 signaling pathway in LE cells. In the present study, the uterine epithelium cKOs ( Stat3 , Gp130 , and Lifr ) also exhibited a high implantation rate and a substantial number of presumed implantation sites after RO8191 treatment (Fig. 3 ). This indicated that RO8191 can affect the interaction between the embryos and the uterus even in the absence of STAT3, Gp130, and LIFR in the uterine epithelium. The size of the implantation sites induced by the RO8191 administration differed among the cKOs mouse strains (Fig. 3 G). The degree of the decidual reaction appears to be different, suggesting that epithelial STAT3, Gp130, and LIFR have different contribution to the decidualization. On the other hand, only Lifr cKO mice showed a larger size of the implantation sites compared to the control. The absence of LIFR might enhance the RO8191-induced signaling transduction, regulating the proliferation of endometrial stromal cell. As conditional deletion of Stat3 , Gp130 , or Lifr in uterine epithelial cells results in defective decidualization [ 20 ],[ 29 ],[ 30 ] , LIFR/Gp130-JAK/STAT3 signaling in the uterine epithelium is indispensable for decidualization in normal pregnancy. However, activation of the LIF-STAT3-Egr1 signaling pathway in the endometrial stroma stimulates the decidualization of endometrial stromal cells through WNT4 regulation [ 31 ] . Importantly, the present study showed that all cKOs uteri exhibited decidualized stromal cells after RO8191 treatment according to histological observations (Fig. 4 ). These results indicate that the activation of the STAT3 pathway by RO8191 in LE cells should play a minor role in the decidualization of uterine stromal cells. Alternatively, RO8191 should directly activate the STAT3 pathway in uterine stromal cells to initiate decidualization. Embryo development in the decidua of Stat3 and Gp130 cKO uterus could not be rescued by RO8191, as no embryos were observed (Fig. 4 C-F), although RO8191 exhibited low toxicity in cell culture [ 21 ] and positive effects in Lifr cKO mice (Fig. 4 G,H). The Stat3 cKO uterus exhibited key features of a receptive endometrium, including partial uterine epithelial breakdown, shedding, and blood vessel development, which are essential processes for embryonic growth (Fig. 4 C, D) [ 32 ] . In contrast, the Gp130 cKO did not show these characteristics. This suggested that embryonic development was already initiated but terminated at different stages of the decidualized reaction in the Stat3 and Gp130 cKO uterus. In the Gp130 cKO uterus, an excessive inflammatory response and embryo death were observed after RO8191 treatment (Fig. 4 F). Decidualization is typically accompanied by leukocyte infiltration, with approximately 15% of uterine decidual cells being immune cells, primarily uterine NK cells, macrophages, and T lymphocytes [ 33 ] . However, abnormal leukocyte infiltration also can lead to the early embryo loss [ 34 ] . Previous research has shown that the absence of epithelial Gp130 reduces the expression of the progesterone receptor and ALOX15, a downstream target of the progesterone receptor [ 20 ] . This reduction leads to defective decidualization and an excessive inflammatory response in Gp130 cKO mice at D4 [ 20 ] . This indicates that RO8191 was ineffective in alleviating the defective decidualization and excessive inflammation in Gp130 cKO mice. The marked failure of decidualization in the Stat3 and Gp130 cKO uteri suggests that STAT3 and Gp130 play crucial but distinct roles in the RO8191-mediated signaling, which regulates decidualization and embryonic development. RO8191 also has the advantage of convenient oral administration in mice, which is patient-friendly for the future application [ 21 ] . In this study, we demonstrated that RO8191 can contribute to embryo implantation in mice. Although the exact mechanism of RO8191 on implantation remains unclear, it is promising that RO8191 has potential for the treatment of implantation failure. Declarations Acknowledgements We thank Dr. T. Daikoku (Kanazawa University) and Dr. S.K. Dey (Cincinnati Children’s Hospital Medical Center) for providing Ltf iCre mice, Dr. S. Akira (Osaka University) for providing Stat3 flox mice, and Dr. W. Muller (University of Manchester) for providing Gp130 flox mice. We also thank Medical Research Council (MRC) for providing Lifr tm1a(EUCOMM)Hmgu . FLPe transgenic mouse strain (RBRC01834) was provided by RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. This work was supported by Grants-in-Aid for Scientific Research from the JSPS (23K23785 to E.H.). This work was partially supported by the Center for Human and Animal Symbiosis Science, Azabu University, and a research project grant awarded by the Azabu University Research Services Division. 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Dual control of LIF expression and LIF receptor function regulate Stat3 activation at the onset of uterine receptivity and embryo implantation. Proceedings of the National Academy of Sciences 98, 8680–8685 (2001). Jang, H. et al. Characterization of interferon α and β receptor IFNAR1 and IFNAR2 expression and regulation in the uterine endometrium during the estrous cycle and pregnancy in pigs. Theriogenology 88 , 166–173 (2017). Catalano, R. D. et al. Inhibition of Stat3 activation in the endometrium prevents implantation: A nonsteroidal approach to contraception. Proc. Natl. Acad. Sci. U S A . 102 , 8585–8590 (2005). Pawar, S. et al. STAT3 Regulates Uterine Epithelial Remodeling and Epithelial-Stromal Crosstalk During Implantation. Mol. Endocrinol. 27 , 1996–2012 (2013). Liang, X. H. et al. Egr1 Protein Acts Downstream of Estrogen-Leukemia Inhibitory Factor (LIF)-STAT3 Pathway and Plays a Role during Implantation through Targeting Wnt4 *. J. Biol. Chem. 289 , 23534–23545 (2014). Whitby, S., Zhou, W. & Dimitriadis, E. Alterations in Epithelial Cell Polarity During Endometrial Receptivity: A Systematic Review. Front. Endocrinol. 11 , (2020). Erlebacher, A. Immunology of the Maternal-Fetal Interface. Annu. Rev. Immunol. 31 , 387–411 (2013). Baines, M. G., Duclos, A. J., Antecka, E. & Haddad, E. K. Decidual infiltration and activation of macrophages leads to early embryo loss. Am. J. Reprod. Immunol. 37 , 471–477 (1997). Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigure1.docx SupplementaryFigures25.docx Cite Share Download PDF Status: Published Journal Publication published 09 Sep, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 18 Dec, 2024 Reviews received at journal 10 Dec, 2024 Reviews received at journal 27 Nov, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers agreed at journal 20 Nov, 2024 Reviewers invited by journal 19 Nov, 2024 Editor assigned by journal 19 Nov, 2024 Editor invited by journal 19 Nov, 2024 Submission checks completed at journal 18 Nov, 2024 First submitted to journal 28 Oct, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5350329","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":388824145,"identity":"1e6a514f-4478-4c60-8daf-28f98de881e8","order_by":0,"name":"Junlan Shu","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Junlan","middleName":"","lastName":"Shu","suffix":""},{"id":388824146,"identity":"faa9f17a-d24b-456c-a2bc-8a55d7c1461e","order_by":1,"name":"Jumpei Terakawa","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Jumpei","middleName":"","lastName":"Terakawa","suffix":""},{"id":388824148,"identity":"6fd324bb-c5e5-49b3-b660-0d20387d15e2","order_by":2,"name":"Satoko Osuka","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Satoko","middleName":"","lastName":"Osuka","suffix":""},{"id":388824149,"identity":"65ddceb1-6bac-46b9-b170-2aaf7e15f658","order_by":3,"name":"Ayako Muraoka","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Ayako","middleName":"","lastName":"Muraoka","suffix":""},{"id":388824150,"identity":"0029eee0-67a4-44a2-a98e-757ab9fa0e7a","order_by":4,"name":"Jiali Ruan","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Jiali","middleName":"","lastName":"Ruan","suffix":""},{"id":388824151,"identity":"ed1dcec4-68e2-4313-9aa3-151b6018fe78","order_by":5,"name":"Atsuo Iida","email":"","orcid":"","institution":"Nagoya University","correspondingAuthor":false,"prefix":"","firstName":"Atsuo","middleName":"","lastName":"Iida","suffix":""},{"id":388824152,"identity":"019abbf5-5a2e-4f8e-b99f-24038e286630","order_by":6,"name":"Junya Ito","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Junya","middleName":"","lastName":"Ito","suffix":""},{"id":388824153,"identity":"f21e37cf-7b56-4f0d-98da-9fe3011f5677","order_by":7,"name":"Eiichi Hondo","email":"data:image/png;base64,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","orcid":"","institution":"Nagoya University","correspondingAuthor":true,"prefix":"","firstName":"Eiichi","middleName":"","lastName":"Hondo","suffix":""}],"badges":[],"createdAt":"2024-10-29 02:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5350329/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5350329/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-18471-3","type":"published","date":"2025-09-09T15:57:32+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71799195,"identity":"d71ce898-c450-4c5f-b0a7-793a208cb3fe","added_by":"auto","created_at":"2024-12-18 16:30:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":248135,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRO8191 induces embryo implantation in delayed implantation (DI) model mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eThe experimental procedure of the artificial DI mouse. Ovariectomy (OVX) and subcutaneous administration of siliconized medroxyprogesterone acetate (MPA) were performed on D3. A single injection of oil or RO8191 was performed on D7. Females were euthanized and dissected on D10. \u003cstrong\u003e(B) \u003c/strong\u003eRepresentative images of the gross uterine morphology.\u003cstrong\u003e \u003c/strong\u003eBlastocysts were recovered from oil-treated mice (white arrowheads). Implantation sites (yellow arrowheads on right panel) were visible in DI mice after RO8191 treatment. Scale bars: 1 cm for uterine images and 200 μm for blastocysts. \u003cstrong\u003e(C, D) \u003c/strong\u003eImplantation rate (C) and the number of implantation sites (D) in the oil and RO8191-treated DI mice. * indicates significantly different (p \u0026lt; 0.0001). \u003cstrong\u003e(E) \u003c/strong\u003eRepresentative histological image of uterine cross-sections of implantation sites on D10 after RO8191 treatment in DI mice. Right panels are higher magnification of left panels indicated by yellow line. Lower panels indicated abnormal embryo development and decidual reaction. Scale bars: 100 μm. dec: decidua;em: embryo. All data relating to embryo implantation induction are presented in Supplementary Figure 2.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-5350329/v1/d5ed5e268a8012e43a859235.png"},{"id":71800295,"identity":"2985efe4-a395-45c2-866c-db0641541edd","added_by":"auto","created_at":"2024-12-18 16:38:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":50257,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis of STAT3 and p-STAT3 for the LE cells after RO8191 treatment.\u003c/strong\u003e The predicted band size is 89 (STAT3α), 69 (STAT3β) and 66 kDa (p-STAT3). The whole image of the original Western blot is presented in Supplemental Figure 1.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-5350329/v1/78bd771dffddf2dd17f189e5.png"},{"id":71799192,"identity":"b36d3b89-3c18-4c15-a14c-0351158136b8","added_by":"auto","created_at":"2024-12-18 16:30:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":315638,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRO8191 induces decidualization in mice with genetic implantation failure\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The experimental procedure of the genetically modified mice. For control, a single oil injection was performed on D4. For cKOs, a single injection of oil or RO8191 was performed on D4. Females were euthanized and dissected on D7. (B-D) Uterine morphology of cKOs mice after the injection of oil or RO8191. The white arrowheads indicate the presumptive implantation site. Scale bars: 1 cm. \u003cstrong\u003e(E)\u003c/strong\u003e The implantation rate in cKOs mice after oil or RO8191 administration. \u003cstrong\u003e(F)\u003c/strong\u003e The number of implantation sites in cKOs mice after the injection of oil or RO8191. \u003cstrong\u003e(G)\u003c/strong\u003e The size of implantation sitesin cKOsmice after oil or RO8191 administration. * indicates significantly different (p \u0026lt; 0.05). All data relating to embryo implantation induction are presented in Supplementary Figure 3-5.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-5350329/v1/897e5ff01852558db0bb5fae.png"},{"id":71799194,"identity":"025341ca-05cc-4f03-990f-53e27c31a710","added_by":"auto","created_at":"2024-12-18 16:30:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":350671,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological characterization of uterine cross-sections from presumed implantation sites on D7\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e Control (\u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e) uterus after sesame oil treatment. \u003cstrong\u003e(C, D)\u003c/strong\u003e \u003cem\u003eStat3\u003c/em\u003e cKO, \u003cstrong\u003e(E, F) \u003c/strong\u003e\u003cem\u003eGp130\u003c/em\u003e cKO, and \u003cstrong\u003e(G, H) \u003c/strong\u003e\u003cem\u003eLifr \u003c/em\u003ecKO uterus after RO8191 treatment. Higher magnification in yellow frames inserted in D and F. Scale bar, 100 µm. dec: decidua; em: embryo; bv: blood vessels; le: luminal epithelium; li: leukocyte infiltration.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-5350329/v1/b78dd659219b4ad000bfabf6.png"},{"id":91359346,"identity":"0ce02145-aa6c-4185-9908-dedfcd0ce091","added_by":"auto","created_at":"2025-09-15 16:05:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1893873,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5350329/v1/09279eca-6c24-4387-aff9-6adfd8def7a3.pdf"},{"id":71799196,"identity":"27bd2b98-5fe2-44bf-a5af-c3adc7451cef","added_by":"auto","created_at":"2024-12-18 16:30:53","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":396316,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5350329/v1/eb60cc5561adc658e21a9453.docx"},{"id":71799197,"identity":"4c89197c-a2f6-4b01-8293-399f4023c3dd","added_by":"auto","created_at":"2024-12-18 16:30:53","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":8864608,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigures25.docx","url":"https://assets-eu.researchsquare.com/files/rs-5350329/v1/2235c3e392521df2be76d5ee.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"RO8191, a new compound for initiating embryo implantation in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEmbryo implantation is a crucial stage in early mammalian gestation. The blastocyst hatches, attaches, adheres, and invades the receptive uterus in humans and mice\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Recovery of embryo implantation is a significant challenge in the field of infertility treatment. Previous studies have shown that the success rate of implantation per embryo hovers around 25% in human populations with normal fertility, according to both natural cycles and assisted reproductive technology indications\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Although the exact molecular mechanisms of blastocyst implantation are not fully understood, existing research has revealed that successful implantation depends on the synchronization of endometrial receptivity with blastocyst activation\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. For example, insufficient endometrial receptivity can lead to recurrent implantation failure (RIF) in clinical pregnancies\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e],[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Endometrial receptivity is controlled by the hormones 17β-estradiol (E2) and progesterone (P4)\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e, which are primarily synthesized by the ovaries.\u003c/p\u003e \u003cp\u003eIn mice, embryo implantation occurs on the fourth day of pregnancy (D4) (D1=the day the vaginal plug is found). This process is triggered by the transient elevation of E2\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e],[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. E2 can stimulate the release of leukemia inhibitory factor (LIF), a cytokine that belongs to the interleukin (IL)-6 family cytokines (including oncostatin M, IL-11, IL-27, ciliary neurotrophic factor [CNTF], and cardiotrophin-1 [CT-1])\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. It is widely accepted that LIF is expressed in the uterine glandular epithelium (GE) during the pre-implantation phase\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e and is considered crucial for implantation\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. The LIF interacts with the LIF receptor (LIFR) on the luminal epithelium and forms a heterodimer with the glycoprotein 130 (Gp130)\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. This complex heterodimer activates the JAK/STAT3 signaling pathway, leading to the phosphorylation of Janus kinase and subsequent phosphorylation of STAT3\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e],[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Following phosphorylation, p-STAT3 translocates to the nucleus, where it mediates the expression of a variety of genes in the cell\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eInfertility due to failure of embryo adhesion has been observed in \u003cem\u003eLif\u003c/em\u003e-deficient mice\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. The importance of LIF for embryo implantation has been further demonstrated by the finding that intraperitoneal administration of a LIF antagonist\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e or anti-LIF antibody\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e in the C57BL/6J (B6) mice prevented embryo implantation, leading to infertility. Intriguingly, injection of recombinant LIF or recombinant CT-1 can induce embryo implantation by activating STAT3 signaling in the uterine epithelium in delayed implantation (DI) mice (ICR or B6 strain)\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. In contrast, CNTF, which belongs to the same IL-6 cytokine family as LIF and CT-1, neither induced embryo implantation nor activated STAT3 phosphorylation in DI mice. Mice with a conditional knockout of \u003cem\u003eStat3\u003c/em\u003e in the uterine epithelium exhibit infertility due to implantation failure\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Similarly, mice with a conditional knockout of either \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e or \u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e in the uterine epithelium are also infertile due to failure of blastocyst implantation. These findings suggest that the epithelial-originated LIFR/Gp130-JAK/STAT3 signaling pathway is essential for successful embryo implantation in mice.\u003c/p\u003e \u003cp\u003eSuccessful embryo implantation can be achieved through activation of STAT3, but to date, no pharmacological agents have been used to activate the STAT3 pathway and induce implantation in mice. The purpose of the present study was to determine whether the pharmacological agents could induce embryo implantation by activating the JAK/STAT3 signaling pathway. We found that RO8191, an interferon-α receptor 2 agonist\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, is a useful STAT3 activator that can induce embryo implantation.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e This study was approved by the Ethics Review Board for Animal Experiments at Nagoya University (approval number: A240034-002) and the Ethical Committee for Vertebrate Experiments at Azabu University (approval number: 230327-9). All experiments were conducted in accord with the relevant guidelines and regulations, including the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.\u003c/p\u003e \u003cp\u003eICR (Japan SLC, Shizuoka, Japan), C57BL/6J (Jackson Laboratory Japan, Kanagawa, Japan), and the following mouse strains, all aged over seven weeks, were used in the experiments: \u003cem\u003eLtf\u003c/em\u003e\u003csup\u003e\u003cem\u003eiCre/+\u003c/em\u003e\u003c/sup\u003e mouse\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eLtf\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1(icre)Tdku\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e/J\u003c/em\u003e, JAX: 026030 \u0026gt;\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003etm2Aki\u003c/em\u003e\u003c/sup\u003e\u0026gt;\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e) mouse\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eIl6st\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1Wme\u003c/em\u003e\u003c/sup\u003e\u0026gt;\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e, and \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e (\u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e)\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1c(EUCOMM)Hmgu\u003c/em\u003e\u003c/sup\u003e\u0026gt;. To obtain the \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox/flox\u003c/em\u003e\u003c/sup\u003e mice (tm1c), \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003etm1a(EUCOMM)Hmgu\u003c/em\u003e\u003c/sup\u003e purchased from the European Mouse Mutant Archive (EMMA) (strain ID: EM:06941), harboring a knockout first allele, were crossed with FLPe transgenic mice\u0026thinsp;\u0026lt;\u0026thinsp;Tg(CAG-flpe)36Ito\u0026gt; (RBRC01834)\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e and FRT-LacZ-neo-FRT cassette was removed and loxP-flanked exon 4 was left. \u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice were crossed with \u003cem\u003eLtf\u003c/em\u003e\u003csup\u003e\u003cem\u003eiCre/+\u003c/em\u003e\u003c/sup\u003e mouse to generate uterine epithelial-specific gene deficient (conditional knockout, cKO) (\u003cem\u003eStat3\u003c/em\u003e cKO, \u003cem\u003eGp130\u003c/em\u003e cKO or \u003cem\u003eLifr\u003c/em\u003e cKO) mice as previously described\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e, respectively. The primers used for genotyping were; 5\u0026prime;-GTTTCCTCCTTCTGGGCTCC-3\u0026prime;, 5\u0026prime;-TTTAGTGCCCAGCTTCCCAG-3\u0026prime; and 5\u0026prime;-CCTGTTGTTCAGCTTGCACC-3\u0026prime; for \u003cem\u003eLtf\u003c/em\u003e\u003csup\u003e\u003cem\u003eiCre\u003c/em\u003e\u003c/sup\u003e; 5\u0026prime;-CCTGAAGACCAAGTTCATCTGTGTGAC-3\u0026prime;, 5\u0026prime;-CACACAAGCCATCAAACTCTGGTCTCC-3\u0026prime; and 5\u0026prime;-GATTTGAGTCAGGGATCCTTATCTTCG-3\u0026prime; for \u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox\u003c/em\u003e\u003c/sup\u003e; 5\u0026prime;-GGCTTTTCCTCTGGTTCTTG-3\u0026prime; and 5\u0026prime;-CAGGAACATTAGGCCAGATG-3\u0026prime; for \u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox\u003c/em\u003e\u003c/sup\u003e; 5\u0026prime;-TGAGAGCACGGAAGCTCTTT-3\u0026prime; and 5\u0026prime;-ACTGCCCGACAAGGTTTTTA-3\u0026prime; for \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003eflox\u003c/em\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAll the mouse strains, except ICR, were maintained in a C57BL/6 strain background and housed in the barrier facility at Azabu University. ICR mice were housed in the barrier facility at Nagoya University. The mice were fed \u003cem\u003ead libitum\u003c/em\u003e under controlled light-dark cycles (12 hours of light followed by 12 hours of dark) at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;3\u0026deg;C. The first day of pregnancy (D1) was determined as the morning when a vaginal plug was observed in the female that had been mated with fertile wildtype males on the previous evening.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eRO8191 treatment in DI mice\u003c/h3\u003e\n\u003cp\u003eThe experimental procedure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. To induce delayed implantation (DI) model mice, plug-positive ICR females were ovariectomized between 1300 and 1530 h on D3 under sevoflurane (193-17791, FujiFilm Wako Pure Chemicals, Osaka, Japan) anesthesia, and medroxyprogesterone acetate (100 \u0026micro;l/head; Pfizer Inc, New York, NY, USA) was injected subcutaneously in the ventral region. The RO8191 (T22142, TargetMol, Boston, MA, USA) was dissolved in sesame oil (196-15385, FujiFilm Wako Pure Chemicals), and a single intraperitoneal (i.p.) injection of RO8191 (400 \u0026micro;g/head; dissolved in sesame oil) or sesame oil was performed at 1300 h on D7. These female mice were euthanized in a carbon dioxide chamber, followed by dissection at 1300 h on D10 to count the number of implantation sites. The saline solution was used to flush the uterine horns to check for the presence of embryos if the uteri did not show the implantation sites. DI mice were excluded from the statistical analysis if they had no implantation sites and no blastocyst was recovered. ICR females injected with sesame oil were used as a control. The implantation rates of two groups (oil and RO8191), which were defined as the ratio of the number of mice with implantation sites to the total number of mice after being injected with oil or RO8191.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eRO8191 treatment in conditional knockout mice\u003c/h3\u003e\n\u003cp\u003eThe experimental procedure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA. Plug-positive females of \u003cem\u003eStat3\u003c/em\u003e, \u003cem\u003eGp130\u003c/em\u003e or \u003cem\u003eLifr\u003c/em\u003e cKO were administered with a single i.p. injection of RO8191 (400 \u0026micro;g/head; dissolved in sesame oil) or sesame oil at between 1330 and 1700 h on D4. Plug-positive females of \u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e or \u003cem\u003eLifr\u003c/em\u003e \u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e were injected with sesame oil as a pregnant control group. The number of implantation sites was counted macroscopically on D7. If there was no implantation site, the uterine horns were flushed with saline solution to retrieve the embryos and determine the success of mating. Mice were excluded from the statistical analysis, if they had no implantation sites and no blastocyst was recovered. Images were saved in PNG format at a resolution of 806x1441 pixels. ImageJ software (version 1.54f; National Institutes of Health, Bethesda, MD, USA) was utilized to measure the areas of uterine bulges. The scale was set using the 'Set Scale' function based on a 1 cm scale bar. The bulges were selected using 'Oval selection'. The measurements were recorded in square centimeters (cm\u0026sup2;). The size of each bulge was measured in the control and cKOs (\u003cem\u003eStat3\u003c/em\u003e, \u003cem\u003eGp130\u003c/em\u003e, and \u003cem\u003eLifr\u003c/em\u003e) uteri on D7. Statistical analysis was conducted using GraphPad Prism (version 10.0; GraphPad Software), with results expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). One-way ANOVA followed by Tukey's multiple comparison test was used to assess group differences, with significance set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. In this study, we defined the bulges in the cKOs uterus as the putative implantation site.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIsolation of LE\u003c/h3\u003e\n\u003cp\u003eAfter the injection of RO8191 or sesame oil for 24 hours, DI mice were sacrificed by inhalation of carbon dioxide, and the uterine horns were collected. For the sesame oil injection group, each uterine horn was rinsed with 0.5 mL saline solution to recover blastocysts, while the uteri without embryos were excluded from sampling and discarded. The uteri were washed three times briefly with saline solution on ice. All uterine horns were opened longitudinally and cut into 2 mm pieces. The uterine pieces were collected in a tube and kept at -80℃ overnight and incubated with RNA stabilization solution (AM7020, Thermo Fisher Scientific, Waltham, MA, USA) at -80℃ for 1 hour. The luminal epithelium (LE) was isolated from the uterine pieces using forceps on the inner surface of the endometrium and collected by gravity sedimentation, washed in RNA stabilization solution, and stored at -80\u0026deg;C prior to protein extraction.\u003c/p\u003e\n\u003ch3\u003eProtein extraction and Western blot analysis\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eProtein extraction and Western blot analysis\u003c/div\u003e \u003cp\u003eThe RIPA lysis buffer containing a 1% protease inhibitor cocktail (25955-24; Nacalai tesque, Kyoto, Japan) was used for total protein isolation. Liver tissue was obtained from nonpregnant ICR mice. The LE or liver was lysed for 5 minutes on ice and centrifuged at 15,000 rpm for 30 minutes at 4\u0026deg;C for protein extraction. Total protein concentrations were determined using the Qubit\u0026trade; protein assay kit (2411539, Thermo Fisher Scientific). LE protein (25 \u0026micro;g) and liver protein (10 \u0026micro;g) were separated on an 8% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto nitrocellulose membranes, which were followed by the blocking with 5% nonfat dry milk (Megmilk Snow Brand, Tokyo, Japan)/TTBS (Tris buffered saline with 0.01%-detergent) for 30 minutes at room temperature (RT). The membranes were then incubated with a primary antibody dilution buffer, consisting of TTBS diluted with 5% bovine serum albumin (010-25783, FujiFilm Wako Pure Chemicals) and primary antibodies, either STAT3 (1:2,000, 79D7, Cell Signaling Technology, Danvers, MA, USA) or phospho-STAT3 (p-STAT3 (Tyr705), 1:2,000, Cell Signaling Technology) overnight at 4\u0026deg;C. After six 5-minte washes in TTBS solution, the membranes were incubated with horseradish peroxidase (HRP) conjugated goat anti-rabbit antibody (PI-1000-1, Vector Labs, Newark, CA, USA) for 1 hour at RT, followed by six 5-minte washes in. The Western BLoT Ultra Sensitive HRP Substrate (#T7104A, Takara, Japan) immersed nitrocellulose membrane was placed in the Lumicube (Liponics, Tokyo, Japan), the aperture was adjusted to f/2.8, and the ISO was set to 12,800 with an exposure time of 30 seconds for photography. ImageJ software (version 1.54f; National Institutes of Health, Bethesda, MD, USA) was utilized to measure the molecular weight.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistological methods\u003c/h2\u003e \u003cp\u003eUterine tissues from the mice were fixed in 4% paraformaldehyde (PFA) solution in phosphate-buffered saline (PBS) (pH 7.4) using standard histological techniques (Presnell and Schreibman, 2013; Suvarna et al., 2013). These tissues were dehydrated through increasing concentrations of ethanol solutions, cleared with xylene, and embedded in paraffin blocks. A plane of transverse sections was cut at 5 \u0026micro;m thickness and stained with the routine hematoxylin and eosin (H\u0026amp;E). Micrographs were captured by BZ-X700 microscopy (Keyence, Osaka, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe parameter data including the implantation sites and implantation rate in the DI and cKOs mice were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM or mean. Statistical analysis was performed by using independent Student\u0026rsquo;s t test at the GraphPad Prism (version 10.0; GraphPad Software). P values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eRO8191 promotes blastocysts implantation in DI mice\u003c/h2\u003e \u003cp\u003eRO8191 has been reported to bind to the interferon-α receptor 2 and promote phosphorylation of STAT3\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. To validate the promotion activity of RO8191 for implantation in mice, RO8191 was injected into the DI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). No implantation site was detected in the control mice, and nonadherent blastocysts were recovered after washing the uterus lumen (n\u0026thinsp;=\u0026thinsp;5) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). On the other hand, distinct implantation sites were observed in approximately 80% (15/19) of the RO8191-injected DI mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). In the negative samples after RO8191 injection (4/19), no implantation site was found, and the blastocysts were recovered. We confirmed the growing embryo in the implantation sites in RO8191-treated mice by the histological observation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, upper panels), although some part of implantation sites showed abnormal embryo development and decidual reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, lower panels). The implantation rate was significantly different between the two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The number of average implantation sites was 6.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.0 in RO8191 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003ePhosphorylation of STAT3 by RO8191\u003c/h2\u003e \u003cp\u003eTo evaluate whether RO8191 initiates implantation by regulating the JAK/STAT3 signaling pathway, the LE cells was collected from DI mice 24 hours after RO8191 injection. STAT3 expression was comparable between the control and RO8191-injected mice by Western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Phosphorylated-STAT3 (p-STAT3) was detectable in the LE after the intraperitoneal injection of RO8191 in DI mice compared to the control. High expression levels of both STAT3 and p-STAT3 were observed in the liver tissue from nonpregnant ICR mice as a control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe effects on RO8191 for cKOs mice\u003c/h2\u003e \u003cp\u003eSince the epithelial-derived LIF-LIFR/Gp130-JAK/STAT3 signaling pathway is critical for establishing embryo implantation in mice, RO8191 was administered to \u003cem\u003eStat3\u003c/em\u003e cKO\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e, \u003cem\u003eGp130\u003c/em\u003e cKO\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e and \u003cem\u003eLifr\u003c/em\u003e cKO\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e],[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e mice to examine whether their implantation failure phenotype can be restored (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As a pregnant control, \u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice were injected with the same dose of sesame oil (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe percentage of implantation rate and the number of implantation sites of the flox mice.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStrain\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eInjection\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eImplantation/n\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eThe number of implantation sites\u003c/p\u003e \u003cp\u003e(mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStat3\u003c/em\u003e \u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5/5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eGp130\u003c/em\u003e \u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOil\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3/3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e9.7\u0026thinsp;\u0026plusmn;\u0026thinsp;0.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eLifr\u003c/em\u003e \u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4/4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eIn the \u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;5), \u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;3), and \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;4) mice after oil injection on D4, implantation sites were lined along with the uteri on D7 and no defect of embryo implantation was found in each mouse strain (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-D and Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The average number of implantation sites was 5.6, 9.7, and 8.3 in \u003cem\u003eStat3\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eGp130\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eLifr\u003c/em\u003e\u003csup\u003e\u003cem\u003ef/f\u003c/em\u003e\u003c/sup\u003e mice, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Implantation sites, including small uterine bulges, were observed in \u003cem\u003eStat3\u003c/em\u003e cKO (2/4), \u003cem\u003eGp130\u003c/em\u003e cKO (4/5), and \u003cem\u003eLifr\u003c/em\u003e cKO (3/3) cKO mice injected with RO8191 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-F). The average number of implantation sites was 2.5, 4.4, and 8.7 in \u003cem\u003eStat3\u003c/em\u003e, \u003cem\u003eGp130\u003c/em\u003e, and \u003cem\u003eLifr\u003c/em\u003e cKO mice, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Administration of sesame oil in cKOs mice did not promote implantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-F). The size of the implantation bulges varied among the different cKOs mouse strains. The average area of implantation bulges was 0.077 cm\u0026sup2;, 0.061 cm\u0026sup2;, 0.039 cm\u0026sup2;, and 0.121 cm\u0026sup2; in control, \u003cem\u003eStat3\u003c/em\u003e, \u003cem\u003eGp130\u003c/em\u003e, and \u003cem\u003eLifr\u003c/em\u003e cKO mice strains, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The areas of the bulges in \u003cem\u003eStat3\u003c/em\u003e cKO and \u003cem\u003eGp130\u003c/em\u003e cKO were notably smaller compared to the control, whereas those in \u003cem\u003eLifr\u003c/em\u003e cKO was larger (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The bulges areas in \u003cem\u003eStat3\u003c/em\u003e cKO mice were markedly larger than those in \u003cem\u003eGp130\u003c/em\u003e cKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eHistological analysis revealed that uterine sections of all cKOs mouse exhibited the decidualized stromal cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-H). In both the control and \u003cem\u003eLifr\u003c/em\u003e cKO uterus, blastocysts were located at the anti-mesometrial side of the crypt (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B, G, H). No blastocyst was observed in the uterus of \u003cem\u003eStat3\u003c/em\u003e and \u003cem\u003eGp130\u003c/em\u003e cKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D, E, F). In the \u003cem\u003eStat3\u003c/em\u003e cKO uterus, LE was broken and shed, and the development of blood vessels was observed at implantation sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Histological analysis of \u003cem\u003eGp130\u003c/em\u003e cKO mice uterus showed that the leukocyte infiltration and embryo death had occurred (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is widely accepted that a transient elevation of E2 in the uterus can induce LIF secretion from the glandular epithelium at D4 of pregnancy in mice. LIF activates the JAK/STAT3 signaling pathway through LIFR/Gp130 and phosphorylation of STAT3 in the uterine epithelium, which is critical for embryo implantation\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e],[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Therefore, uterine epithelial cKO mice lacking \u003cem\u003eStat3\u003c/em\u003e, \u003cem\u003eGp130\u003c/em\u003e, or \u003cem\u003eLifr\u003c/em\u003e exhibit defects in STAT3 signaling, resulting in implantation failure\u003csup\u003e[\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u0026ndash;[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Although the detailed mechanisms of downstream STAT3 signaling remain elusive, rescuing implantation failure through STAT3 activation offers considerable potential for future applications.\u003c/p\u003e \u003cp\u003eThe purpose of this study was to test whether activation of STAT3 signaling by RO8191 can initiate embryo implantation. RO8191 functions similarly to type I interferons (IFNs) as a ligand: it binds to the IFN-α receptor 2 (IFNAR2) as a homodimer, phosphorylates and activates the STAT protein family, including STAT1, STAT2, STAT3, STAT5, and STAT6, and induces IFN-inducible gene expression\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e, while type I IFNs phosphorylate STAT proteins by inducing heterodimerization of IFNAR1 and IFNAR2. In the present study, DI mice injected with RO8191 showed high implantation rates, a substantial number of implantation sites, succeed embryo development and decidualization (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e), indicating its potential as a STAT3 activator. On the other hand, individual physiological differences among the DI mice may have led to abnormal embryo development and decidualization at some implantation sites. RO8191 may bind the IFNAR2, which is known to be expressed in the luminal epithelium\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e, and/or an unknown receptor, to activate STAT3 and regulate gene expression, and eventually rescue embryo implantation in DI mice.\u003c/p\u003e \u003cp\u003ePrevious research has shown that mouse LE cells treated with LIF \u003cem\u003ein vitro\u003c/em\u003e exhibited a clear single band of p-STAT3 by Western blot analysis\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e and DI mice injected with LIF also showed a high expression level of p-STAT3\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Consistent with these findings, p-STAT3 was found in the uterine LE of DI mice 24 hours after RO8191 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003e), while it was absent in the control, suggesting that RO8191 can directly activate the STAT3 signaling pathway in LE cells.\u003c/p\u003e \u003cp\u003eIn the present study, the uterine epithelium cKOs (\u003cem\u003eStat3\u003c/em\u003e, \u003cem\u003eGp130\u003c/em\u003e, and \u003cem\u003eLifr\u003c/em\u003e) also exhibited a high implantation rate and a substantial number of presumed implantation sites after RO8191 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This indicated that RO8191 can affect the interaction between the embryos and the uterus even in the absence of STAT3, Gp130, and LIFR in the uterine epithelium. The size of the implantation sites induced by the RO8191 administration differed among the cKOs mouse strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). The degree of the decidual reaction appears to be different, suggesting that epithelial STAT3, Gp130, and LIFR have different contribution to the decidualization. On the other hand, only \u003cem\u003eLifr\u003c/em\u003e cKO mice showed a larger size of the implantation sites compared to the control. The absence of LIFR might enhance the RO8191-induced signaling transduction, regulating the proliferation of endometrial stromal cell.\u003c/p\u003e \u003cp\u003eAs conditional deletion of \u003cem\u003eStat3\u003c/em\u003e, \u003cem\u003eGp130\u003c/em\u003e, or \u003cem\u003eLifr\u003c/em\u003e in uterine epithelial cells results in defective decidualization\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e],[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e],[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e, LIFR/Gp130-JAK/STAT3 signaling in the uterine epithelium is indispensable for decidualization in normal pregnancy. However, activation of the LIF-STAT3-Egr1 signaling pathway in the endometrial stroma stimulates the decidualization of endometrial stromal cells through WNT4 regulation\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Importantly, the present study showed that all cKOs uteri exhibited decidualized stromal cells after RO8191 treatment according to histological observations (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These results indicate that the activation of the STAT3 pathway by RO8191 in LE cells should play a minor role in the decidualization of uterine stromal cells. Alternatively, RO8191 should directly activate the STAT3 pathway in uterine stromal cells to initiate decidualization.\u003c/p\u003e \u003cp\u003eEmbryo development in the decidua of \u003cem\u003eStat3\u003c/em\u003e and \u003cem\u003eGp130\u003c/em\u003e cKO uterus could not be rescued by RO8191, as no embryos were observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-F), although RO8191 exhibited low toxicity in cell culture\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e and positive effects in \u003cem\u003eLifr\u003c/em\u003e cKO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG,H). The \u003cem\u003eStat3\u003c/em\u003e cKO uterus exhibited key features of a receptive endometrium, including partial uterine epithelial breakdown, shedding, and blood vessel development, which are essential processes for embryonic growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D)\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In contrast, the \u003cem\u003eGp130\u003c/em\u003e cKO did not show these characteristics. This suggested that embryonic development was already initiated but terminated at different stages of the decidualized reaction in the \u003cem\u003eStat3\u003c/em\u003e and \u003cem\u003eGp130\u003c/em\u003e cKO uterus. In the \u003cem\u003eGp130\u003c/em\u003e cKO uterus, an excessive inflammatory response and embryo death were observed after RO8191 treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Decidualization is typically accompanied by leukocyte infiltration, with approximately 15% of uterine decidual cells being immune cells, primarily uterine NK cells, macrophages, and T lymphocytes\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. However, abnormal leukocyte infiltration also can lead to the early embryo loss\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Previous research has shown that the absence of epithelial Gp130 reduces the expression of the progesterone receptor and ALOX15, a downstream target of the progesterone receptor\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. This reduction leads to defective decidualization and an excessive inflammatory response in \u003cem\u003eGp130\u003c/em\u003e cKO mice at D4\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. This indicates that RO8191 was ineffective in alleviating the defective decidualization and excessive inflammation in \u003cem\u003eGp130\u003c/em\u003e cKO mice. The marked failure of decidualization in the \u003cem\u003eStat3\u003c/em\u003e and \u003cem\u003eGp130\u003c/em\u003e cKO uteri suggests that STAT3 and Gp130 play crucial but distinct roles in the RO8191-mediated signaling, which regulates decidualization and embryonic development.\u003c/p\u003e \u003cp\u003eRO8191 also has the advantage of convenient oral administration in mice, which is patient-friendly for the future application\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In this study, we demonstrated that RO8191 can contribute to embryo implantation in mice. Although the exact mechanism of RO8191 on implantation remains unclear, it is promising that RO8191 has potential for the treatment of implantation failure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Dr. T. Daikoku (Kanazawa University) and Dr. S.K. Dey (Cincinnati Children’s Hospital Medical Center) for providing \u003cem\u003eLtf\u003csup\u003eiCre\u003c/sup\u003e\u003c/em\u003e mice, Dr. S. Akira (Osaka University) for providing \u003cem\u003eStat3\u003csup\u003eflox\u003c/sup\u003e\u003c/em\u003emice, and Dr. W. Muller (University of Manchester) for providing \u003cem\u003eGp130\u003csup\u003eflox\u003c/sup\u003e\u003c/em\u003emice. We also thank Medical Research Council (MRC) for providing\u0026nbsp;\u003cem\u003eLifr\u003csup\u003etm1a(EUCOMM)Hmgu\u003c/sup\u003e\u003c/em\u003e. FLPe transgenic\u0026nbsp;mouse strain (RBRC01834) was provided by RIKEN BRC through the National BioResource Project of the MEXT/AMED, Japan. This work was supported by Grants-in-Aid for Scientific Research from the JSPS (23K23785 to E.H.). This work was partially supported by the Center for Human and Animal Symbiosis Science, Azabu University, and a research project grant awarded by the Azabu University Research Services Division.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.H. planned the experiments. E.H. J.S. J.T.,\u0026nbsp;and J.R. performed the experiments and made all figures. J.S. J.T. S.O. A.M. J.R. A.I. J.I. and E.H. analyzed the data. J.S. J.T. A.I. J.I and E.H. wrote the manuscript. All authors agreed the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAuthors declare no conflict of interests for this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData is provided within the manuscript or supplementary information files.\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMacklon, N. S., Geraedts, J. P. M. \u0026amp; Fauser, B. C. J. M. Conception to ongoing pregnancy: the \u0026lsquo;black box\u0026rsquo; of early pregnancy loss. \u003cem\u003eHum. Reprod. Update\u003c/em\u003e. \u003cb\u003e8\u003c/b\u003e, 333\u0026ndash;343 (2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiudice, L. C. Potential biochemical markers of uterine receptivity. \u003cem\u003eHum. 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Immunol.\u003c/em\u003e \u003cb\u003e37\u003c/b\u003e, 471\u0026ndash;477 (1997).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"RO8191, STAT3, Implantation, Uterus, Mice","lastPublishedDoi":"10.21203/rs.3.rs-5350329/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5350329/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDuring early pregnancy in mice, leukemia inhibitory factor (LIF) regulates embryo implantation by activating the JAK/STAT3 signaling pathway. The STAT3 pathway has been recognized to play a critical role in embryo implantation. However, it is not clear whether STAT3 activation itself can cause induction of embryo implantation. In this study, the effects of RO8191, a potential STAT3 activator, on embryo implantation were investigated through a series of studies with different mouse models. We found that RO8191 can induce embryo implantation by activating the STAT3 pathway in delayed implantation mice. Furthermore, RO8191 can initiate decidualization, which is essential for embryo implantation, even in uterine epithelial-specific \u003cem\u003eStat3\u003c/em\u003e, \u003cem\u003eGp130\u003c/em\u003e, or \u003cem\u003eLifr \u003c/em\u003econditional knockout (cKO) mice that exihbits infertility due to embryo implantation failure. Histomorphological observations revealed successful embryo implantation and embryonic development in \u003cem\u003eLifr\u003c/em\u003e cKO mice. Increased epithelial detachment and vascularization were observed in \u003cem\u003eStat3 \u003c/em\u003ecKO mice, and excessive inflammatory response and embryo death were observed in \u003cem\u003eGp130\u003c/em\u003ecKO mice. These results suggest that STAT3, Gp130 and LIFR each play a distinct role in embryo implantation and development. Although the specific mechanisms of RO8191 are not fully understood, this study providedinsights to support the application of RO8191 in treating recurent implantation failure.\u003c/p\u003e","manuscriptTitle":"RO8191, a new compound for initiating embryo implantation in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-18 16:30:48","doi":"10.21203/rs.3.rs-5350329/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-18T16:51:46+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-11T03:49:01+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-27T15:40:05+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95951337522612005763965687020938001569","date":"2024-11-20T10:12:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"313016575022406771864939060007721564097","date":"2024-11-20T09:03:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-11-19T23:16:43+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-19T22:52:11+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-11-19T22:02:48+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-18T05:24:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-10-29T01:56:55+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3f4c782f-f853-4d05-b7a6-c9dec90045e1","owner":[],"postedDate":"December 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41438774,"name":"Biological sciences/Physiology/Reproductive biology/Reproductive disorders"},{"id":41438775,"name":"Biological sciences/Physiology/Reproductive biology"}],"tags":[],"updatedAt":"2025-09-15T16:05:40+00:00","versionOfRecord":{"articleIdentity":"rs-5350329","link":"https://doi.org/10.1038/s41598-025-18471-3","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-09-09 15:57:32","publishedOnDateReadable":"September 9th, 2025"},"versionCreatedAt":"2024-12-18 16:30:48","video":"","vorDoi":"10.1038/s41598-025-18471-3","vorDoiUrl":"https://doi.org/10.1038/s41598-025-18471-3","workflowStages":[]},"version":"v1","identity":"rs-5350329","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5350329","identity":"rs-5350329","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}