Seminal plasma exosome-derived miR-26-5p promotes embryo implantation and development by regulating decidual macrophage polarization via PTEN / PI3K / AKT signaling pathway

Seminal plasma exosome-derived miR-26-5p promotes embryo implantation and development by regulating decidual macrophage polarization via PTEN / PI3K / AKT signaling pathway | 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 Seminal plasma exosome-derived miR-26-5p promotes embryo implantation and development by regulating decidual macrophage polarization via PTEN / PI3K / AKT signaling pathway Yan Zhang, Xiaolin Chen, Jie Li, Xin Chen, Jing Zhao, Qing Liu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4940193/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract The immunomodulatory effects of seminal plasma(SP) on the maternal immune system play an important role in the implantation and development of the embryo. Decidual macrophages(dMΦs) are one of the major immune cells in the maternal-fetal immune microenvironment, and their M2-type polarization facilitates the establishment and maintenance of pregnancy. However, the role of SP on the polarization of dMΦs is unknown. In this study, we investigated the role and mechanism of SP on the polarization of dMΦs by gene chip sequencing as well as in vitro and in vivo experiments. The results revealed that SP promoted dMΦs M2-type polarization. Gene chip sequencing revealed that miR-26-5p was highly expressed in seminal exosomes(SEs) which could act on PTEN/PI3K/AKT signaling pathway and significantly promote MΦs M2 polarization. Moreover, SEs supplementation significantly reduced embryo resorption in spontaneously aborted mice. In conclusion, our study demonstrated that the SEs derived miR-26-5p in SP promoted the M2 polarization of dMΦs by targeting PTEN/PI3K/AKT signaling pathway, which created an immune-tolerant environment conducive to embryo implantation and development. This study provided new ideas for clinical SP-assisted therapy to improve pregnancy outcomes. Biological sciences/Molecular biology Earth and environmental sciences/Biogeochemistry seminal plasma seminal exosomes macrophage polarity miR-26-5p embryo implantation and development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 INTRODUCTION For the mother, the embryo is a semi-allograft with the paternal antigen. Therefore, the appropriate immune state at the maternal-fetal interface is very important for embryo implantation and early embryo development( 1 ). The maternal-fetal interface is composed of trophoblast cells, decidual stromal cells and decidual immune cells. Decidual immune cells mainly include natural killer cells (NKs), macrophages (MΦs), T cells and dendritic cells (DC) which secrete the corresponding cytokines involved in the regulation of immune homeostasis at the maternal-fetal interface. Decidual macrophages(dMΦs), the second largest population in decidual immune cells, play a crucial role in creating a maternal tolerance environment and regulating tissue remodeling during blastocyst implantation and placental development( 2 ). MΦs exhibit a high degree of plasticity and can adapt their phenotype in response to environmental stimuli( 3 ). M1-dMΦs are potent effector cells that induce pro-infammatory T helper 1(Th1) cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). By contrast, M2-dMΦs attenuate these Th1 responses by producing anti- infammatory Th2 cytokines, such as IL-10 ( 4 ). Furthermore, inducible nitric oxide synthase(iNOS) produced by M1-dMΦs can inhibit migration and cell trophoblast invasion by inducing higher levels of NO production, however recombinant human arginase-1 secreted by M2-dMΦ can inhibit immune cytotoxicity and promote endometrial decidualization and angiogenesis( 5 ). Thus, it is now widely accepted that M2-type polarization of dMΦs enriched at the maternal–fetal interface would be conducive to promoting the immune tolerance of semi-allograft embryos, remodeling local tissues and blood vessels, and benefiting other pregnancy-related physiological processes( 6 ). Seminal plasma (SP) has previously been considered as a carrier only to transport sperm, but researches now suggest that SP also has an auxiliary role in embryo implantation and early embryonic development( 7 – 9 ). Assisted reproductive technologies(ARTs) have been extensively employed in the treatment of infertility. Clinical meta-studies find low quality evidence that the application of SP as a therapeutic approach to improve the clinical pregnancy rate in cycles of ART( 10 ). There have been some experimental studies showing that human SP can stimulate the female immune response to provoke a controlled inflammatory response that facilitates embryo implantation, and promotes generation of immune tolerance for pregnancy, such as regulating the levels of dendritic cell (DC) or uNK cells related cytokines and chemokines( 11 , 12 ). Animal experiments also confirm that cytokines synthesized in the male accessory glands are transferred to the female at insemination, activating changes in gene expression that lead to modifications in structure and function of the female tissues. The consequences are increased fertilization rates, conditioning of the female immune response to tolerate the conceptus, and changes in the endometrium that facilitate embryo development and implantation.( 13 ). However, whether SP can regulate dMΦs polarity has not yet been reported. Seminal exosomes (SEs) are one of the main mediators of SP to perform their reproductive regulatory functions and play regulatory functions mainly through the DNA, RNA, and proteins they contain( 14 ). For examples: SEs can be internalized by human endometrial stromal cells (eSCs) and subsequently induce them to produce IL-6 and IL-8 which are involved in the immunoregulation of embryo implantation( 15 ); age-related alterations of SEs may be partially responsible for lower implantation rates in the aged-SP group compared with those in the young-SP group, which were mediated by uterine immunomodulation( 16 ). Phosphatase and tensin homolog (PTEN), a tumor suppressor gene, regulates many biological processes, including proliferation, survival, cellular architecture, motility, energy metabolism, and genomic stability( 17 ). PTEN can also participate in regulating embryo implantation and development by regulating trophoblast invasion, promoting endometrial epithelium cells (EECs) proliferation, and inducing endometrial stromal cells (ESCs) apoptosis at the maternal-fetal interface( 18 – 20 ). However, few articles has reported its regulation on MΦs in female reproductive system. This topic aims to explore the regulation function and mechanism of SP on dMΦs, which is conducive to the development of new targets for intervention to improve reproductive outcomes and may also provide new ideas for SP-assisted treatment of clinical infertility. RESULTS 1.SEs promote the polarization of macrophages towards M2 phenotype Electron microscopy and NTA revealed that particles isolated from SP contained exosomes (SEs) with diameters ranging from 30–150nm(Fig.1A,B) and WB experiment confirmed that SEs expressed exosome-specific-markers CD9, CD63 and HSP70(Fig.1C). SEs were stained with green fluorescent dye PKH67, and then co-cultured with THP-1cells for 24 hours. The green fluorescent was exhibited in the cytoplasm and diffused as small spots around the cell nucleus by confocal microscopy (Fig.1D) confirming that SEs could be uptaken by THP-1 cells. To investigate the potential regulation of SEs on MΦs polarization, SEs and controls were separately co-cultured with THP-1 cells. Flow cytometry results showed that the ratio of M2 (CD206 +) / M1 (CD86 +) was increased after THP-1 cells treated with SEs (Fig. 2A), implying that SEs may induce M2-MΦs polarization. Subsequently, detection of protein markers associated with MΦs polarization revealed that co-culture with SEs increased the expression of the M2-MΦs-related proteins (Arginase-1 and IL-10), consistent with a decrease in the M1-MΦs-related proteins (iNOS and IFN- γ) (Fig. 2B). The above results comprehensively indicated that SEs could promote the differentiation of MΦs to M2 type, induce the secretion of anti-inflammatory factors and inhibit the expression of inflammatory factors, thus creating an immune tolerance environment conducive to embryo implantation. 2. SEs promote M2 polarization through PTEN/PI3K/AKT signaling pathway Studies have shown that PTEN/PI3K/AKT signaling pathway plays an important role in regulating cell biological behavior(19). In the study, WB experiment discovered that the expression of PTEN/PI3K/AKT signaling pathway was altered after MΦs co-cultured with SEs (Fig 3A), which suggested that SEs promoted MΦs to M2 polarization through PTEN / PI3K / AKT signaling pathway. The similar results were found in human decidual tissue. During the IVF assisted pregnancy process, barrier contraception was usually used to avoid unintended pregnancy, thus lacking the direct contact regulation effect of SP on the uterus. We collected decidual tissues from patients with spontaneous abortion after natural pregnancy（NP group）and IVF assisted pregnancy (IVF group), determined the PTEN expression using immunohistochemistry(IHC) assay. The PTEN expression in the IVF group was significantly higher than that in the NP group (P<0.05) (Fig. 3B). From this result, it could be speculated that SP mainly the SEs has some regulatory effects on the expression of PTEN in the decidua. 3.miR-26a-5p is critical for SEs to promote macrophage M2 polarization As SEs primarily manifest their biological influence by delivering bioactive compounds, including miRNAs, we sought to investigate the plausible ingredients of SEs for macrophage polarization. The GeneChipTM sequencing results showed that miR-26a-5p, which had a targeted modulation effect with PTEN, was abundant in the SEs (Fig.4, Table S1). The expression of miR-26a-5p increased in THP-1 cells after co-culture with SEs confirming that miR-26a-5p was highly expressed in SEs and can be transported into THP-1 cells (Fig.5A). The increase of miR-26a-5p expression in THP-1 cells by the transfection with miR-26a-5p mimics laid the foundation for subsequent experiments (Fig. S1). Flow cytometry assay showed that THP-1 cells were polarized to M2 and increased the M2 / M1 ratio after transfection of miR-26a-5p mimic compared with the control group and miR-26a-5p inhibitor group(Fig.5B). Meanwhile, M2-MΦs-related proteins Arginase-1 and IL-10 were increased, while M1-MΦs-related protein iNOS and IFN- γ were decreased(Fig.5C). WB experiment further showed that SEs transported the loaded miR-26a-5p to THP-1 cells by acting on PTEN / PI3K / AKT signaling pathway(Fig.5C). Collectedly，SEs could transport the loaded miR-26a-5p to THP-1 cells, and promoted its polarization to M2 by acting on PTEN / PI3K / AKT signaling pathway, which was conducive to immune tolerance 4. SEs help to improve pregnancy outcome in spontaneously aborted mice. The procedures of animal experiment were shown as Figure 6A. SEs labeled with red fluorescent PKH26 were detected at the maternal-fetal interface of mice after administered by transvaginal injection which confirmed that SEs could reach the maternal uterus through sexual intercourse and directly regulated the cell function at the maternal-fetal interface(Fig. 6B). Then, we used spontaneous abortion mouse model to test the protective effect of SEs on pregnancy. There was a significant decrease in embryo resorption rate after vaginal injection of SEs to spontaneous abortion mice which proved that SEs can ameliorate embryo loss(Fig.6C, Table1). Mechanically, in the SE group, miR-26a-5p expression was significantly increased, accompanying with a significantly decrease in PTEN expression at the maternal-fetal interface compared with the control group(Fig.6D-E), which was very similar to the results of human decidua. Altered PTEN/PI3K/AKT and M1/M2-MΦs-related proteins level indicated that SEs regulated PTEN/PI3K/AKT signaling pathway by targeted transmission of their endogenous miR-26a-5p to MΦs, accordingly promoted MΦs polarization to M2, thereby shaped an immune tolerance state to alleviate abortion(Fig.6F). Table1. The embryo resorption rate after vaginal injection of SEs in mice with spontaneous abortions. control group（n=4） SE group（n=4） P Value Surviving fetuses 20 32 Resorbed fetuses 9 4 Resorption rate (%) 31.03% 11.11 P<0.05 Discussion The success of pregnancy mainly depends on immune tolerance of the mother for the semi-allogeneic fetus. Therefore, immunological status at the maternal-fetal interface is critical for the establishment and maintenance of pregnancy( 21 ). dMΦs, the second largest community at the maternal-fetal interface, are proposed to be involved in immune tolerance required for a successful pregnancy. MΦs exhibit a high degree of plasticity and can adapt their phenotype in response to environmental stimuli. The M1 populations refer to the classically activated MΦs and display the capacity to present antigens to the adaptive immune system which are more effective at antigen clearance. Compared to M1 phenotype, M2 populations are alternatively activated which have immunosuppressive capacities( 22 – 24 ). The polarization and functions of dMΦs can be triggered by signals present in the surrounding environment, such as seminal fluid or trophoblast-derived miRNA( 25 – 27 ). SP has been viewed as simply a vehicle to carry sperm to fertilize the oocyte before, but evidence is now building that attributes of seminal fluid other than sperm fertilizing capability influence reproductive outcomes in mammals( 28 ). Increasing number of molecular biology studies have revealed that SP can regulate the cells function at the maternal and fetal interface: ①Impaired modulation of immune response and improper placental development due to altered cytokine levels in seminal components may be the contributing paternal factors inrecurrent pregnancy loss (RPL)( 29 );② Exposure of endometrial epithelial cells (eECs) and endometrial stromal fibroblasts (eSFs) to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death.( 30 )༛③IL-11 is one of the most potently SP-induced genes in eSFs and is important for SP-facilitated decidualization( 31 ); ④Low expression of GDF-15 and overexpressed C3 in the SEs of RPL patients may distort maternal immune response to paternal antigens leading to impaired decidualization( 32 ). Clinical meta-studies also have shown that supplement with SP is beneficial to improve the clinical pregnancy rate in the process of assisted reproduction( 10 ). And this effectiveness has also been demonstrated in a variety of animal experiments( 33 ). In light of the observed ability of SEs to regulate macrophage polarization, we successfully extracted exosomes from healthy SP and co-cultured them with THP-1 cells. SEs were found to be phagocytosed by THP-1 cells and induced their transition to M2-type macrophages, accompanied by increased expression of M2-associated proteins (arginase-1 and IL-10), thus creating a state of immune tolerance that promoted embryo implantation and development. This study demonstrated for the first time that SP, particularly SEs, can mediate uterine immunomodulation through directly regulating MΦs polarization, in addition to decidual DC maturation( 16 ), CD11c + antigen presenting cells (APC) and paternal antigen-specific regulatory T cells (Tregs) development( 34 , 35 ). PTEN/PI3K/AKT signaling pathway is one of the highly conserved important molecules in regulating cell biological function at the maternal-fetal interface, such as alleviating the inflammatory damage of bovine eECs( 36 ), promoting eECs proliferation and inducing eSFs apoptosis( 19 ). The IHC experiments of clinical specimens in this experiment suggested that SP has regulatory effects on decidual PTEN expression. In vitro testing revealed that SEs promote MΦs polarization toward M2 phenotype through PTEN/PI3K/AKT signaling pathway. Based on the above results, we speculated that SP, mainly SEs, regulated decidual macrophage M2 polarization through the PTEN / PI3K / AKT signaling pathway and promoted immune tolerance. miRNAs were identifed as crucial constituents of exosomes, signifcantly determining the impact of exosomes on target cells. In order to uncover the molecular mechanisms underlying the impact of the SEs on macrophage polarization, the GeneChipTM sequencing was applied to reveal the abundance of diferent miRNAs present in the SEs. miR-26a-5p, which has been recognized having the targeted modulation effect on PTEN, was abundant in the SEs( 37 – 39 ). miR-26a-5p can directly inhibit PTEN translation by targeting its 3'-UTR, which in turn activates the PI3K/AKT pathway( 18 ). After transfected THP-1 cells with SEs, miR-26a-5p was significantly increased in THP-1 cells, which further confirming that miR-26a-5p was enriched in the SEs. A corresponding decrease in the expression levels of PTEN and M2-MΦs polarization were also found. The above results proved that SEs transported miR26a-5p to MΦs and promoted M2 polarization through PTEN / PI3K / AKT signaling pathway. Native or engineered exosomes are low toxicity and low immunogenicity, therefore they are effective tool for delivering small-molecule drugs and biological therapeutics into cells and tissue, having enormous potential for therapeutic applications( 40 , 41 ). Increased embryo absorption rate and exorbitant proinflammatory cytokines were found in RPL mice (CBA/J females paired with DBA/2 males)( 42 ), however SEs transvaginal application markedly improved the poor conditions above. We unfurled the remarkable therapeutic ability of SEs in abortion-prone mice, and this was achieved by targeting MΦs-M2 polarity thus favoring immune tolerance at the maternal-fetal interface. MATERIALS AND METHODS SEs extraction All procedures involving participants in this study were approved by the Clinical Trial Ethics Committee of Renmin Hospital of Wuhan University (Wuhan, China, CTEC number: WDRY2021-K044). To extract SEs, seminal fluid was centrifuged at 800g for 15 minutes at 25°C to separate spermatozoa, and then centrifuged at 10,000g for 30 minutes at 4°C to remove cell debris and other impurities. The supernatant was in turn centrifuged at 10,000g for 90 minutes to pellet microvesicles (MVs). Remaining supernatant was ultracentrifuged at 100,000g for 70 minutes and the exosome-containing pellets were washed with PBS for two times. SEs ultimately resuspended in PBS and stored at − 80°C for the following steps. The size and purity of the isolated SEs were determined using nanoparticle tracking analyzer (NTA, ZetaView 7PMX 120, Particle Metrix, Germany), transmission electron microscope (TEM, JEOL, Tokyo, Japan), and Western blot analysis. The antibodies used were as follows: anti-CD9 (1:1000, ab92726, Abcam, USA), anti-CD63 (1:1000, ab216130, Abcam, USA), anti-HSP70 (1:2000, ab181606, Abcam, USA). Cell culture Monocyte THP1 was purchased from Wuhan Procell Life Science and Technology Co., Ltd., China, and cultured in RPMI-1640 medium (Gibco, USA) which contained 10% FBS (Gibco, USA) and 1% penicillin–streptomycin (Servicebio, Wuhan) at 37°C in a humidified incubator under 5% CO2. THP1 monocytes were matured into M0 macrophages with 100ng/mL of phospholipid 12-myristic acid 13-acetate (PMA, Sigma-Aldrich, USA) for 48h to macrophage polarization. Cellular internalization of SEs SEs were labelled with PKH67 according to the manufacturer’s instructions(Sigma-Aldrich, USA). Shortly, SEs were added to diluent C, and PKH67 were added to diluent C. The mixture was incubated at room temperature. An equal volume of FBS was added to stop the dyeing reaction and bind the excess dye. Excess dye was removed by centrifuging the SEs at 400g at 25°C for 10 minutes.The nuclei were stained with DAPI (0.5 µg/mL, Invitrogen, USA). The labelled SEs were incubated with THP-1 cells for 48 hours. The internalization of SEs by THP-1 cells was observed using the fluorescence microscope (BX53, Olympus, Japan). Western Blot(WB)assay Proteins from cell lysates were separated by Radioimmunoprecipitation assay (RIPA) (Servicebio, Wuhan) lysis buffer and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skimmed milk and incubated with appropriate primary antibodies at 4°C overnight. Thereafter, the PVDF membranes were incubated with horseradish peroxidaseconjugated secondary antibodies. Protein blots were visualized by using the ECL-Plus western blotting detectionsystem (Thermo Fisher Scientific, USA). The antibodies used were as follows: rabbit anti-CD9 (1:1000, ab92726, Abcam, USA), rabbit anti-CD63 (1:1000, ab216130, Abcam, USA), rabbit anti-HSP70 (1:1000, ab181606, Abcam, USA), rabbit anti-PTEN(1:1000, ab267787, Abcam, USA), rat anti-p-AKT (1:1000, ab38449, Abcam, USA), rat anti-p-PI3K(1:500, ab182651, Abcam, USA), rabbit anti-Arginase-1 (1:1000, ab133543, Abcam, USA), rabbit anti-iNOS (1:1000, ab178945, Abcam, USA), rabbit anti-IL-10 (1:1000, ab133575, Abcam, USA), rabbit anti-IFN-γ (1:1000, ab267369, Abcam, USA). Flow cytometry Cells were suspended in the staining buffer (PBS + 3% BSA). Phycoerythrin (PE)-conjugated anti-human CD68 antibody (eBioscience, USA), PE-Cy7-conjugated anti-human CD86 and CD206 antibody were used for staining. All the data were acquired using FACS Canto II, BD Company, USA, and processed using FlowJo (Tree Star, Ashland, OR, USA). Clinical sample collection All participants were recruited from the Reproductive Medical Center at the Renmin Hospital of Wuhan University from June, 2021 to June, 2022. First-trimester human decidual tissues were obtained from patients with spontaneous abortion after natural pregnancy(NP group, n = 10) or IVF assisted pregnancy(IVF group, n = 10) Spontaneous abortion caused by fetal chromosome abnormalities, endocrine disorders or metabolic abnormalities, uterine malformations, autoimmune diseases were excluded. All patients undergoing IVF treatment were required to use barrier contraception to reduce unplanned pregnancy. Immunohistochemistry(IHC) Decidual tissues were collected from spontaneous abortion after natural pregnancy and IVF assisted pregnancy. These tissues were fixed in 10% formaldehyde at 37°C for 6h, followed by routine deparaffinization and dehydration for immunohistochemistry. Formalin-fixed paraffin-embedded tissues were cut into 4 µm sections. 3% hydrogen peroxide was used to block the endogenous peroxidase activity, and the non-specific binding was blocked with 5% bovine serum albumin for 20 minutes. The sections were incubated at 37°C with rabbit anti-PTEN antibody (1:100, ab267787, abcam, USA) for 4°C overnight. After washed in PBS, the sections were then incubated with HRP-labelled goat anti-rabbit IgG (1:500, AS1107, ASPEN, USA) secondary antibodies for 30 minutes at room temperature, followed by wash with PBS three times for 5 minutes. The color was developed with a DAB kit (Dako Cytomation, Glostrup, Denmark). Positive signals were visualized as brown. Mean fuorescence intensity (IntDen/Area) was quantitatively analyzed using Image J software. Quantitative Real-Time PCR (qRT-PCR) The total RNA in the tissues was isolated using TRIzol Reagent (ELK Biotechnology, China), according to the manufacturer’s instructions. The cDNA was performed on the ABI7900 system (Applied Biosystems, USA) for mRNA expression. The reverse transcription kit (ELK Biotechnology, China) was used to reverse-transcribe the RNA. The relative RNA quantifcation was performed using the comparative 2 − ΔΔCt method. Cell transfection In all cell transfections, 5×10 5 cells were plated on 35mm plates. 5µl oligonucleotides including miR-26a-5p mimics or miR-26a-5p inhibitor were diluted in 250µl Opti-Medium (Life Technology, USA) and mixed with 5µl Lipofectamine 2000 (Invitrogen,Thermo Fisher Scientific, Inc., USA) according to the manufacturer’s instructions. These mixture was then added with 500 µl dye solution and 1500µl basal medium. All of the culture media were completely replaced after 6 hours. Animal experiments All animal operations were approved by the ethics committee for laboratory animal welfare (IACUC) of Renmin Hospital of Wuhan University (Approval number: No. WDRM animal (f) No. 20201207). 6–8 weeks of age ICR mice, CBA/J female mice and DBA/2 male mice were obtained from the Animal Experiment Center of Wuhan University. ICR female mice mated ICR male mice at 1:1 for normal pregnant group; CBA / J female mice mated with DBA / 2 male mice at 1:1 for spontaneous abortion group. Overall, 8 spontaneous abortion mice were randomly divided into two groups as follows: naive-control and SEs treatment. Pregnant mare serum gonadotropin (PMSG, China) 10IU was given at 8pm on the first day, and human chorionic gonadotropin (HCG, China) 10IU was given at 8pm on the third day to induce superovulation. The day on which the vaginal plug appeared in female mice was recorded as the 0.5 day (D0.5) of pregnancy. For the SE groups, three shots of 100µl PHK26 dye labeled SEs (1ug/ul in PBS)were injected by vaginal on D0.5, D3.5 and D6.5. The naive-control group received a vaginal injection of PBS. On D14.5, the mice were euthanized via CO2 inhalation. The placental tissues, including decidual tissues and the fetus, were collected for the following experiments.All experimental procedures were conducted in conformity with the institutional guidelines issued by the ARRIVE guidelines. Conclusions After coitus, SP has a direct contact regulation effect on the female reproductive tract, and the SEs are one of the main media playing the SP regulation function. In this study, we found that SP, namely SEs promote the polarization of MΦs towards M2 phenotype through PTEN/PI3K/AKT signaling pathway. miR-26a-5p is the key regulatory molecules in SEs playing the regulatory role. The regulatory effect of SEs on MΦs helps to construct an immune-tolerant environment that promotes embryo cultivation and development(Fig. 7 ). This protective effect was confirmed in abortive mouse model trials. Thus, SEs are therefore expected as a novel treatment to improve pregnancy outcomes. Declarations Author Contributions: Conceptualization: Zhuoni Xiao and Xiaolin Chen; methodology: Yan zhang, Xin Chen, Qin Liu, Xinyu Wang and Xiaoling Li; validation: Qin Liu, Xinyu Wang and Xiaoling Li; investigation: Jie Li and Jing Zhao; formal analysis:Yan zhang, Xin Chen; writing—original draft preparation:Yan zhang and Xiaolin Chen; writing—review and editing: Zhuoni Xiao ; visualization:Jie Li and Jing Zhao; project administration:Yan zhang and Xiaolin Chen; funding acquisition:Zhuoni Xiao; All authors have read and agreed to thepublished version of the manuscript. Funding: This work was supported by The Interdisciplinary Innovative Talents Foundation from Renmin Hospital of Wuhan University (JCRCGW-2022-003). Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Clinical Trial Ethics Committee of Renmin Hospital of Wuhan University (Wuhan, China, CTEC number: WDRY2021-K044) and the ethics committee for laboratory animal welfare (IACUC) of Renmin Hospital of Wuhan University (Approval number: No. WDRM animal (f) No. 20201207) Informed Consent Statement: Informed consent was obtained from all subjects involved in the study Conflicts of Interest: The authors declare no conflict of interest. Data availability: The data supporting these fndings is available upon request from the corresponding author, [email protected] . References Yang, F., Zheng, Q. & Jin, L. Dynamic Function and Composition Changes of Immune Cells During Normal and Pathological Pregnancy at the Maternal-Fetal Interface. Front. Immunol. 10 , 2317 (2019). Locati, M., Curtale, G. & Mantovani, A. Diversity, Mechanisms, and Significance of Macrophage Plasticity. Annu. Rev. Pathol. 15 , 123–147 (2020). Wang, L. L. et al. Decorin promotes decidual M1-like macrophage polarization via mitochondrial dysfunction resulting in recurrent pregnancy loss. Theranostics . 12 (17), 7216–7236 (2022). Hu, J. J. et al. Lactobacillus murinus alleviate intestinal ischemia/reperfusion injury through promoting the release of interleukin-10 from M2 macrophages via Toll-like receptor 2 signaling. Microbiome . 10 (1), 38 (2022). Kieler, M., Hofmann, M. & Schabbauer, G. More than just protein building blocks: how amino acids and related metabolic pathways fuel macrophage polarization. FEBS J. 288 (12), 3694–3714 (2021). Zhang, Y. H., He, M., Wang, Y. & Liao, A. H. Modulators of the Balance between M1 and M2 Macrophages during Pregnancy. Front. Immunol. 8 , 120 (2017). Shen, Q. et al. Immune Regulation of Seminal Plasma on the Endometrial Microenvironment: Physiological and Pathological Conditions. Int. J. Mol. Sci. 24 (19), 14639 (2023). Ahmadi, H. et al. Composition and effects of seminal plasma in the female reproductive tracts on implantation of human embryos. Biomed. Pharmacother . 151 , 113065 (2022). Schjenken, J. E. & Robertson, S. A. The Female Response to Seminal Fluid. Physiol. Rev. 100 (3), 1077–1117 (2020). Ata, B. et al. Application of seminal plasma to female genital tract prior to embryo transfer in assisted reproductive technology cycles (IVF, ICSI and frozen embryo transfer). Cochrane Database Syst. Rev. 2 (2), CD11809 (2018). Craenmehr, M. H. C. et al. Effect of seminal plasma on dendritic cell differentiation in vitro depends on the serum source in the culture medium. J. Reprod. Immunol. Feb , 137:103076 (2020). Taima, A. et al. A semen-based stimulation method to analyze cytokine production by uterine CD56bright natural killer cells in women with recurrent pregnancy loss. J. Reprod. Immunol. 142 , 103206 (2020). Robertson, S. A. Seminal fluid signaling in the female reproductive tract: lessons from rodents and pigs. J. Anim. Sci. 85 (13 Suppl), E36–44 (2007). Machtinger, R., Laurent, L. C. & Baccarelli, A. A. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. Hum. Reprod. Update . 22 (2), 182–193 (2016 Mar-Apr). Paktinat, S. et al. Seminal exosomes induce interleukin-6 and interleukin-8 secretion by human endometrial stromal cells. Eur. J. Obstet. Gynecol. Reprod. Biol. Apr , 235:71–76 (2019). Wang, D. et al. Seminal Plasma and Seminal Plasma Exosomes of Aged Male Mice Affect Early Embryo Implantation via Immunomodulation. Front. Immunol. 12 , 12723409 (2021 Oct). Chen, C. Y. et al. PTEN: Tumor Suppressor and Metabolic Regulator. Front Endocrinol (Lausanne). Jul 9 :9338. (2018). Chen, L. et al. CircSTAM inhibits migration and invasion of trophoblast cells by regulating miR-148a-5p/PTEN axis. J. Assist. Reprod. Genet. 40 (1), 201–210 (2023). Zhang, L. et al. miR-26a promoted endometrial epithelium cells (EECs) proliferation and induced stromal cells (ESCs) apoptosis via the PTEN-PI3K/AKT pathway in dairy goats. J. Cell. Physiol. 233 (6), 4688–4706 (2018). Terakawa, J. et al. Ovarian insufficiency and CTNNB1 mutations drive malignant transformation of endometrial hyperplasia with altered PTEN/PI3K activities. Proc. Natl. Acad. Sci. U S A . 116 (10), 4528–4537 (2019). Zhang, Y., Liu, Z. & Sun, H. Fetal-maternal interactions during pregnancy: a 'three-in-one' perspective. Front. Immunol. 14 , 1198430 (2023). PrabhuDas, M. et al. Immune mechanisms at the maternal-fetal interface: perspectives and challenges. Nat. Immunol. 16 (4), 328–334 (2015). Ding, J. et al. M2 macrophage-derived G-CSF promotes trophoblasts EMT, invasion and migration via activating PI3K/Akt/Erk1/2 pathway to mediate normal pregnancy. J. Cell. Mol. Med. 25 (4), 2136–2147 (2021). Ding, J. et al. Granulocyte colony-stimulating factor in reproductive-related disease: Function, regulation and therapeutic effect. Biomed. Pharmacother . Jun , 150:112903 (2022). Wang, N., Liang, H. & Zen, K. Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front. Immunol. 5 , 614 (2014). Yang, J. et al. Trophoblast-derived miR-410-5p induces M2 macrophage polarization and mediates immunotolerance at the fetal-maternal interface by targeting the STAT1 signaling pathway. J. Transl Med. 22 (1), 19 (2024). Roertson, S. A. et al. Role of high molecular weight seminal vesicle proteins in eliciting the uterine inflammatory response to semen in mice. J. Reprod. Fertil. 107 (2), 265–277 (1996). Rodriguez-Martinez, H. et al. Seminal Plasma: Relevant for Fertility? Int. J. Mol. Sci. 22 (9), 4368 (2021). Jena, S. R. et al. Comparative proteome profiling of seminal components reveal impaired immune cell signalling as paternal contributors in recurrent pregnancy loss patients. Am. J. Reprod. Immunol. 89 (2), e13613 (2023). Chen, J. C. et al. Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts. Hum. Reprod. 29 (6), 1255–1270 (2014). George, A. F. et al. Seminal Plasma Promotes Decidualization of Endometrial Stromal Fibroblasts In Vitro From Women With and Without Inflammatory Disorders in a Manner Dependent on Interleukin-11 Signaling. Hum. Reprod. 35 (3), 617–640 (2020). Jena, S. R. et al. Paternal contributors in recurrent pregnancy loss: Cues from comparative proteome profiling of seminal extracellular vesicles. Mol. Reprod. Dev. 88 (1), 96–112 (2021). Schjenken, J. E. & Robertson, S. A. Seminal fluid and immune adaptation for pregnancy–comparative biology in mammalian species. Reprod. Domest. Anim. 49 (Suppl 3), 27–36 (2014). Shima, T. et al. Uterine CD11c + cells induce the development of paternal antigen-specific Tregs via seminal plasma priming. J. Reprod. Immunol. 141 , 103165 (2020). Robertson, S. A. et al. Seminal fluid drives expansion of the CD4 + CD25 + T regulatory cell pool and induces tolerance to paternal alloantigens in mice. Biol. Reprod. 80 (5), 1036–1045 (2009). Liu, J., Liang, Q. & Wang, T. IFN-τ mediated miR-26a targeting PTEN to activate PI3K/AKT signalling to alleviate the inflammatory damage of bEECs. Sci. Rep. 12 (1), 9410 (2022). Jiang, X. et al. Extracellular vesicles derived from human ESC-MSCs target macrophage and promote anti-inflammation process, angiogenesis, and functional recovery in ACS-induced severe skeletal muscle injury. Stem Cell. Res. Ther. 14 (1), 331 (2023). Gao, P. et al. Long noncoding RNA LINC-PINT retards the abnormal growth of airway smooth muscle cells via regulating the microRNA-26a-5p/PTEN axis in asthma. Int. Immunopharmacol. 99 , 107997 (2021). Wang, R. Q. et al. Lnc-GAN1 expression is associated with good survival and suppresses tumor progression by sponging mir-26a-5p to activate PTEN signaling in non-small cell lung cancer. J. Exp. Clin. Cancer Res. 40 (1), 9 (2021). Zhang, B. et al. Engineered EVs with pathogen proteins: promising vaccine alternatives to LNP-mRNA vaccines. J. Biomed. Sci. 31 (1), 9 (2024). Lokumcu, T. et al. Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. ACS Nano Jan 11. (2024). Liu, Z. et al. Bushen Antai recipe alleviates embryo absorption by enhancing immune tolerance and angiogenesis at the maternal-fetal interface via mobilizing MDSCs in abortion-prone mice. Phytomedicine . 123 , 155164 (2024). Additional Declarations No competing interests reported. Supplementary Files Fig.S1.tif SFigure.rar TableS1.pdf Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 14 Oct, 2024 Reviews received at journal 24 Sep, 2024 Reviews received at journal 20 Sep, 2024 Reviewers agreed at journal 11 Sep, 2024 Reviewers agreed at journal 10 Sep, 2024 Reviewers invited by journal 10 Sep, 2024 Editor assigned by journal 10 Sep, 2024 Editor invited by journal 03 Sep, 2024 Submission checks completed at journal 02 Sep, 2024 First submitted to journal 19 Aug, 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. 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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-4940193","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":360972994,"identity":"510a477c-35ab-4f69-9aed-403149414a1f","order_by":0,"name":"Yan Zhang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Yan","middleName":"","lastName":"Zhang","suffix":""},{"id":360972995,"identity":"b36306f7-4575-4756-836a-1ed5078555d5","order_by":1,"name":"Xiaolin Chen","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaolin","middleName":"","lastName":"Chen","suffix":""},{"id":360972996,"identity":"0e78d51c-9c1a-4562-bfd0-c3730703149b","order_by":2,"name":"Jie Li","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Li","suffix":""},{"id":360972997,"identity":"a64da8e5-94af-4535-b345-ceb955e8dd61","order_by":3,"name":"Xin Chen","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Chen","suffix":""},{"id":360972998,"identity":"5c440225-4ee1-40bd-b986-275ed9c66e51","order_by":4,"name":"Jing Zhao","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhao","suffix":""},{"id":360972999,"identity":"35f34781-3738-4b85-b9c0-9f26a2745364","order_by":5,"name":"Qing Liu","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Qing","middleName":"","lastName":"Liu","suffix":""},{"id":360973000,"identity":"bbfc7b47-1cc7-4ac7-a27e-f8f26f14a018","order_by":6,"name":"Xiaoling Li","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoling","middleName":"","lastName":"Li","suffix":""},{"id":360973001,"identity":"49b8e37c-6599-4fee-99a4-32300c771999","order_by":7,"name":"Xinyu Wang","email":"","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":false,"prefix":"","firstName":"Xinyu","middleName":"","lastName":"Wang","suffix":""},{"id":360973004,"identity":"6c77768a-815b-45d8-82ee-375400e26a73","order_by":8,"name":"Zhuoni Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwklEQVRIiWNgGAWjYFACxoYDCQYSPPwMPCAeMzFamA8+eFBgISPZQLwWtmTDBx8qbAwOEKvFfNoZMwmQw4xv5B6TYKiwTmxgP3sArxaZ2zkQLWY38tIkGM6kJzbw5CXg1SIhDdMC0svYdjixQYLHgDgtxrNBWv4RpSUt2QCkxQCkl7GBKC3JBx+AtEjcf2NskXAs3biNJ4eQlsSGgz/+1Nnz95wxvPGhxlq2n/0Mfi2oIAGI2UhQPwpGwSgYBaMABwAAkvE9yXGngREAAAAASUVORK5CYII=","orcid":"","institution":"Renmin Hospital of Wuhan University","correspondingAuthor":true,"prefix":"","firstName":"Zhuoni","middleName":"","lastName":"Xiao","suffix":""}],"badges":[],"createdAt":"2024-08-19 17:21:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4940193/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4940193/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-92880-2","type":"published","date":"2025-03-17T15:58:03+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":65889481,"identity":"d8e305ef-af6a-4866-ba96-bd14431850e3","added_by":"auto","created_at":"2024-10-04 04:41:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":825031,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCellular internalization of SEs .\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A,B) The shape and diameter of SE were observed by transmission electron microscopy and\u003c/p\u003e\n\u003cp\u003eNanoparticle Tracking Analysis (NTA)(Scale bar, 100 nm). (C) Western blot analysis revealed the\u003c/p\u003e\n\u003cp\u003eexpression of exosome-specific-markers CD9,CD63 and HSP70 in SEs. (D) SEs were labeled\u003c/p\u003e\n\u003cp\u003ewith PKH67 green fluorescent dye,and then co-cultured with THP-1 cells for 24 hours.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/4b67e9f47583275bcabfc5e1.png"},{"id":65889480,"identity":"6f33c268-a46a-4a7e-b4d7-dfe03c89af45","added_by":"auto","created_at":"2024-10-04 04:41:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":657561,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEs promote the polarization of macrophages towards M2 phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The percentages of M1(CD86+) and M2(CD206+) were detected by flow cytometry. (B)\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was used to examine the expression of M1/M2-MΦs-related\u003c/p\u003e\n\u003cp\u003eproteins(iNOS/IFN-γ and Arginase-1/IL-10). Values were listed as the mean ±\u003c/p\u003e\n\u003cp\u003eSD,*P\u0026lt;0.05,**P\u0026lt;0.01,***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/713ccd4bbe237012a3f771a1.png"},{"id":65889484,"identity":"ebeb57f7-7d2a-45dd-96fe-c22cc3ce3152","added_by":"auto","created_at":"2024-10-04 04:41:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":342263,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEs promote M2 polarization through PTEN/PI3K/AKT signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analysis was used to examine the expression of PTEN/phosphorylated\u003c/p\u003e\n\u003cp\u003ePI3K/AKT(p-PI3K and p-AKT). Values were listed as the mean SD,*P\u0026lt;0.05,**P\u0026lt;0.01,***P\u0026lt;0.001.(B) The decidual tissues from patients with spontaneous abortion after natural pregnancy (NP group) or IVF assisted pregnancy (IVF group). The expression of PTEN was detected by IHC and compared by Image J. Graphs, mean±SD. Magnification, ×100 and ×400. Scale bars = 50μm; ***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/033b97a2d145b598ce0515d7.png"},{"id":65889653,"identity":"7a9b3dfa-ec4b-4112-9dbe-059e2b073a3c","added_by":"auto","created_at":"2024-10-04 04:49:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":176364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of miR-26a-5p of SE was abundant the GeneChipTM sequencing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The GeneChipTM sequencing obtained the gene number of miRNAs in SEs samples. (B)\u003c/p\u003e\n\u003cp\u003eClustering heat map of 335 miRNAs expressed in SEs. (C) The levels of miR-26a-5p were\u003c/p\u003e\n\u003cp\u003eabundant in the SEs.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/47b949d97ad4dcb73717042d.png"},{"id":65889486,"identity":"1a3e1cc7-c61d-491e-9e63-991c49229ffa","added_by":"auto","created_at":"2024-10-04 04:41:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":262930,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-26a-5p is critical for SEs to promote macrophage M2 polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(B) RT-PCR assays measured the levels of miR-26a-5p in THP-1 cells treated with SE and control\u003c/p\u003e\n\u003cp\u003egroup. (B) The percentages of M1(CD86 + ) and M2(CD206 + ) were detected by flow cytometry\u003c/p\u003e\n\u003cp\u003eafter THP-1 cells transfection with controls/miR-26a-5p mimics/miR-26a-5p inhibitor. (C)Western blot analysis was used to examine the expression of M1/M2-MΦs-related\u003c/p\u003e\n\u003cp\u003eproteins(iNOS/IFN-γ and Arginase-1/IL-10).and PTEN/phosphorylated PI3K/AKT(p-PI3K and\u003c/p\u003e\n\u003cp\u003ep-AKT). Values were listed as the mean±SD,*P\u0026lt;0.05,**P\u0026lt;0.01,***P\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/c5b5beb03090dd8eeb3e7981.png"},{"id":65890097,"identity":"aa2cece0-5a71-4560-b037-c794a4fb1fe9","added_by":"auto","created_at":"2024-10-04 04:57:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":969109,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEs help to improve pregnancy outcome in spontaneously aborted mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) The procedures of animal experiment. (B) SEs labeled with red fluorescent PKH26 could be\u003c/p\u003e\n\u003cp\u003eobserved at the maternal-fetal interface. (C) Compared the embryonic absorption rate of SE group\u003c/p\u003e\n\u003cp\u003eand control group in spontaneously aborted mice. Abortion sites were the necrotic appearance. The\u003c/p\u003e\n\u003cp\u003eembryonic absorption rate was calculated as the ratio of resorption sites to total implantation sites.\u003c/p\u003e\n\u003cp\u003e(D,E) RT-PCR assays measured the levels of miR-26a-5p and IHC assays were performed to\u003c/p\u003e\n\u003cp\u003edetect the expression of PTEN at the maternal-fetal interface in SE group and control group. (F)\u003c/p\u003e\n\u003cp\u003eWestern blot analysis was used to examine the expression of M1/M2-MΦs-related\u003c/p\u003e\n\u003cp\u003eproteins(iNOS/IFN-γ and Arginase-1/IL-10) and PTEN/phosphorylated PI3K/AKT(p-PI3K and\u003c/p\u003e\n\u003cp\u003ep-AKT). Values were listed as the mean±SD,*P\u0026lt;0.05,**P\u0026lt;0.01,***P\u0026lt;0.0\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/ba78d5f644502a23ec5860a4.png"},{"id":65889654,"identity":"f457db13-0aba-4912-a641-a8655d2eb717","added_by":"auto","created_at":"2024-10-04 04:49:45","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":118502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of SEs-derived miR-26a-5p regulate the polarization of\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003edecidual MΦs through the PTEN / PI3K / AKT pathway.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter intercourse, SEs reach the uterus with the seminal plasma. After swallowed by endometrial\u003c/p\u003e\n\u003cp\u003emacrophages, SEs release the contained miR-26a-5p, target the PTEN / PI3K / AKT signaling\u003c/p\u003e\n\u003cp\u003epathway, promote macrophage polarization toward M2 phenotype, and release inflammatory\u003c/p\u003e\n\u003cp\u003einhibitors Arginase-1 and IL-10, thus shaping an immune tolerance microenvironment conducive\u003c/p\u003e\n\u003cp\u003eto embryonic implantation and development.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/0f8d3f92a69bdd50f7e1c04e.png"},{"id":79120516,"identity":"4d4f878a-9d1c-4da3-a3d2-be9adc70a22f","added_by":"auto","created_at":"2025-03-24 16:09:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4380347,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/79db67a7-42c7-45c5-b3e0-afe46b6032a5.pdf"},{"id":65889501,"identity":"3d837ef2-6ca9-428e-8343-ae7dde9daeea","added_by":"auto","created_at":"2024-10-04 04:41:45","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14745850,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.S1.tif","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/7bfd8a4e48658238ca989d9e.tif"},{"id":65889500,"identity":"9bf5d7df-f6ff-4fae-9384-4ad9044d2c66","added_by":"auto","created_at":"2024-10-04 04:41:45","extension":"rar","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1017293,"visible":true,"origin":"","legend":"","description":"","filename":"SFigure.rar","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/2a2b73fc4b88cca705e1ada1.rar"},{"id":65889483,"identity":"6eca50d6-e94c-4a90-bb19-57d33d00ed57","added_by":"auto","created_at":"2024-10-04 04:41:45","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":82329,"visible":true,"origin":"","legend":"","description":"","filename":"TableS1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4940193/v1/ca85bef04248814537e8746f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Seminal plasma exosome-derived miR-26-5p promotes embryo implantation and development by regulating decidual macrophage polarization via PTEN / PI3K / AKT signaling pathway","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eFor the mother, the embryo is a semi-allograft with the paternal antigen. Therefore, the appropriate immune state at the maternal-fetal interface is very important for embryo implantation and early embryo development(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). The maternal-fetal interface is composed of trophoblast cells, decidual stromal cells and decidual immune cells. Decidual immune cells mainly include natural killer cells (NKs), macrophages (MΦs), T cells and dendritic cells (DC) which secrete the corresponding cytokines involved in the regulation of immune homeostasis at the maternal-fetal interface.\u003c/p\u003e \u003cp\u003eDecidual macrophages(dMΦs), the second largest population in decidual immune cells, play a crucial role in creating a maternal tolerance environment and regulating tissue remodeling during blastocyst implantation and placental development(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). MΦs exhibit a high degree of plasticity and can adapt their phenotype in response to environmental stimuli(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). M1-dMΦs are potent effector cells that induce pro-infammatory T helper 1(Th1) cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). By contrast, M2-dMΦs attenuate these Th1 responses by producing anti- infammatory Th2 cytokines, such as IL-10 (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Furthermore, inducible nitric oxide synthase(iNOS) produced by M1-dMΦs can inhibit migration and cell trophoblast invasion by inducing higher levels of NO production, however recombinant human arginase-1 secreted by M2-dMΦ can inhibit immune cytotoxicity and promote endometrial decidualization and angiogenesis(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Thus, it is now widely accepted that M2-type polarization of dMΦs enriched at the maternal\u0026ndash;fetal interface would be conducive to promoting the immune tolerance of semi-allograft embryos, remodeling local tissues and blood vessels, and benefiting other pregnancy-related physiological processes(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeminal plasma (SP) has previously been considered as a carrier only to transport sperm, but researches now suggest that SP also has an auxiliary role in embryo implantation and early embryonic development(\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Assisted reproductive technologies(ARTs) have been extensively employed in the treatment of infertility. Clinical meta-studies find low quality evidence that the application of SP as a therapeutic approach to improve the clinical pregnancy rate in cycles of ART(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThere have been some experimental studies showing that human SP can stimulate the female immune response to provoke a controlled inflammatory response that facilitates embryo implantation, and promotes generation of immune tolerance for pregnancy, such as regulating the levels of dendritic cell (DC) or uNK cells related cytokines and chemokines(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Animal experiments also confirm that cytokines synthesized in the male accessory glands are transferred to the female at insemination, activating changes in gene expression that lead to modifications in structure and function of the female tissues. The consequences are increased fertilization rates, conditioning of the female immune response to tolerate the conceptus, and changes in the endometrium that facilitate embryo development and implantation.(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). However, whether SP can regulate dMΦs polarity has not yet been reported.\u003c/p\u003e \u003cp\u003eSeminal exosomes (SEs) are one of the main mediators of SP to perform their reproductive regulatory functions and play regulatory functions mainly through the DNA, RNA, and proteins they contain(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). For examples: SEs can be internalized by human endometrial stromal cells (eSCs) and subsequently induce them to produce IL-6 and IL-8 which are involved in the immunoregulation of embryo implantation(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e); age-related alterations of SEs may be partially responsible for lower implantation rates in the aged-SP group compared with those in the young-SP group, which were mediated by uterine immunomodulation(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePhosphatase and tensin homolog (PTEN), a tumor suppressor gene, regulates many biological processes, including proliferation, survival, cellular architecture, motility, energy metabolism, and genomic stability(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). PTEN can also participate in regulating embryo implantation and development by regulating trophoblast invasion, promoting endometrial epithelium cells (EECs) proliferation, and inducing endometrial stromal cells (ESCs) apoptosis at the maternal-fetal interface(\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). However, few articles has reported its regulation on MΦs in female reproductive system.\u003c/p\u003e \u003cp\u003eThis topic aims to explore the regulation function and mechanism of SP on dMΦs, which is conducive to the development of new targets for intervention to improve reproductive outcomes and may also provide new ideas for SP-assisted treatment of clinical infertility.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003e1.SEs promote the polarization of macrophages towards M2 phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eElectron microscopy and\u0026nbsp;NTA\u0026nbsp;revealed that particles isolated from\u0026nbsp;SP\u0026nbsp;contained\u0026nbsp;exosomes\u0026nbsp;(SEs)\u0026nbsp;with diameters ranging from 30\u0026ndash;150nm(Fig.1A,B) and\u0026nbsp;WB experiment\u0026nbsp;confirmed\u0026nbsp;that SEs expressed exosome-specific-markers\u0026nbsp;CD9,\u0026nbsp;CD63 and HSP70(Fig.1C).\u0026nbsp;SEs were stained with green fluorescent dye PKH67, and then co-cultured with THP-1cells for 24 hours. The green fluorescent was exhibited in the cytoplasm and diffused as small spots around the cell nucleus by confocal microscopy (Fig.1D)\u0026nbsp;confirming that SEs could be uptaken by THP-1 cells.\u003c/p\u003e\n\u003cp\u003eTo investigate the potential regulation of SEs on M\u0026Phi;s polarization, SEs and controls were separately co-cultured with THP-1 cells. Flow cytometry results showed that the ratio of M2 (CD206 +) / M1 (CD86 +) was increased after THP-1 cells treated with SEs (Fig. 2A), implying that SEs may induce M2-M\u0026Phi;s polarization. Subsequently, detection of protein markers associated with M\u0026Phi;s polarization revealed that co-culture with SEs increased the expression of the M2-M\u0026Phi;s-related proteins (Arginase-1 and IL-10), consistent with a decrease in the M1-M\u0026Phi;s-related proteins (iNOS and IFN- \u0026gamma;) (Fig. 2B). The above results comprehensively indicated that SEs could promote the differentiation of M\u0026Phi;s to M2 type, induce the secretion of anti-inflammatory factors and inhibit the expression of inflammatory factors, thus creating an immune tolerance environment conducive to embryo implantation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. SEs promote M2 polarization through PTEN/PI3K/AKT signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudies have shown that PTEN/PI3K/AKT signaling pathway plays an important role in regulating cell biological behavior(19). In the study, WB experiment discovered that the expression of PTEN/PI3K/AKT signaling pathway\u0026nbsp;was altered after\u0026nbsp;M\u0026Phi;s\u0026nbsp;co-cultured with SEs (Fig 3A), which suggested that SEs promoted\u0026nbsp;M\u0026Phi;s\u0026nbsp;to M2 polarization through PTEN / PI3K / AKT signaling pathway. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe similar results were found in human decidual tissue. During the IVF assisted pregnancy process, barrier contraception was usually used to avoid unintended pregnancy, thus lacking the direct contact regulation effect of SP on the uterus. We collected decidual tissues from patients with spontaneous abortion after natural pregnancy（NP group）and IVF assisted pregnancy (IVF group), determined the PTEN expression using immunohistochemistry(IHC) assay. The PTEN expression in the IVF group was significantly higher than that in the NP group (P\u0026lt;0.05) (Fig. 3B). From this result, it could be speculated that SP mainly the SEs has some regulatory effects on the expression of PTEN in the decidua.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.miR-26a-5p is critical for SEs to promote macrophage M2 polarization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs SEs primarily manifest their biological influence by delivering bioactive compounds, including miRNAs, we sought to investigate the plausible ingredients of SEs for macrophage polarization. The GeneChipTM sequencing results showed that miR-26a-5p, which had a targeted modulation effect with PTEN, was abundant in the SEs (Fig.4, Table S1). The expression of miR-26a-5p increased in THP-1 cells after co-culture with SEs confirming that miR-26a-5p was highly expressed in SEs and can be transported into THP-1 cells (Fig.5A). The increase of miR-26a-5p expression in THP-1 cells by the transfection with miR-26a-5p mimics laid the foundation for subsequent experiments (Fig. S1).\u003c/p\u003e\n\u003cp\u003eFlow cytometry assay showed that THP-1 cells were polarized to M2 and increased the M2 / M1 ratio after transfection of miR-26a-5p mimic compared with the control group and miR-26a-5p inhibitor group(Fig.5B). Meanwhile, M2-M\u0026Phi;s-related proteins Arginase-1 and IL-10 were increased, while M1-M\u0026Phi;s-related protein iNOS and IFN- \u0026gamma; were decreased(Fig.5C). WB experiment further showed that SEs transported the loaded miR-26a-5p to THP-1 cells by acting on PTEN / PI3K / AKT signaling pathway(Fig.5C). Collectedly，SEs could transport the loaded miR-26a-5p to THP-1 cells, and promoted its polarization to M2 by acting on PTEN / PI3K / AKT signaling pathway, which was conducive to immune tolerance\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.\u003c/strong\u003e\u003cstrong\u003eSEs help to improve pregnancy outcome in spontaneously aborted mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe procedures of animal experiment were shown as Figure 6A. SEs labeled with red fluorescent PKH26 were detected at the maternal-fetal interface of mice after administered by transvaginal injection which confirmed that SEs could reach the maternal uterus through sexual intercourse and directly regulated the cell function at the maternal-fetal interface(Fig. 6B).\u003c/p\u003e\n\u003cp\u003eThen, we used spontaneous abortion mouse model to test the protective effect of SEs on pregnancy. There was a significant decrease in embryo resorption rate after vaginal injection of SEs to spontaneous abortion mice which proved that SEs can ameliorate embryo loss(Fig.6C, Table1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMechanically, in the SE group, miR-26a-5p expression was significantly increased, accompanying with a significantly decrease in PTEN expression at the maternal-fetal interface compared with the control group(Fig.6D-E), which was very similar to the results of human decidua.\u003c/p\u003e\n\u003cp\u003eAltered PTEN/PI3K/AKT and M1/M2-M\u0026Phi;s-related proteins level indicated that SEs regulated PTEN/PI3K/AKT signaling pathway by targeted transmission of their endogenous miR-26a-5p to M\u0026Phi;s, accordingly promoted M\u0026Phi;s polarization to M2, thereby shaped an immune tolerance state to alleviate abortion(Fig.6F).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable1. The embryo resorption rate after vaginal injection of SEs in mice with spontaneous abortions.\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" align=\"\" width=\"88%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.3968%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003econtrol group（n=4）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003eSE group（n=4）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003eP \u0026nbsp;Value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.3968%;\"\u003e\n \u003cp\u003eSurviving fetuses\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003e32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.3968%;\"\u003e\n \u003cp\u003eResorbed fetuses\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003e9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd style=\"width: 24.3968%;\"\u003e\n \u003cp\u003eResorption rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003e31.03%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003e11.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 25.2011%;\"\u003e\n \u003cp\u003eP\u0026lt;0.05\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe success of pregnancy mainly depends on immune tolerance of the mother for the semi-allogeneic fetus. Therefore, immunological status at the maternal-fetal interface is critical for the establishment and maintenance of pregnancy(\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e). dM\u0026Phi;s, the second largest community at the maternal-fetal interface, are proposed to be involved in immune tolerance required for a successful pregnancy. M\u0026Phi;s exhibit a high degree of plasticity and can adapt their phenotype in response to environmental stimuli. The M1 populations refer to the classically activated M\u0026Phi;s and display the capacity to present antigens to the adaptive immune system which are more effective at antigen clearance. Compared to M1 phenotype, M2 populations are alternatively activated which have immunosuppressive capacities(\u003cspan class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e). The polarization and functions of dM\u0026Phi;s can be triggered by signals present in the surrounding environment, such as seminal fluid or trophoblast-derived miRNA(\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eSP has been viewed as simply a vehicle to carry sperm to fertilize the oocyte before, but evidence is now building that attributes of seminal fluid other than sperm fertilizing capability influence reproductive outcomes in mammals(\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e). Increasing number of molecular biology studies have revealed that SP can regulate the cells function at the maternal and fetal interface: ①Impaired modulation of immune response and improper placental development due to altered cytokine levels in seminal components may be the contributing paternal factors inrecurrent pregnancy loss (RPL)(\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e);② Exposure of endometrial epithelial cells (eECs) and endometrial stromal fibroblasts (eSFs) to SP in vitro increases expression of genes and secreted proteins associated with cellular migration, proliferation, viability and inhibition of cell death.(\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e)༛③IL-11 is one of the most potently SP-induced genes in eSFs and is important for SP-facilitated decidualization(\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e); ④Low expression of GDF-15 and overexpressed C3 in the SEs of RPL patients may distort maternal immune response to paternal antigens leading to impaired decidualization(\u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e). Clinical meta-studies also have shown that supplement with SP is beneficial to improve the clinical pregnancy rate in the process of assisted reproduction(\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e). And this effectiveness has also been demonstrated in a variety of animal experiments(\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e). In light of the observed ability of SEs to regulate macrophage polarization, we successfully extracted exosomes from healthy SP and co-cultured them with THP-1 cells. SEs were found to be phagocytosed by THP-1 cells and induced their transition to M2-type macrophages, accompanied by increased expression of M2-associated proteins (arginase-1 and IL-10), thus creating a state of immune tolerance that promoted embryo implantation and development. This study demonstrated for the first time that SP, particularly SEs, can mediate uterine immunomodulation through directly regulating M\u0026Phi;s polarization, in addition to decidual DC maturation(\u003cspan class=\"CitationRef\"\u003e16\u003c/span\u003e), CD11c\u0026thinsp;+\u0026thinsp;antigen presenting cells (APC) and paternal antigen-specific regulatory T cells (Tregs) development(\u003cspan class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003ePTEN/PI3K/AKT signaling pathway is one of the highly conserved important molecules in regulating cell biological function at the maternal-fetal interface, such as alleviating the inflammatory damage of bovine eECs(\u003cspan class=\"CitationRef\"\u003e36\u003c/span\u003e), promoting eECs proliferation and inducing eSFs apoptosis(\u003cspan class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e\n\u003cp\u003eThe IHC experiments of clinical specimens in this experiment suggested that SP has regulatory effects on decidual PTEN expression. In vitro testing revealed that SEs promote M\u0026Phi;s polarization toward M2 phenotype through PTEN/PI3K/AKT signaling pathway. Based on the above results, we speculated that SP, mainly SEs, regulated decidual macrophage M2 polarization through the PTEN / PI3K / AKT signaling pathway and promoted immune tolerance.\u003c/p\u003e\n\u003cp\u003emiRNAs were identifed as crucial constituents of exosomes, signifcantly determining the impact of exosomes on target cells. In order to uncover the molecular mechanisms underlying the impact of the SEs on macrophage polarization, the GeneChipTM sequencing was applied to reveal the abundance of diferent miRNAs present in the SEs. miR-26a-5p, which has been recognized having the targeted modulation effect on PTEN, was abundant in the SEs(\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e). miR-26a-5p can directly inhibit PTEN translation by targeting its 3\u0026apos;-UTR, which in turn activates the PI3K/AKT pathway(\u003cspan class=\"CitationRef\"\u003e18\u003c/span\u003e). After transfected THP-1 cells with SEs, miR-26a-5p was significantly increased in THP-1 cells, which further confirming that miR-26a-5p was enriched in the SEs. A corresponding decrease in the expression levels of PTEN and M2-M\u0026Phi;s polarization were also found. The above results proved that SEs transported miR26a-5p to M\u0026Phi;s and promoted M2 polarization through PTEN / PI3K / AKT signaling pathway.\u003c/p\u003e\n\u003cp\u003eNative or engineered exosomes are low toxicity and low immunogenicity, therefore they are effective tool for delivering small-molecule drugs and biological therapeutics into cells and tissue, having enormous potential for therapeutic applications(\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e). Increased embryo absorption rate and exorbitant proinflammatory cytokines were found in RPL mice (CBA/J females paired with DBA/2 males)(\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e), however SEs transvaginal application markedly improved the poor conditions above. We unfurled the remarkable therapeutic ability of SEs in abortion-prone mice, and this was achieved by targeting M\u0026Phi;s-M2 polarity thus favoring immune tolerance at the maternal-fetal interface.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSEs extraction\u003c/h2\u003e \u003cp\u003eAll procedures involving participants in this study were approved by the Clinical Trial Ethics Committee of Renmin Hospital of Wuhan University (Wuhan, China, CTEC number: WDRY2021-K044). To extract SEs, seminal fluid was centrifuged at 800g for 15 minutes at 25\u0026deg;C to separate spermatozoa, and then centrifuged at 10,000g for 30 minutes at 4\u0026deg;C to remove cell debris and other impurities. The supernatant was in turn centrifuged at 10,000g for 90 minutes to pellet microvesicles (MVs). Remaining supernatant was ultracentrifuged at 100,000g for 70 minutes and the exosome-containing pellets were washed with PBS for two times. SEs ultimately resuspended in PBS and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for the following steps.\u003c/p\u003e \u003cp\u003eThe size and purity of the isolated SEs were determined using nanoparticle tracking analyzer (NTA, ZetaView 7PMX 120, Particle Metrix, Germany), transmission electron microscope (TEM, JEOL, Tokyo, Japan), and Western blot analysis. The antibodies used were as follows: anti-CD9 (1:1000, ab92726, Abcam, USA), anti-CD63 (1:1000, ab216130, Abcam, USA), anti-HSP70 (1:2000, ab181606, Abcam, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eMonocyte THP1 was purchased from Wuhan Procell Life Science and Technology Co., Ltd., China, and cultured in RPMI-1640 medium (Gibco, USA) which contained 10% FBS (Gibco, USA) and 1% penicillin\u0026ndash;streptomycin (Servicebio, Wuhan) at 37\u0026deg;C in a humidified incubator under 5% CO2. THP1 monocytes were matured into M0 macrophages with 100ng/mL of phospholipid 12-myristic acid 13-acetate (PMA, Sigma-Aldrich, USA) for 48h to macrophage polarization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCellular internalization of SEs\u003c/h2\u003e \u003cp\u003eSEs were labelled with PKH67 according to the manufacturer\u0026rsquo;s instructions(Sigma-Aldrich, USA). Shortly, SEs were added to diluent C, and PKH67 were added to diluent C. The mixture was incubated at room temperature. An equal volume of FBS was added to stop the dyeing reaction and bind the excess dye. Excess dye was removed by centrifuging the SEs at 400g at 25\u0026deg;C for 10 minutes.The nuclei were stained with DAPI (0.5 \u0026micro;g/mL, Invitrogen, USA). The labelled SEs were incubated with THP-1 cells for 48 hours. The internalization of SEs by THP-1 cells was observed using the fluorescence microscope (BX53, Olympus, Japan).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot(WB)assay\u003c/h2\u003e \u003cp\u003eProteins from cell lysates were separated by Radioimmunoprecipitation assay (RIPA) (Servicebio, Wuhan) lysis buffer and transferred to polyvinylidene fluoride (PVDF) membranes. The PVDF membranes were blocked with 5% skimmed milk and incubated with appropriate primary antibodies at 4\u0026deg;C overnight. Thereafter, the PVDF membranes were incubated with horseradish peroxidaseconjugated secondary antibodies. Protein blots were visualized by using the ECL-Plus western blotting detectionsystem (Thermo Fisher Scientific, USA). The antibodies used were as follows: rabbit anti-CD9 (1:1000, ab92726, Abcam, USA), rabbit anti-CD63 (1:1000, ab216130, Abcam, USA), rabbit anti-HSP70 (1:1000, ab181606, Abcam, USA), rabbit anti-PTEN(1:1000, ab267787, Abcam, USA), rat anti-p-AKT (1:1000, ab38449, Abcam, USA), rat anti-p-PI3K(1:500, ab182651, Abcam, USA), rabbit anti-Arginase-1 (1:1000, ab133543, Abcam, USA), rabbit anti-iNOS (1:1000, ab178945, Abcam, USA), rabbit anti-IL-10 (1:1000, ab133575, Abcam, USA), rabbit anti-IFN-γ (1:1000, ab267369, Abcam, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eCells were suspended in the staining buffer (PBS\u0026thinsp;+\u0026thinsp;3% BSA). Phycoerythrin (PE)-conjugated anti-human CD68 antibody (eBioscience, USA), PE-Cy7-conjugated anti-human CD86 and CD206 antibody were used for staining. All the data were acquired using FACS Canto II, BD Company, USA, and processed using FlowJo (Tree Star, Ashland, OR, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eClinical sample collection\u003c/h2\u003e \u003cp\u003eAll participants were recruited from the Reproductive Medical Center at the Renmin Hospital of Wuhan University from June, 2021 to June, 2022. First-trimester human decidual tissues were obtained from patients with spontaneous abortion after natural pregnancy(NP group, n\u0026thinsp;=\u0026thinsp;10) or IVF assisted pregnancy(IVF group, n\u0026thinsp;=\u0026thinsp;10) Spontaneous abortion caused by fetal chromosome abnormalities, endocrine disorders or metabolic abnormalities, uterine malformations, autoimmune diseases were excluded. All patients undergoing IVF treatment were required to use barrier contraception to reduce unplanned pregnancy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry(IHC)\u003c/h2\u003e \u003cp\u003eDecidual tissues were collected from spontaneous abortion after natural pregnancy and IVF assisted pregnancy. These tissues were fixed in 10% formaldehyde at 37\u0026deg;C for 6h, followed by routine deparaffinization and dehydration for immunohistochemistry. Formalin-fixed paraffin-embedded tissues were cut into 4 \u0026micro;m sections. 3% hydrogen peroxide was used to block the endogenous peroxidase activity, and the non-specific binding was blocked with 5% bovine serum albumin for 20 minutes. The sections were incubated at 37\u0026deg;C with rabbit anti-PTEN antibody (1:100, ab267787, abcam, USA) for 4\u0026deg;C overnight. After washed in PBS, the sections were then incubated with HRP-labelled goat anti-rabbit IgG (1:500, AS1107, ASPEN, USA) secondary antibodies for 30 minutes at room temperature, followed by wash with PBS three times for 5 minutes. The color was developed with a DAB kit (Dako Cytomation, Glostrup, Denmark). Positive signals were visualized as brown. Mean fuorescence intensity (IntDen/Area) was quantitatively analyzed using Image J software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative Real-Time PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eThe total RNA in the tissues was isolated using TRIzol Reagent (ELK Biotechnology, China), according to the manufacturer\u0026rsquo;s instructions. The cDNA was performed on the ABI7900 system (Applied Biosystems, USA) for mRNA expression. The reverse transcription kit (ELK Biotechnology, China) was used to reverse-transcribe the RNA. The relative RNA quantifcation was performed using the comparative 2\u0026thinsp;\u0026minus;\u0026thinsp;ΔΔCt method.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eCell transfection\u003c/h2\u003e \u003cp\u003eIn all cell transfections, 5\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells were plated on 35mm plates. 5\u0026micro;l oligonucleotides including miR-26a-5p mimics or miR-26a-5p inhibitor were diluted in 250\u0026micro;l Opti-Medium (Life Technology, USA) and mixed with 5\u0026micro;l Lipofectamine 2000 (Invitrogen,Thermo Fisher Scientific, Inc., USA) according to the manufacturer\u0026rsquo;s instructions. These mixture was then added with 500 \u0026micro;l dye solution and 1500\u0026micro;l basal medium. All of the culture media were completely replaced after 6 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eAnimal experiments\u003c/h2\u003e \u003cp\u003eAll animal operations were approved by the ethics committee for laboratory animal welfare (IACUC) of Renmin Hospital of Wuhan University (Approval number: No. WDRM animal (f) No. 20201207). 6\u0026ndash;8 weeks of age ICR mice, CBA/J female mice and DBA/2 male mice were obtained from the Animal Experiment Center of Wuhan University. ICR female mice mated ICR male mice at 1:1 for normal pregnant group; CBA / J female mice mated with DBA / 2 male mice at 1:1 for spontaneous abortion group. Overall, 8 spontaneous abortion mice were randomly divided into two groups as follows: naive-control and SEs treatment. Pregnant mare serum gonadotropin (PMSG, China) 10IU was given at 8pm on the first day, and human chorionic gonadotropin (HCG, China) 10IU was given at 8pm on the third day to induce superovulation. The day on which the vaginal plug appeared in female mice was recorded as the 0.5 day (D0.5) of pregnancy. For the SE groups, three shots of 100\u0026micro;l PHK26 dye labeled SEs (1ug/ul in PBS)were injected by vaginal on D0.5, D3.5 and D6.5. The naive-control group received a vaginal injection of PBS. On D14.5, the mice were euthanized via CO2 inhalation. The placental tissues, including decidual tissues and the fetus, were collected for the following experiments.All experimental procedures were conducted in conformity with the institutional guidelines issued by the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eAfter coitus, SP has a direct contact regulation effect on the female reproductive tract, and the SEs are one of the main media playing the SP regulation function. In this study, we found that SP, namely SEs promote the polarization of M\u0026Phi;s towards M2 phenotype through PTEN/PI3K/AKT signaling pathway. miR-26a-5p is the key regulatory molecules in SEs playing the regulatory role. The regulatory effect of SEs on M\u0026Phi;s helps to construct an immune-tolerant environment that promotes embryo cultivation and development(Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). This protective effect was confirmed in abortive mouse model trials. Thus, SEs are therefore expected as a novel treatment to improve pregnancy outcomes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: Zhuoni Xiao and Xiaolin Chen; methodology: Yan zhang, Xin Chen, Qin Liu, Xinyu Wang and Xiaoling Li; validation: Qin Liu, Xinyu Wang and Xiaoling Li; investigation: Jie Li and Jing Zhao; formal analysis:Yan zhang, Xin Chen; writing\u0026mdash;original draft preparation:Yan zhang and Xiaolin Chen; writing\u0026mdash;review and editing: Zhuoni Xiao ; visualization:Jie Li and Jing Zhao; project administration:Yan zhang and Xiaolin Chen; funding acquisition:Zhuoni Xiao; All authors have read and agreed to thepublished version of the manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by The Interdisciplinary Innovative Talents Foundation from Renmin Hospital of Wuhan University (JCRCGW-2022-003).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Clinical Trial Ethics Committee of Renmin Hospital of Wuhan University (Wuhan, China, CTEC number: WDRY2021-K044) and the ethics committee for laboratory animal welfare (IACUC) of Renmin Hospital of Wuhan University (Approval number: No. WDRM animal (f) No. 20201207)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Informed consent was obtained from all subjects involved in the study\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest:\u0026nbsp;\u003c/strong\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u0026nbsp;\u003c/strong\u003eThe data supporting these fndings is available upon request from the corresponding author, [email protected].\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYang, F., Zheng, Q. \u0026amp; Jin, L. Dynamic Function and Composition Changes of\u0026ensp;Immune\u0026ensp;Cells During Normal and Pathological Pregnancy at the\u0026ensp;Maternal-Fetal\u0026ensp;Interface. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e10\u003c/b\u003e, 2317 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLocati, M., Curtale, G. \u0026amp; Mantovani, A. Diversity,\u0026ensp;Mechanisms, and\u0026ensp;Significance\u0026ensp;of\u0026ensp;Macrophage\u0026ensp;Plasticity. \u003cem\u003eAnnu. Rev. Pathol.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 123\u0026ndash;147 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, L. L. et al. Decorin promotes\u0026ensp;decidual\u0026ensp;M1-like\u0026ensp;macrophage\u0026ensp;polarization via mitochondrial dysfunction resulting in recurrent pregnancy loss. \u003cem\u003eTheranostics\u003c/em\u003e. \u003cb\u003e12\u003c/b\u003e (17), 7216\u0026ndash;7236 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHu, J. J. et al. Lactobacillus murinus alleviate intestinal ischemia/reperfusion injury through promoting the release of interleukin-10 from M2 macrophages via Toll-like receptor 2 signaling. \u003cem\u003eMicrobiome\u003c/em\u003e. \u003cb\u003e10\u003c/b\u003e (1), 38 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKieler, M., Hofmann, M. \u0026amp; Schabbauer, G. More than just protein building blocks: how amino acids and related metabolic pathways fuel macrophage polarization. \u003cem\u003eFEBS J.\u003c/em\u003e \u003cb\u003e288\u003c/b\u003e (12), 3694\u0026ndash;3714 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Y. H., He, M., Wang, Y. \u0026amp; Liao, A. H. Modulators of the Balance between M1 and M2\u0026ensp;Macrophages\u0026ensp;during Pregnancy. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e8\u003c/b\u003e, 120 (2017).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen, Q. et al. Immune Regulation of Seminal Plasma on the Endometrial Microenvironment: Physiological and Pathological Conditions. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e24\u003c/b\u003e (19), 14639 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhmadi, H. et al. Composition and effects of seminal plasma in the female reproductive tracts on implantation of human embryos. \u003cem\u003eBiomed. Pharmacother\u003c/em\u003e. \u003cb\u003e151\u003c/b\u003e, 113065 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchjenken, J. E. \u0026amp; Robertson, S. A. The Female Response to Seminal Fluid. \u003cem\u003ePhysiol. Rev.\u003c/em\u003e \u003cb\u003e100\u003c/b\u003e (3), 1077\u0026ndash;1117 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAta, B. et al. Application of seminal plasma to female genital tract prior to embryo transfer in assisted reproductive technology cycles (IVF, ICSI and frozen embryo transfer). \u003cem\u003eCochrane Database Syst. Rev.\u003c/em\u003e \u003cb\u003e2\u003c/b\u003e (2), CD11809 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCraenmehr, M. H. C. et al. Effect of seminal plasma on dendritic cell differentiation in vitro depends on the serum source in the culture medium. \u003cem\u003eJ. Reprod. Immunol.\u003c/em\u003e \u003cb\u003eFeb\u003c/b\u003e, 137:103076 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaima, A. et al. A\u0026ensp;semen-based stimulation method to analyze cytokine production by uterine CD56bright\u0026ensp;natural\u0026ensp;killer\u0026ensp;cells\u0026ensp;in women with recurrent pregnancy loss. \u003cem\u003eJ. Reprod. Immunol.\u003c/em\u003e \u003cb\u003e142\u003c/b\u003e, 103206 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobertson, S. A. Seminal fluid signaling in the female reproductive tract: lessons from rodents and pigs. \u003cem\u003eJ. Anim. Sci.\u003c/em\u003e \u003cb\u003e85\u003c/b\u003e (13 Suppl), E36\u0026ndash;44 (2007).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMachtinger, R., Laurent, L. C. \u0026amp; Baccarelli, A. A. Extracellular vesicles: roles in gamete maturation, fertilization and embryo implantation. \u003cem\u003eHum. Reprod. Update\u003c/em\u003e. \u003cb\u003e22\u003c/b\u003e (2), 182\u0026ndash;193 (2016 Mar-Apr).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePaktinat, S. et al. Seminal exosomes induce interleukin-6 and interleukin-8 secretion by human endometrial stromal cells. \u003cem\u003eEur. J. Obstet. Gynecol. Reprod. Biol.\u003c/em\u003e \u003cb\u003eApr\u003c/b\u003e, 235:71\u0026ndash;76 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, D. et al. Seminal Plasma and Seminal Plasma Exosomes of Aged Male Mice Affect Early Embryo Implantation via Immunomodulation. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 12723409 (2021 Oct).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, C. Y. et al. PTEN: Tumor Suppressor and Metabolic Regulator. Front Endocrinol (Lausanne). Jul \u003cb\u003e9\u003c/b\u003e:9338. (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, L. et al. CircSTAM inhibits migration and invasion of\u0026ensp;trophoblast\u0026ensp;cells by regulating miR-148a-5p/PTEN\u0026ensp;axis. \u003cem\u003eJ. Assist. Reprod. Genet.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e (1), 201\u0026ndash;210 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, L. et al. miR-26a promoted endometrial epithelium cells (EECs) proliferation and induced stromal cells (ESCs) apoptosis via the PTEN-PI3K/AKT pathway in dairy goats. \u003cem\u003eJ. Cell. Physiol.\u003c/em\u003e \u003cb\u003e233\u003c/b\u003e (6), 4688\u0026ndash;4706 (2018).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTerakawa, J. et al. Ovarian insufficiency and CTNNB1 mutations drive malignant transformation of\u0026ensp;endometrial\u0026ensp;hyperplasia with altered\u0026ensp;PTEN/PI3K activities. \u003cem\u003eProc. Natl. Acad. Sci. U S A\u003c/em\u003e. \u003cb\u003e116\u003c/b\u003e (10), 4528\u0026ndash;4537 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, Y., Liu, Z. \u0026amp; Sun, H. Fetal-maternal\u0026ensp;interactions during pregnancy: a 'three-in-one' perspective. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e, 1198430 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePrabhuDas, M. et al. Immune mechanisms at the maternal-fetal interface: perspectives and challenges. \u003cem\u003eNat. Immunol.\u003c/em\u003e \u003cb\u003e16\u003c/b\u003e (4), 328\u0026ndash;334 (2015).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing, J. et al. M2 macrophage-derived G-CSF promotes trophoblasts EMT, invasion and migration via activating PI3K/Akt/Erk1/2 pathway to mediate normal pregnancy. \u003cem\u003eJ. Cell. Mol. Med.\u003c/em\u003e \u003cb\u003e25\u003c/b\u003e (4), 2136\u0026ndash;2147 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing, J. et al. Granulocyte colony-stimulating factor in reproductive-related disease: Function, regulation and therapeutic effect. \u003cem\u003eBiomed. Pharmacother\u003c/em\u003e. \u003cb\u003eJun\u003c/b\u003e, 150:112903 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, N., Liang, H. \u0026amp; Zen, K. Molecular\u0026ensp;mechanisms\u0026ensp;that\u0026ensp;influence\u0026ensp;the\u0026ensp;macrophage\u0026ensp;m1-m2\u0026ensp;polarization\u0026ensp;balance. \u003cem\u003eFront. Immunol.\u003c/em\u003e \u003cb\u003e5\u003c/b\u003e, 614 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang, J. et al. Trophoblast-derived miR-410-5p induces M2 macrophage polarization and mediates immunotolerance at the fetal-maternal interface by targeting the STAT1 signaling pathway. \u003cem\u003eJ. Transl Med.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e (1), 19 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoertson, S. A. et al. Role of high molecular weight seminal vesicle proteins in eliciting the uterine inflammatory response to semen in mice. \u003cem\u003eJ. Reprod. Fertil.\u003c/em\u003e \u003cb\u003e107\u003c/b\u003e (2), 265\u0026ndash;277 (1996).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRodriguez-Martinez, H. et al. Seminal Plasma: Relevant for Fertility? \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e22\u003c/b\u003e (9), 4368 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJena, S. R. et al. Comparative proteome profiling of seminal components reveal impaired immune cell signalling as paternal contributors in recurrent pregnancy loss patients. \u003cem\u003eAm. J. Reprod. Immunol.\u003c/em\u003e \u003cb\u003e89\u003c/b\u003e (2), e13613 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen, J. C. et al. Seminal plasma induces global transcriptomic changes associated with cell migration, proliferation and viability in endometrial epithelial cells and stromal fibroblasts. \u003cem\u003eHum. Reprod.\u003c/em\u003e \u003cb\u003e29\u003c/b\u003e (6), 1255\u0026ndash;1270 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGeorge, A. F. et al. Seminal Plasma Promotes Decidualization of Endometrial Stromal Fibroblasts In Vitro From Women With and Without Inflammatory Disorders in a Manner Dependent on Interleukin-11 Signaling. \u003cem\u003eHum. Reprod.\u003c/em\u003e \u003cb\u003e35\u003c/b\u003e (3), 617\u0026ndash;640 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJena, S. R. et al. Paternal contributors in recurrent pregnancy loss: Cues from comparative proteome profiling of seminal extracellular vesicles. \u003cem\u003eMol. Reprod. Dev.\u003c/em\u003e \u003cb\u003e88\u003c/b\u003e (1), 96\u0026ndash;112 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchjenken, J. E. \u0026amp; Robertson, S. A. Seminal fluid and immune adaptation for pregnancy\u0026ndash;comparative biology in mammalian species. \u003cem\u003eReprod. Domest. Anim.\u003c/em\u003e \u003cb\u003e49\u003c/b\u003e (Suppl 3), 27\u0026ndash;36 (2014).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShima, T. et al. Uterine CD11c\u0026thinsp;+\u0026thinsp;cells induce the development of paternal antigen-specific Tregs via seminal plasma priming. \u003cem\u003eJ. Reprod. Immunol.\u003c/em\u003e \u003cb\u003e141\u003c/b\u003e, 103165 (2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRobertson, S. A. et al. Seminal fluid drives expansion of the CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;T regulatory cell pool and induces tolerance to paternal alloantigens in mice. \u003cem\u003eBiol. Reprod.\u003c/em\u003e \u003cb\u003e80\u003c/b\u003e (5), 1036\u0026ndash;1045 (2009).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, J., Liang, Q. \u0026amp; Wang, T. IFN-τ mediated miR-26a targeting\u0026ensp;PTEN\u0026ensp;to activate\u0026ensp;PI3K/AKT\u0026ensp;signalling to alleviate the inflammatory damage of bEECs. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e (1), 9410 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang, X. et al. Extracellular vesicles derived from human ESC-MSCs target\u0026ensp;macrophage\u0026ensp;and promote anti-inflammation process, angiogenesis, and functional recovery in ACS-induced severe skeletal muscle injury. \u003cem\u003eStem Cell. Res. Ther.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (1), 331 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao, P. et al. Long noncoding RNA LINC-PINT retards the abnormal growth of airway smooth muscle cells via regulating the microRNA-26a-5p/PTEN\u0026ensp;axis in asthma. \u003cem\u003eInt. Immunopharmacol.\u003c/em\u003e \u003cb\u003e99\u003c/b\u003e, 107997 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang, R. Q. et al. Lnc-GAN1 expression is associated with good survival and suppresses tumor progression by sponging\u0026ensp;mir-26a-5p\u0026ensp;to activate\u0026ensp;PTEN\u0026ensp;signaling in non-small cell lung cancer. \u003cem\u003eJ. Exp. Clin. Cancer Res.\u003c/em\u003e \u003cb\u003e40\u003c/b\u003e (1), 9 (2021).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang, B. et al. Engineered EVs with pathogen proteins: promising vaccine alternatives to LNP-mRNA vaccines. \u003cem\u003eJ. Biomed. Sci.\u003c/em\u003e \u003cb\u003e31\u003c/b\u003e (1), 9 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLokumcu, T. et al. Proteomic, Metabolomic, and Fatty Acid Profiling of Small Extracellular Vesicles from Glioblastoma Stem-Like Cells and Their Role in Tumor Heterogeneity. \u003cem\u003eACS Nano\u003c/em\u003e Jan 11. (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, Z. et al. Bushen Antai recipe alleviates embryo absorption by enhancing immune tolerance and angiogenesis at the maternal-fetal interface via mobilizing MDSCs in abortion-prone\u0026ensp;mice. \u003cem\u003ePhytomedicine\u003c/em\u003e. \u003cb\u003e123\u003c/b\u003e, 155164 (2024).\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":"seminal plasma, seminal exosomes, macrophage polarity, miR-26-5p, embryo implantation and development","lastPublishedDoi":"10.21203/rs.3.rs-4940193/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4940193/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe immunomodulatory effects of seminal plasma(SP) on the maternal immune system play an important role in the implantation and development of the embryo. Decidual macrophages(dMΦs) are one of the major immune cells in the maternal-fetal immune microenvironment, and their M2-type polarization facilitates the establishment and maintenance of pregnancy. However, the role of SP on the polarization of dMΦs is unknown. In this study, we investigated the role and mechanism of SP on the polarization of dMΦs by gene chip sequencing as well as in vitro and in vivo experiments. The results revealed that SP promoted dMΦs M2-type polarization. Gene chip sequencing revealed that miR-26-5p was highly expressed in seminal exosomes(SEs) which could act on PTEN/PI3K/AKT signaling pathway and significantly promote MΦs M2 polarization. Moreover, SEs supplementation significantly reduced embryo resorption in spontaneously aborted mice. In conclusion, our study demonstrated that the SEs derived miR-26-5p in SP promoted the M2 polarization of dMΦs by targeting PTEN/PI3K/AKT signaling pathway, which created an immune-tolerant environment conducive to embryo implantation and development. This study provided new ideas for clinical SP-assisted therapy to improve pregnancy outcomes.\u003c/p\u003e","manuscriptTitle":"Seminal plasma exosome-derived miR-26-5p promotes embryo implantation and development by regulating decidual macrophage polarization via PTEN / PI3K / AKT signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-04 04:41:40","doi":"10.21203/rs.3.rs-4940193/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-14T09:32:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-24T22:15:41+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-09-20T13:57:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170047689088101111627526099527304859193","date":"2024-09-11T20:35:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"81288760694895755372695939828133038926","date":"2024-09-10T19:47:53+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-09-10T19:32:47+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-09-10T19:27:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-03T14:19:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-03T03:27:51+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-08-19T17:17:39+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":"1fd2ae9b-79ed-4b20-8f53-e66ced261572","owner":[],"postedDate":"October 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":38402565,"name":"Biological sciences/Molecular biology"},{"id":38402566,"name":"Earth and environmental sciences/Biogeochemistry"}],"tags":[],"updatedAt":"2025-03-24T16:02:57+00:00","versionOfRecord":{"articleIdentity":"rs-4940193","link":"https://doi.org/10.1038/s41598-025-92880-2","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-17 15:58:03","publishedOnDateReadable":"March 17th, 2025"},"versionCreatedAt":"2024-10-04 04:41:40","video":"","vorDoi":"10.1038/s41598-025-92880-2","vorDoiUrl":"https://doi.org/10.1038/s41598-025-92880-2","workflowStages":[]},"version":"v1","identity":"rs-4940193","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4940193","identity":"rs-4940193","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}