MicroRNA participates in embryo implantation by modulating endometrial tolerance in sows during peri-implantation period.

During each estrous cycle, sows release approximately 20–25 oocytes. However, the litter size at birth is reduced to 10–15 piglets. This reduction is largely attributable to the natural loss of pig embryos, which mainly occurs in two stages: one is the peri-implantation period (10–30 days gestation), where the loss of embryos accounts for approximately 30% of the total gestational loss, and the second trimester (50–70 days gestation), which accounts for approximately 10%-15% of the total gestational loss ( 1 ). During the peri-implantation period, the endometrial receptivity was changed due to significant changes in cytokines. Only the synchronous development of endometrium and embryo can ensure a suitable environment for the embryo, and synchronous development starts from the secretion of estrogen by the embryo ( 2 ). This article reviews the mechanism of miRNA regulation of embryo implantation in porcine endometrium, aiming to provide a reference for the development of more effective nutritional strategies to regulate miRNA expression and improve the survival of porcine embryos during peri-implantation period.

During the peri-implantation period, the embryo carries paternal antigens (Swine leukocyte antigen, SLA and Y chromosome-encoded male-specific histocompatibility antigen, H-Y antigen) that stimulate the mother’s immune system ( 101 ). If the immune system remains intact, the embryo will be killed due to immune rejection, but the reality is that the embryo can still survive because the mother’s immune system is partially shielded. During pregnancy, natural killer cells (NK) and T cells from the mother are recruited into the endometrium, and NK cells in pregnant sows are three times higher than in non-pregnant sows, and T cells are mainly involved in the establishment of the placenta ( 102 , 103 ). Chemokines act as signaling molecules during pregnancy, alerting the maternal immune system to local immune responses at newly formed endometrial blood vessels and recruiting immune cells to the implantation site, which can disrupt endometrial angiogenesis and lead to fetal death. Previous studies have shown that porcine chemokines are involved in the recruitment of these immune cells and the establishment of the immune tolerance environment in utero . Cysteine-X-cysteine motif chemokine ligand 2 (CXCL2), CXCL5, CXCL11, and CXCL12 are chemokines required for immune cell recruitment, and CXCL9 and CXCL10 are involved in the establishment of the immune tolerance environment of swine uterus ( 104 ). CXCL9, CXCL10, CXCL11, and their receptor CXCR3 are localized in stromal cells, endothelial cells, or vascular smooth muscle cells of the swine endometrium and are most richly expressed on day 15 of gestation. Interferon-γ (IFN-γ) can increase the abundance of CXCL9, CXCL10 and CXCL11, and promote the migration of NK and T cells ( 105 ). Dual-luciferase reporter gene detection results confirmed that miR-9 can target CXCR4 and CXCL11 in pigs, indicating that miR-9 may play a role in recruitment of immune cells during embryo implantation by regulating the expression of CXCR4 and CXCL11 genes ( 106 ). In addition, CXCL12 regulates the aggregation of white blood cells into the abdominal cavity and tissue growth in endometriosis, and 35 miRNAs expression changes were found in endometrial stromal cells treated with CXCL12. The target genes of these miRNAs are mainly involved in chemokines of immune cells, inflammatory and immune responses, and pathological processes of human endometriosis lesions ( 107 ). Around 12 days of swine gestation, the embryo elongates and releases Interleukin-1 beta (IL-1β), and after elongation, interferon IFN-γ and small amounts of IFN-δ are released, which play an important role in the embryo’s attachment to the uterus. In addition to these pro-inflammatory factors, there are IL-6, IL-18, leukemia inhibitory factor (LIF) and Tumor necrosis factor-α (TNF-α), These pro-inflammatory factors create a pro-inflammatory microenvironment in the endometrium, which can receive additional tissue nutrients to nourish the pre-implantation embryo, endometrial estrogen and prostaglandin synthesis. IL-1β can activate p38MAPK and ERK1/2 signaling pathways to promote epithelial cell proliferation ( 108 ). IFN-γ is thought to promote the structural changes of endometrium during implantation by regulating the expression of tight junction protein ZO-1 in swine uterine surface epithelial cells ( 109 ). Receptors for IL-6 and LIF were expressed in both embryonic and uterine surface epithelial cells of porcine on days 10–14 of gestation, indicating that these cytokines play an important role in the implantation process ( 110 , 111 ). The pro-inflammatory cytokine IL-18, released from the pig endometrium at 15–18 days gestation, may stimulate the release of IFN-γ in the embryo ( 112 ). MiRNAs can regulate the maternal immune system during the peri-implantation period by regulating the expression of these pro-inflammatory factors. In LPS-induced bovine endometritis, miR-193a-3p exacerbates inflammation by upregulating pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) ( 113 ). Conversely, several miRNAs exhibit anti-inflammatory properties: miR-488 and miR-26a suppress IL-1β, IL-6, and TNF-α expression ( 114 , 115 ). miR-148a specifically inhibits IL-1β and TNF-α production ( 116 ). miR-424-5p and miR-24-3p attenuate LPS-induced inflammation by blocking cytokine secretion (IL-1β, IL-6, IL-8, TNF-α) and inactivating the NF-ĸB pathway, with miR-24-3p showing conserved effects in murine models ( 117 , 118 ). The anti-inflammatory action of miRNAs extends to: miR-643, which reduces IL-1β/IL-6 secretion and NF-κB activation in human endometrial epithelial cells ( 119 ). Exosomal miR-218 from inflamed bovine endometrium, which maintains immune homeostasis by suppressing IL-6, IL-1β, TNF-α, and chemokines (MIP-1α/β) ( 120 ). Notably, IFN-γ-treated human endometrial mesenchymal stem cells secrete exosomes containing differentially expressed miR-150-5p and miR-196b-5p, which participate in IL-6/8/12 signaling transduction ( 121 ). The above research results indicate that, miRNAs can affect embryo implantation by regulating CXCR4, CXCL11, IL, TNF-α and other immune-related factors. However, the regulation of miRNAs on the expression of these pro-inflammatory factors in porcine endometrium has not been reported.

Recent high-throughput studies have uncovered dynamic miRNA expression profiles at the porcine embryo-maternal interface. During early pregnancy, elongating embryos secrete estrogen (primarily E 2 ) as the key signal for maternal recognition. While exogenous E 2 administration significantly elevates endometrial E 2 concentrations by day 10 of pregnancy, it does not alter miRNA expression patterns in either the endometrium ( 21 ) or blastocysts ( 22 ). Comparative studies reveal significant miRNA expression differences in the endometrium between high-fecundity Meishan and Large White pigs during the peri-implantation period. On day 12 of pregnancy, strain-specific miRNAs predominantly regulate p53 and Wnt signaling pathways, which are critical for successful implantation ( 23 ). Later in gestation (days 15-50), differentially expressed miRNAs primarily function in endometrial remodeling (including cellular proliferation, migration, apoptosis, cytoskeletal organization, and angiogenesis) and cell communication (particularly hormone response and cell-matrix adhesion) ( 24 ).Wessels et al. identified distinct miRNA between maternal and fetal tissues during early pregnancy, with miR-10a, miR-27a, miR-29c, miR-323 and miR-331-5p showing maternal-specific expression patterns potentially linked to early pregnancy loss ( 25 ). The maternal immune system undergoes significant adaptation during this period, as evidenced by trophoblast miRNA profiles between days 12–20 that modulate inflammatory processes and cellular immunity. Notably, miR-150, miR-296-5p and miR-19a exhibit were differentially expressed in the endometrium of arrested embryos and healthy embryos on day 20 ( 26 ). These findings suggest that miRNAs play an important regulatory role in angiogenesis and immune cell development at the maternal-fetal interface, so they may be essential for embryo survival.

The uterus serves as the primary site for embryonic growth and development, and consists of three parts: uterine body, cervix, and two separate uterine horns. Endometrium is a dynamic and complex tissue composed of multiple cells targeted by steroid hormones, even during non-pregnancy, and plays a crucial role in the continuation of animals ( 15 , 16 ). Between day 10 and 13 after fertilization, the endometrium undergoes partial changes in structure and function, and its state changes from a non-receptive state to a receptive state, allowing embryo implantation and pregnancy to begin, which is called endometrium receptivity ( 17 ). The early development of pig embryos is complex. Following fertilization, the zygote forms in the ampulla-isthmus junction of the oviduct and begins cleaving, reaching the two-cell stage at ~26 hours and the four- to eight-cell stage by 48–56 hours, when it enters the uterus. By day 5, the embryo develops into a blastocyst, composed of a blastocoel cavity, trophoblast layer, and inner cell mass. After hatching from the zona pellucida on day 7, the embryo undergoes rapid expansion, growing from 0.5–1 mm (day 7) to 2–6 mm (day 10). Between days 10–11, dramatic morphological remodeling occurs: the spherical embryo (10–15 mm) elongates into a tubular (15 × 50 mm) and then a filamentous structure (1 × 100–200 mm). This transformation maximizes the contact area between the trophoblast and uterine endometrium, facilitating nutrient uptake and supporting subsequent embryo development ( 18 ). Endometrial receptivity is closely related to embryo implantation, and its receptivity will greatly affect the success rate of embryo implantation and further impact embryo implantation. Many embryos have implantation failure clinically, which is related to endometrial receptivity intolerance or insufficient receptivity ( 19 , 20 ). Endometrial receptivity is affected by a variety of factors, such as hormones, cytokines, angiogenesis, immune factors, etc., and these factors are also regulated by a variety of miRNAs, thus producing interactions, thereby regulating endometrial receptivity and affecting embryo survival.

In conclusion, miRNAs regulate endometrial receptivity during the peri-implantation period in pigs by modulating the secretion of hormones such as E 2 , P 4 , PGF 2α , and PGE 2 . By regulating the expression of ITGB, IGF, RBP4, ACP, CAD, MMP and FGF, the embryo adhesion process was affected. MiRNAs are differentially expressed at the implantation site of the embryo, which strictly controls the formation of blood vessels and thus regulates the implantation of the embryo. However, it is very regrettable that there are few studies on miRNAs in pig immune system shielding so far. In the future, we can study the precise regulation and expression of miRNAs in the immune system in many fields such as human conception and animal reproduction, so as to promote the high expression of miRNAs conducive to embryo survival, reduce the occurrence of fetal diseases after pregnancy and delivery, improve the litter size and embryo survival rate of livestock and poultry, and provide a basis for human and animal embryo health.

Extracellular vesicles are nanoscale particles secreted by organisms, which are encapsulated by phospholipid bilayers and contain components of donor cells ( 27 ). EVs can carry different molecules, such as proteins (including common EV markers like CD63 and CD9, along with other cellular proteins), different RNA species (e.g., miRNA), DNA, and membrane-associated components, facilitating intercellular signaling ( 28 ). In pigs, EVs were first observed at the maternal-fetal interface on day 16 of pregnancy ( 29 ), and have since been isolated from various fluids including uterine fluid and seminal plasma ( 30 ). During porcine peri-implantation, active EV secretion occurs in endometrial and trophoblast cells. Notably, miRNA-containing EVs were detected in uterine fluid by days 14-16, demonstrating their role in maternal-fetal communication. For example, uterine cavity-derived EV miR-125b regulates LIF/LIFR expression in endometrial epithelium by day 12 ( 11 ). Trophoblast-derived EVs carrying angiogenic miRNAs (miR-16, miR-17-5p, etc.) stimulate endothelial proliferation, suggesting roles in placental vascularization ( 31 ). These findings establish EVs as crucial mediators of embryo-maternal crosstalk during implantation.

MicroRNAs (miRNAs) are a class of non-coding single-stranded RNA with a length of 18–25 nucleotides, which is widely distributed in animals, plants, protozoa and viruses. For most miRNAs, their biosynthesis begins with the catalytic transcription of RNA polymerase II, producing primary miRNAs (pri-miRNAs) with caps and poly (A) tails ( 3 – 5 ). The DROSHA-DGCR8 complex then cleave pri-miRNAs in the nucleus to generate hairpin microRNA precursors (pre-miRNAs). These pre-miRNAs are exported to the cytoplasm through the Exportin-5 and RAN-GTP complexes, and under the action of RNase III enzyme DICER1, the terminal loop is removed to generate a mature miRNA double chain with a size of about 22 bp ( 6 ). Subsequently, the double strands were loaded onto the Argonaute (Ago) protein to form miRISC (RISC, RNA-induced silencing complex). When binding to Ago protein, only one chain of Ago protein is stably bound, while the other chain is lost ( 7 ). Mature miRNAs degrade mRNA or inhibit its translation by complementary pairing with the 3’untranslated region (3’UTR) of the corresponding mRNA. The mechanism of miRNAs generation is presented in the Figure 1 . Process of miRNA generation. It has been reported that DICER1 and AGO2 genes involved in miRNA synthesis or transport may have serious effects on reproductive function and embryonic development ( 8 , 9 ). Stowe H.M. et al. demonstrated that DICER1 is expressed in porcine early embryos and plays a critical functional role ( 10 ). Additionally, Krawczynski et al. revealed tissue-specific expression patterns of miRNA biogenesis and transport-related genes in pig embryos, trophoblasts, and endometrium ( 11 , 12 ). Uterine glands and luminal epithelium are the only sites of DICER1 and AGO2 immune response, and miRNA biosynthesis seems likely to occur in these two sites during early pregnancy in pigs. During days 11–12 of pregnancy, porcine conceptuses exhibited upregulated expression of XPO5, DICER1, TNRC6A, and AGO2, coinciding with blastocyst transformation from spherical to tubular and filamentous morphologies. This period was also marked by elevated transcriptomic activity and increased estradiol (E 2 ) secretion. On day 16 of pregnancy, the expression of AGO4 and DGCR8 increased significantly in the endometrium, accompanied by a slight increase in DICER1 and AGO3 ( 13 , 14 ). In summary, miRNA plays an important role in the regulation of pig peri-implantation period.