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

doi:10.21203/rs.3.rs-4940193/v1

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

https://doi.org/10.21203/rs.3.rs-4940193/v1

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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

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.

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

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.

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, 5×10⁵ 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.

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.

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].

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No competing interests reported.

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

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Abstract

Figures

INTRODUCTION

RESULTS

Discussion

MATERIALS AND METHODS

SEs extraction

Cell culture

Cellular internalization of SEs

Western Blot(WB)assay

Flow cytometry

Clinical sample collection

Immunohistochemistry(IHC)

Quantitative Real-Time PCR (qRT-PCR)

Cell transfection

Animal experiments

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1