IntroductionToll-like receptors (TLRs) are well-known ligand-bound transmembrane proteins belonging to the pattern-recognition receptor (PRR) family, playing a central role in the innate immunity1. While TLRs are primarily recognized for their role in detecting microbial components, their involvement extends beyond immunity to include diverse functions in health and disease, such as inflammation, tissue homeostasis, and cancer2,3. In mammals, ten TLRs have been identified and are classified into two groups based on their cellular localization: cell membrane TLRs (TLR1, TLR2, TLR4, TLR5, and TLR6) and intracellular vesicle TLRs (TLR3, TLR7, TLR8, and TLR9)4. Additionally, TLR10 is expressed on the cell membrane in certain species such as humans, although its function remains unclear5. Other TLRs, like TLR11, TLR12, and TLR13, are not universally expressed across mammalian species6. Among these receptors, TLR2 and TLR4 are particularly well-studied due to their unique and broad roles in innate immunity7.TLRs can recognize both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). Specifically, TLR2 and TLR4 recognize a wide range of microbial components, including those from bacteria, fungi, viruses, and parasites8,9,10. Their expression extends across various tissues, including immune cells (e.g., macrophages and dendritic cells (DCs)), epithelial cells, and reproductive tissues11,12. Beyond their immune functions, these receptors are implicated in non-immune processes such as tissue homeostasis, metabolism, reproduction, and pathogenesis of diseases like cancer and chronic inflammatory conditions13,14,15,16. While TLR2 and TLR4 are often the first lines of defense against extracellular pathogens, intracellular TLRs play a critical role in combating intracellular infections, particularly viral infections17. Thus, both extracellular and intracellular TLRs are essential components of the host’s defense against a wide range of pathogens.In addition to PAMPs, TLR2 and TLR4 are activated by a diverse array of endogenous DAMPs released from damaged or stressed cells, to initiate immune signaling. DAMPs include a wide range of molecules, consisting of nucleic acids, proteins, ions, glycans, and metabolites. Several DAMPs have been reported to activate TLRs, and specifically for TLR2 and TLR4, key examples include PAUF, API5, RPS3, HMGB1, DNA-binding protein, heat shock proteins, DJ-1, extra domain A-fibronectin, tenascin C, oxidized lipids in the extracellular matrix, S100 proteins, and cold-inducible RNA-binding protein18. These proteins are also expressed in the reproductive tract and can influence TLR signaling19. In the context of reproductive biology, DAMPs are likely to play a significant role in several key processes, including ovulation, decidualization, parturition, sperm functions, and sperm–female reproductive tract (FRT) interactions. The precise roles of specific DAMPs in these processes are actively being investigated, highlighting the complex interplay between innate immunity and reproductive physiology.Recent evidence highlights the important role of TLR2 and TLR4 in reproductive health, where both physiological and pathological inflammation play pivotal roles. Physiological inflammation is requisite for processes like uterine clearance and embryo implantation, while excessive or pathological inflammation can lead to severe tissue damage and infertility. The pathways mediating these processes often overlap, making it essential to understand the interplay between physiological and pathological inflammation. While TLR2 and TLR4’s roles in innate immunity are well-characterized, their combined impact on reproductive biology remains underexplored. This review aims to address this gap by examining the roles of TLR2 and TLR4 in reproduction, their interactions within the environment of the reproductive tract and their importance in both inflammatory and immune responses, and reproductive outcomes.Structure and signaling of TLR2 and TLR4Generally, cell membrane TLRs are considered type I integral transmembrane proteins, consisting of three main domains: the N-terminal domain, the transmembrane domain, and the C-terminal domain20. The N-terminal domain, located outside the cytoplasm, forms an ectodomain responsible for recognizing pathogen ligands. The transmembrane domain is a single helix embedded in the cell membrane, while the C-terminal domain resides in the cytoplasm and mainly activates signal transduction adapters to initiate downstream signaling21. This signaling is mediated through the Toll/IL-1 receptor (TIR) homologous domains.To activate key adapters such as MyD88, MAL/TIRAP, TRIF, TRAM, and SARM, the TIR domains of two TLRs must interact22. For this interaction to occur, the TIR domains require close physical association, which necessitates the formation of TLR dimers (either heterodimers or homodimers)23,24,25. This dimerization is triggered by the binding of ligands or bridging molecules. The necessity of ligands or bridging molecules arises from the inherent lack of affinity between TLRs in their dimeric forms20,23,24,25,26. Bridging ligands stabilize the dimeric structure by interacting with both TLRs and maintaining their closeness, thereby facilitating effective signaling.TLR2 heterodimersTo activate TLR2 for selective cellular signaling or responses, such as cytokine expression, TLR2 must form dimers. These include heterodimers with TLR1 and TLR6 or homodimers with another TLR2, although homodimerization remains a topic of ongoing debate27. Some studies have also reported that TLR2 can form heterodimers with TLR4 and TLR1028,29. As explained above, due to the lack of strong intrinsic affinity between TLR2 and other TLRs, a bridging ligand is essential to stabilize the dimeric forms. Computational studies combined with binding free energy (BFE) calculations in human, mouse, and bovine species have demonstrated the absence of spontaneous interactions between TLRs in dimer forms20,26. Conversely, these studies indicated strong interactions between bridging ligands and TLR2 dimers, highlighting the critical role of such molecules in stabilizing these complexes20,26.The type of ligand determines the specific dimerization of TLR2. For example, triacylated lipopeptides derived from Gram-negative bacteria or mycoplasma are recognized by TLR2/1 heterodimers, while diacylated lipopeptides and lipoteichoic acid from Gram-positive bacteria and mycoplasma are detected by TLR2/6 heterodimers10,30,31,32. Additionally, low-endotoxic atypical lipopolysaccharides (LPS) have been reported to form TLR2/4 heterodimers, whereas Helicobacter pylori LPS induces TLR2/10 dimerization33,34. Interestingly, certain TLR2 ligands, such as triacylated and diacylated lipopeptides, also bind to TLR10 to form TLR2/10 heterodimers. Proteoglycans have been identified as ligands for TLR2/2 homodimers27. Beyond natural ligands, synthetic ligands can act as bridging molecules to stabilize TLR2 dimers. Pam3CSK4 (PAM3), a synthetic triacylated LP, is a well-known TLR2/1 ligand (i.e., agonist)23. Similarly, Pam2CSK4 (PAM2) and fibroblast-stimulating lipopeptide 1 are recognized as TLR2/6 ligands24,35. Diprovocim is another synthetic ligand that activates both TLR2/1 and TLR2/2 dimers36.Regarding the inflammatory response mediated by TLR2 dimerization, both pro-inflammatory and anti-inflammatory responses can be induced depending on the cell type and the origin of the ligand30. While the heterodimerization of TLR2 with TLR1 or TLR6 is predominantly associated with pro-inflammatory responses, the anti-inflammatory reaction can act as an immune regulator in certain contexts, particularly in TLR2/6 interactions37. DePaolo et al. reported that the virulence factor LcrV from Yersinia pestis activates TLR2/6, leading to the induction of interleukin 10 (IL-10) in DC, which subsequently becomes tolerogenic37. In contrast, when TLR2 heterodimerizes with TLR1, it promotes IL-12p40 production (a pro-inflammatory cytokine), resulting in inflammatory DC and T cell differentiation37.TLR4 homodimersTo activate TLR4 and initiate selective cellular signaling (either MyD88-dependent or -independent), it must form a homodimer. This homodimerization is triggered by ligands such as LPS. Specifically, TLR4 interacts with a co-receptor protein called myeloid differentiation factor 2 (MD2), which is responsible for detecting LPS25. LPS is an important component of the outer membrane of Gram-negative bacteria, consisting of three main parts: lipid A (a hydrophobic domain and the primary endotoxin), a core oligosaccharide, and the O-antigen (a repeating hydrophilic polysaccharide)38. LPS binds to a hydrophobic pocket in MD2, where five of its six lipid chains fit snugly. The sixth chain interacts directly with TLR4. This binding induces the formation of a symmetrical “M-shaped” complex comprising two TLR4-MD2-LPS units25. This multimerization is essential for activating downstream immune signaling. Additionally, LPS undergoes subtle shifts during binding, allowing its phosphate groups to interact with positively charged regions of TLR4 and MD2. Simultaneously, MD2 undergoes minor structural adjustments to stabilize the interaction, ensuring effective immune activation25.Similar to TLR2, both pro-inflammatory and anti-inflammatory responses have been reported in TLR4-mediated processes. Numerous studies have shown that activation of TLR4 by a variety of ligands can induce pro-inflammatory cytokines through the NF-κB pathway39. Anti-inflammatory responses mediated by TLR4 have also been documented40. For instance, certain probiotic bacteria, such as Bacillus subtilis, can modulate the immune response via a TLR4-dependent mechanism. This occurs through the action of exopolysaccharides, which promote the induction of anti-inflammatory M2 macrophages or inhibitory DCs40.TLR2 and TLR4 signalingTLRs can utilize both MyD88-dependent and MyD88-independent pathways40,41. The MyD88-dependent pathway is crucial for activating NF-κB and MAP kinase pathways in response to TLR signaling40. Conversely, the MyD88-independent pathway is associated with IRF-3 and IRF-7 activation, ultimately leading to the production of interferon-beta (IFN-β), which has antiviral effects42. Both receptors can utilize the MyD88-dependent pathway to activate NF-κB and MAP kinase pathways for pro-inflammatory cytokine production43. This process involves a set of intracellular TIR-domain-containing adapters, such as MyD88 and TIRAP, which are initiated by a direct interaction between these two adapters44. The key difference between TLR2 and TLR4 is that TLR4 employs the MyD88-independent pathway. This pathway, unique to TLR4 and TLR3, requires the TIR-domain-containing adapter TRAM45. In this signaling cascade, the TRIF adapter plays a central role, enabling TLR4 to mediate IFN-β production. Notably, TLR2 is incapable of inducing IFN-β expression, a function specific to TLR446.Co-receptors involved in dimerization of TLR2 and TLR4There is experimental evidence suggesting that TLR1 and TLR6 interact with both TLR2 and TLR4, although with distinct effects (Fig. 1). In epithelial cells, the distinct roles of TLR2/1 and TLR2/6 heterodimers are evident in modulating immune responses and maintaining epithelial integrity during inflammation47. TLR2/1 signaling, involving TLR1 as a co-receptor, plays a protective role by limiting excessive inflammation and supporting epithelial homeostasis (Fig. 1). Notably, TLR1 deficiency leads to a sharp increase in pro-inflammatory cytokine production (e.g., TNF and IL-1β), resulting in severe tissue damage47. In contrast, TLR2/6 signaling, mediated by TLR6, is linked to a robust inflammatory response that, while effective against microbial threats, can worsen tissue injury (Fig. 1). Deletion of TLR6 significantly reduces levels of inflammatory cytokines, including TNF, IL-1β, and IL-17A, and mitigates epithelial damage, highlighting its role in driving a pro-inflammatory cascade47. Beyond its interaction with TLR2, TLR4 is also proposed to form heterodimers with TLR1 and TLR6. A report in human endothelial cells suggests that TLR1 associates with TLR4 through a distinct mechanism, primarily suppressing TLR4-mediated LPS signaling48. This suppression is linked to extracellular, rather than intracellular, signaling. Thus, it can be concluded that TLR1 plays a central role in the interplay between TLR2 and TLR4 (Fig. 1). Specifically, TLR1 primarily acts by activating TLR2 to induce a mild inflammatory response while suppressing TLR4 to restrain potentially harmful innate immune activation26,48. Also, TLR4 can form a heterodimer with TLR6, which plays a significant role in neuroinflammation and is implicated in Alzheimer’s disease49. Shmuel-Galia et al. identified the critical involvement of transmembrane domains (TMDs) in the TLR4/6 heterodimer, particularly when stimulated by amyloid-beta peptides49. Using a peptide-based interference approach, the authors demonstrated that TMD-derived peptides selectively inhibited TLR4-TLR6 dimerization without affecting TLR2-TLR6 interactions (Fig. 1). This disruption effectively reduced microglia-mediated inflammatory responses, highlighting a potential therapeutic strategy for controlling neuroinflammation49. Additionally, TLR2 and TLR4 can interact directly as a heterodimer. Studies by Francisco et al. showed that TLR2/4 heterodimers recognize atypical LPS, though the inflammatory response is weaker compared to the TLR4/4 homodimer34. In summary, the functional divergence between TLR2 and TLR4 signaling hinges on TLR1’s distinct roles. TLR2 necessitates heterodimerization with TLR1 to activate the MyD88-dependent pathway, driving pro-inflammatory cytokine production20,26. Conversely, TLR1 appears to exert a regulatory, potentially inhibitory, influence on TLR4-mediated inflammatory responses.Fig. 1: The effect of different TLR dimerizations on immune responses.TLR1 forms dimers with TLR2 and TLR4, resulting in mild inflammation and ultimately promoting cellular homeostasis. In contrast, TLR6, when paired with TLR2 or TLR4, leads to severe inflammation and tissue damage. Additionally, TLR4 homodimerization induces higher levels of inflammation compared to TLR2/TLR4 heterodimerization.Full size imageRole of TLR2 and TLR4 in male reproductive tract (MRT)Expression of TLR2 and TLR4 throughout male reproductive tract and spermSeveral studies have confirmed that TLR2 and TLR4 are expressed in the male reproductive tract (MRT), including the testis, epididymis, vas deferens, and accessory glands in mammals, particularly humans and rodents50,51. Additionally, these TLRs are expressed in sperm in certain mammalian species, such as humans and mice52,53. The expression of TLR2 in bull sperm was also confirmed by Akthar et al.54. While there is limited information regarding the specific roles of TLR2 and TLR4 in the MRT, Saeidi et al. investigated their involvement in detail50. Their findings highlighted the crucial role of TLRs, particularly TLR2 and TLR4, in protecting spermatozoa within the MRT. They observed that TLRs are expressed in testicular sperm extraction (TESE) samples. Notably, TLR2 expression was relatively lower in TESE samples without sperm compared to those with sperm50. Based on these findings, the authors suggested that TLRs, especially TLR2, may influence the developmental processes during spermatogenesis (Fig. 2)50. Although the specific ligand for activating TLR2 is not yet identified, some endogenous TLR ligands (DAMPs) are known to be produced at different stages of spermatogenesis, particularly by Sertoli cells. Among these, HMGB1 has been suggested as a potential host-derived ligand for TLR2, possibly playing a role in the developmental processes of spermatogenesis55.Fig. 2: The effect of TLR dimerization on functions in the male reproductive tract (MRT).In the testis, TLR2 and TLR4 activation have distinct effects on spermatogenesis. TLR4/4 homodimerization and TLR2/6 heterodimerization negatively impact spermatogenesis, whereas TLR2 activation (hypothesized as TLR2/1 dimerization, though not yet confirmed) appears to enhance this function. In prostate cancer, TLR4, along with TLR1 and TLR6, plays a role, but TLR2 does not. It is hypothesized that TLR4/1 dimerization may suppress, while TLR4/6 dimerization may promote prostate cancer, requiring further experimental validation. Regarding mammalian sperm, TLR2/1 activation enhances sperm hyperactivation, while TLR4/4 dimerization leads to sperm cell death. There is no information on TLR2/6 activation and its effects on sperm kinetics. The figure has been created using BioRender.com software.Full size imageIn addition, studies using rat models have shown that administering LPS at different doses in testicular tissue is directly linked to spermatogenic damage56. Low doses of LPS can lead to mild inflammation and localized inhibition of Leydig cell function with minimal spermatogenic damage (Fig. 2). However, severe inflammation caused by high doses of LPS can result in significant pathological changes in spermatogenic function, likely mediated through TLR4 activation57. Furthermore, TLR2, TLR4, and TLR6 expressions have been confirmed in Sertoli cells. Researchers have noted that while the activation of these TLRs is critical for pathogen defense, the activation of TLR2/6 (via lipopeptides) and TLR4 (via LPS), but possibly not TLR1, may inhibit spermatogenesis (Fig. 2)58. In the prostate, both TLR4 and TLR2 are expressed in epithelial cells. Some studies have indicated that variants of TLR1, TLR4, and TLR6 are associated with an increased risk of prostate cancer in humans59. These findings highlight the importance of the interaction between TLR1 and TLR6 with TLR459. Notably, TLR1 is known to associate with TLR4 and effectively inhibit signaling. Thus, TLR1 may act as a mediator in the development or progression of prostate cancer (Fig. 2).Impact on sperm functionAs discussed, TLR2 and TLR4 have been reported to be expressed in the sperm of various mammalian species, including humans, mice, and bovines52,53,54. This raises an important question: what roles do TLR2 and TLR4 play in sperm function?Our group recently discovered that the activation of TLR2 in bovine sperm can lead to hyperactivation, which facilitates sperm penetration into the mucus and uterine glands (Fig. 2)54,60. Additionally, TLR2 in mammalian sperm has been shown to recognize lipopeptide ligands derived from Gram-positive bacteria52. Furthermore, activation of TLR2 and TLR4 pathways in mammalian sperm is associated with inducing apoptosis52. Interestingly, TLR2 activation in bovine sperm prior to in vitro fertilization (IVF) significantly improved blastocyst rates and embryo cleavage rates, suggesting that TLR2 activation can enhance IVF outcomes61.Regarding TLR4, its expression in sperm enables the recognition of LPS. However, this results in decreased sperm kinetic parameters, such as motility52. A study on human sperm found that exposure to LPS to activate TLR4 led to reductions in motility and progressive motility, although sperm viability, capacitation, and acrosome reactions were unaffected (Fig. 2)62. In this study, human sperm were challenged with LPS, and the effects were assessed after 1 and 12 h62. Similarly, in our unpublished data, we observed that TLR4 activation in bovine sperm also led to a decline in kinetic parameters.While the primary function of TLRs is often associated with pathogen recognition, emerging evidence suggests their involvement in diverse physiological processes of sperm. TLRs may mediate interactions between sperm and the epithelial cells of the FRT, influencing sperm adhesion, migration, and capacitation. TLR signaling could contribute to the selective recruitment of sperm to specific regions of the FRT, potentially guided by DAMPs or other endogenous ligands. Additionally, TLR activation may modulate intracellular signaling pathways within sperm, affecting the timing and efficiency of the acrosome reaction and hyperactivation, both essential for successful fertilization, by regulating calcium influx. Furthermore, TLRs on sperm may play a role in establishing immune tolerance at the maternal-fetal interface by influencing the local cytokine environment, preventing excessive immune responses to the introduction of sperm into the FRT and ensuring a balanced immune reaction that supports fertilization63.Until now, the activation of TLRs has primarily been discussed in the context of PAMPs. The question now arises whether endogenous molecules can affect TLR2 and TLR4 on sperm in both the male and FRTs. Although there is no definitive answer to this question at present, some DAMP molecules may potentially activate these TLRs on sperm, thereby enhancing their physiological function, particularly in the FRT. There is substantial evidence that DAMP-activated TLRs are abundant in the FRT, suggesting that these DAMPs could activate TLRs on sperm and improve their function19. However, further investigations are needed to identify the specific molecules involved.In summary, the activation of TLR2 and TLR4 in mammalian sperm produces contrasting effects. TLR2 activation enhances sperm kinetic parameters, thereby improving sperm function. In contrast, TLR4 activation negatively affects these parameters, suggesting distinct roles for these receptors in sperm function (Fig. 2). Therefore, evidence suggests that TLR2 on spermatozoa is not merely a remnant of their progenitor cells but play an active role in mediating interactions with epithelial cells, including penetration into the uterine glands, potentially triggering local inflammatory responses through engagement with specific DAMP ligands. Additionally, TLR2 activation may enhance the ability of sperm to penetrate the zona pellucida (ZP).Role in female reproductive tract (FRT)Numerous studies have indicated that TLR2 and TLR4 are expressed in various parts of the FRT. In humans, TLR1, TLR2, TLR4, and TLR6 have been reported in the fallopian tubes, uterine endometrium, cervix, and ectocervix (expressed in epithelial and leukocytes cells)11. However, the intensity of TLR2 and TLR4 expression varies significantly depending on the specific region of the FRT. For TLR2, the highest expression is observed in the fallopian tube and cervical tissues, followed by the endometrium and ectocervix. In contrast, TLR4 expression gradually decreases along the tract, with the highest levels in the fallopian tubes and endometrium, and lower levels in the cervix and ectocervix11. This differential expression pattern of TLR2 and TLR4 is attributed to the distinct regulation of inflammation and immunity within different regions of the FRT11. Similarly, in other species such as bovine and mice, TLR2 and TLR4 are expressed throughout the FRT64,65,66.Regarding their roles, it has been reported that these molecules are critical for immunity in the FRT and directly influence pregnancy. Specifically, the upregulation or downregulation of TLR2 and TLR4 can significantly impact pregnancy outcomes67. The dynamic regulation of TLR2/4 expression is essential for normal reproductive processes. The regulation of these TLRs is important for sperm function and interaction with the FRT. Dysregulated TLR2/4 expression, often triggered by microbial infections or other pathological stimuli, can lead to excessive or inappropriate inflammatory responses. This dysregulation can impair immune tolerance, increase susceptibility to infections, and may contribute to adverse pregnancy outcomes such as preterm labor, preeclampsia, and recurrent pregnancy loss68. Chronic inflammation due to TLR upregulation can lead to pathologies such as endometriosis. Although the exact factors responsible for the regulation of TLR2 and TLR4 expression remain unclear, it has been suggested that the diversity of bacterial ligands activating TLR2, such as peptidoglycan from Gram-positive bacteria, bacterial lipopeptides, and zymosan, may modulate their expression in cervical tissue to help prevent ascending reproductive tract infections67. So far, the exact mechanism behind the differential expression of TLR2 and TLR4 in the cervix during pregnancy has not been fully elucidated. However, one possible factor is that sex hormones, which are associated with pregnancy, may modulate the expression of TLR2 and TLR467. Immunohistochemistry studies have shown that early pregnancy is associated with increased expression of TLR2 and TLR4 receptors in cervical tissue. The upregulation of TLR2 and TLR4 in the lower FRT has been reported to play a pivotal role in modulating innate immune and inflammatory mechanisms in the ectocervix during pregnancy67. Additionally, experimental evidence suggests that elevated inflammation mediated via TLR signaling may be implicated in premature birth67. Moreover, it can be hypothesized that cytokines and growth factors regulate TLR expression within the FRT. Therefore, it is clear that the up-/down-regulation of TLR2 and TLR4 in the reproductive tract can have significant physiological consequences, influencing both normal immune function and pregnancy outcomes.TLR2 and TLR4 in the uterus, oviduct and oocytesIn this section, we will explain the roles of TLR2 and TLR4 in three main parts of the FRT: the uterus, oviduct, and oocytes. Both TLR2 and TLR4 play critical roles in pathogen recognition within the uterus14,26,69,70. In bovine models, this interaction with pathogens can trigger severe inflammation, often leading to tissue damage due to excessive Prostaglandin E2 production via MyD88 and p38 MAPK pathways71. In humans, bacterial infections in the endometrium have been effectively treated using TLR2 and TLR4 inhibitors, highlighting their importance in managing such conditions72.Additionally, TLR2 and TLR4 contribute to physiological inflammation in the endometrium, although their roles differ by species63,73. In bovines, physiological inflammation induced by sperm is predominantly mediated by TLR2, while in mice, TLR4 plays a more significant role63,73. As discussed above, TLR2 requires heterodimerization with TLR1 or TLR6 to induce inflammation. In the endometrium, TLR2/1 activation results in weak, mild, and physiological inflammation, whereas TLR2/6 induces a strong, severe inflammatory response, potentially causing tissue damage (Fig. 3)26.Fig. 3: The effect of TLR dimerization on functions in the female reproductive tract (FRT).In the uterus, activation of TLR2/6 and TLR4/4 under pathological conditions results in severe and prolonged inflammation, whereas TLR2/1 activation induces mild inflammation (sperm employ TLR2/1 under physiological conditions). During different stages of the estrous cycle, TLR4 expression is significantly downregulated in the follicular phase to protect sperm from immune attack and in the luteal phase (downregulation of TLR4 and TLR2) to support embryo implantation. In the oviduct, TLR4 activation induces pro-inflammatory responses, while sperm employ TLR2/1 to stimulate anti-inflammatory responses. In the ovary and oocyte, TLR2 and TLR4 activation in surface epithelial and granulosa cells triggers inflammation, a major cause of infertility in mammals. However, TLR2/1 and TLR4/4 activation in oocytes promotes sperm capacitation, enhancing fertilization success. The figure has been created using BioRender.com software.Full size imageIn human endometritis, elevated gene expression of TLR2 and TLR4 in endometrium has been observed, suggesting their involvement in endometritis74. In fact, an association between the increased expression of TLR2 and TLR4 and the development of endometriosis has been demonstrated, indicating that the upregulation of these receptors may contribute to inflammation and, consequently, endometritis74. The higher expression of TLR2 and TLR4 in endometritis is attributed to the presence of microbial ligands, including components from both Gram-positive and Gram-negative bacteria, particularly those recognized by TLR2 in the FRT74. It has been hypothesized that these microbial components may either directly upregulate TLR expression in resident endometrial cells or promote the recruitment of immune cells that express TLR2 and TLR4, thereby increasing overall expression levels at the tissue level. However, it remains unclear whether this elevated expression is due to upregulation within resident endometrial cells or increased infiltration of immune cells expressing these receptors. This distinction has not been clearly addressed in current studies, and further investigation is needed to determine which mechanism predominates in this pathological context.Similar findings have been reported in other mammals, such as in canines. In canine endometrium, TLR2 and TLR4 expression levels vary across the estrous cycle (Fig. 3). Previous studies indicated that TLR4 activation can trigger strong inflammation, compared to TLR2. TLR4 expression is absent during estrus, likely to prevent inflammation during mating and sperm deposition75. However, given the limited inflammatory effect of TLR2 activation, its expression during this phase presents no significant complications. During early diestrus, both TLR2 and TLR4 expression levels decrease, possibly to support embryo implantation (Fig. 3)75. During diestrus, when embryo implantation and uterine receptivity occur, the endometrium must undergo anti-inflammatory regulation76. As a result, the downregulation of TLRs, particularly TLR4 and also TLR2, appears to be a strategic adaptation. However, high TLR4 expression, but not TLR2, has been associated with uterine infections in canine77. These observations indicate a protective role for TLR2, in maintaining uterine homeostasis by balancing protective and pathological inflammation77.TLR2 and TLR4 are also expressed in the oviduct, where they play roles in both physiological and pathological inflammation11,65,78. In bovine models, TLR2 and TLR4 are involved in recognizing pathogen-associated molecules, particularly LPS, to induce pro-inflammatory responses. However, ovarian steroids and luteinizing hormone significantly inhibit LPS-induced TLR4 and TLR2 activation, promoting immune balance and enhancing homeostasis (Fig. 3)65. A similar mechanism of inflammation induction through TLR4 has been observed in other mammalian species, such as rabbits79. In humans, TLR4 inhibition using betulonic acid has been shown to downregulate pro-inflammatory cytokines, leading to enhanced cell proliferation and reduced apoptosis80. Interestingly, in bovine oviducts, TLR2, rather than TLR4, plays a crucial role in sperm recognition (Fig. 3)78. Activation of TLR4 in the oviduct prior to sperm interaction negatively affects sperm motility and function81. Unlike the bovine uterus, sperm interaction with the oviduct induces an anti-inflammatory response (Fig. 3)78. This anti-inflammatory effect is blocked by TLR2/1 antagonists, suggesting that the TLR2/1 signaling pathway in the oviduct contributes to the regulation of inflammation78.Both TLR2 and TLR4 are expressed in the ovary and oocytes across various species. In humans and murine ovaries, the expression of these TLRs has been reported, with TLRs being highly expressed in the surface epithelial cells of normal ovaries. Interestingly, their expression is also observed in ovarian epithelial tumors82. In murine models, TLR2 and TLR4 expression remain consistent across different stages of the estrous cycle82. Similarly, in rabbits, TLR2 and TLR4 expression have been documented, with significant changes noted after LPS treatment83. In bovine species, TLR2 and TLR4 are expressed in granulosa cells of the ovary84. Activation of these TLRs using synthetic bacterial lipoproteins and LPS in granulosa cells triggers the MAPK14 and MAPK3/1 pathways, leading to increased production of pro-inflammatory cytokines such as IL1-b, IL6, IL10, and TNF84. This production of pro-inflammatory cytokines, heightened cytokine activity, can contribute to infertility in cattle (Fig. 3)84. In oocytes, TLR2 and TLR4 are expressed, and their activation can lead to the release of cytokines (Fig. 3). These cytokines, in turn, enhance sperm capacitation, ultimately contributing to improved fertility85.TLR2 and TLR4 in immune cells of the female reproductive tractIt is well-established that TLR activation within the FRT can initiate immune responses, leading to the production of inflammatory mediators. Immune cells are central to these processes, and considerable research has focused on profiling their populations across different FRT compartments to elucidate their roles in reproduction. Complementary to these efforts is the identification of specific TLR ligands and their mechanisms of action. However, studies directly investigating the effects of TLR activation in FRT-resident immune cells and their subsequent impact on reproductive outcomes remain limited.A diverse array of immune cells, including macrophages, DCs, natural killer (NK) cells, neutrophils, mast cells, and T cells, populate the FRT86, with their relative abundance varying significantly between different anatomical locations87. Furthermore, the immune cell landscape within the reproductive tract exhibits dynamic fluctuations throughout the estrous/menstrual cycle. Notably, many immune cell types reach peak abundance during the estrus and metestrus phases, aligning with their critical roles in responding to seminal fluid components (including potential paternal antigens) and invading pathogens introduced around the time of insemination/intercourse88. Studies in women during the menstrual cycle have revealed that T cells, antigen-presenting cells, and neutrophils are prevalent throughout the FRT87. In contrast, NK cells are predominantly localized to the uterus and are generally scarce in the vagina, while B cells and monocytes are present at lower levels compared to other immune cell types, particularly T cells87 (Fig. 4). These immune cells perform dual functions: providing immune defense against pathogens in the lower FRT and establishing immune tolerance towards sperm and the developing embryo/fetus in the upper FRT.Fig. 4: Immune cell profiling in the female reproductive tract (FRT).This figure illustrates the distribution of immune cells in the FRT. T cells, antigen-presenting cells (APCs), and neutrophils are abundant throughout the FRT, while natural killer (NK) cells are predominantly found in the uterus. In contrast, B cells are present at much lower levels compared to other immune cells. Regarding cytokine production and TLR activation, TLR4 stimulation in FRT immune cells results in a relatively low level of cytokine production, suggesting limited activation. However, TLR2 activation in NK cells, especially in the uterus, leads to a marked increase in cytokine production. For other regions of the FRT, data on the effects of TLR2 activation and cytokine response remain limited. The number of arrows represents the relative frequency of each immune cell type or level of cytokine production in different regions of the FRT. Arrows with a question mark indicate a hypothesis based on previous studies showing that TLR2 activation in T cells can enhance cytokine production; however, this has not been confirmed in the FRT. A lone question mark indicates that no information is available. Therefore, we propose that activation of TLR2 in FRT-resident T cells may similarly lead to increased cytokine production.Full size imageRegarding TLR responsiveness in FRT immune cells, Benjelloun et al. reported that TLR4 activation in these cells elicits a comparatively moderate cytokine response compared to peripheral blood mononuclear cells, suggesting a tighter regulatory control of TLR4-mediated inflammation within the FRT87. While data on TLR2 activation in FRT immune cells (Fig. 4) is still emerging, evidence from other tissues indicates differential responsiveness, with T cells and NK cells generally exhibiting a stronger response to TLR2 than TLR489,90. Specifically, TLR2 ligation in T cells can enhance cytokine production and act as a costimulatory signal for T cell proliferation89. Similarly, NK cells typically express higher levels of TLR2 than TLR4 and are more sensitive to TLR2 stimulation90. Notably, uterine NK cells, a dominant immune population in the uterus, strongly express TLR2 and weakly express TLR4, and their activation can lead to the production of cytokines such as IFN-γ91.This underscores the intricate interplay between TLR signaling and immune responses within the reproductive tract, highlighting the crucial role of resident immune cells in regulating reproductive processes. TLR-activated signaling pathways in the FRT involve key immune cell types such as macrophages, DCs, and neutrophils, which are integral to various reproductive events: (1) Decidualization and embryo implantation: Macrophages and DCs play critical roles in establishing immune tolerance and facilitating successful implantation by modulating cytokine production and T cell responses92. (2) Uterine clearance: Neutrophils and macrophages are essential for the efficient clearance of excess sperm, cellular debris and pathogens post-partum or during menstruation, driven by TLR-mediated activation26,70. (3) Follicular development and ovulation: TLR signaling in follicular immune cells can influence the local cytokine environment, impacting granulosa cell function and ultimately, ovulation93.Effects of triggering TLR2 and TLR4 on fertility and pregnancy outcomesIn this section, we discuss how the activation or inhibition of TLR2 and TLR4 can affect fertility and pregnancy outcomes. In bovine, altering the immune response in any part of the FRT, including the uterus, oviduct, oocyte, and sperm, significantly impacts pregnancy. These areas are critical for sperm cross-talk with FRT, embryo competence, and embryo-maternal interactions.Starting with the uterus, it plays a dual role: facilitating sperm immune cross-talk and supporting embryo implantation. Sperm triggers a mild, transient inflammation by interacting with innate immune receptors, including TLRs94. This inflammation triggers epithelial cells to release cytokines, attracting immune cells such as neutrophils and macrophages into the uterine lumen94,95,96. These immune cells help clear excess sperm and pathogens, a process known as uterine clearance, which is essential for creating an ideal uterine environment for embryo receptivity and implantation94. A balanced, moderate inflammation is crucial for this process. Recent studies have shown that in bovine species, sperm-induced inflammation is mediated through the TLR2/1 signaling pathway, which ensures a mild and transient response26. However, in mice, TLR4 has been reported to be involved in sperm-mediated signaling, suggesting species-specific differences in TLR involvement63.So far, there is no definitive information on which molecules from sperm or semen activate TLR2/1 signaling to induce weak inflammation. To address this question, several possibilities can be considered. One possibility is that certain sperm-derived molecules, such as lipoproteins, lipopeptides, glycans, triglycerides, and phospholipids, may serve as potential candidates. Additionally, seminal plasma-derived molecules, particularly DAMPs, could also fall into this category. It is also important to note that the ligands and receptors involved may differ across species. In contrast, TLR2/6 or TLR4 activation leads to stronger, prolonged inflammation that can damage uterine tissues (Fig. 3)73.In artificial insemination, TLR2 is vital for initiating inflammation and aiding uterine clearance. Notably, semen extenders, particularly those containing egg yolk, can induce mild inflammation via the TLR2/1 pathway (activated by triglycerides) without interfering with sperm-induced inflammation97. This emphasizes the importance of TLR2/1 activation in promoting mild, transient inflammation while avoiding the severe responses associated with TLR4 or TLR2/6 activation97. Such balanced inflammation has been shown to improve pregnancy outcomes. Regarding embryo transfer, activating TLR2/1 signaling in sperm before IVF enhances cleavage and blastocyst rates in cumulus-oocyte complexes or cumulus-free oocytes but not in ZP-free oocytes61. In contrast, inhibiting this pathway reduces these rates61. Overall, the role of TLR2/1 signaling is critical at multiple stages of the reproductive process to ensure successful pregnancy outcomes.Regarding the expression of TLR2 and TLR4 in uterine cells during embryo/blastocyst communication with the uterus, it has been reported that TLR4 is significantly downregulated in endometrial epithelial cells (Fig. 3)98. This downregulation plays a crucial role in modulating immune responses during early pregnancy, creating a receptive uterine environment for implantation and protecting the conceptus (embryo/fetus and associated membranes). While immune tolerance in classical immunology is primarily associated with antigen-specific regulatory T cells (adaptive immunity), in pregnancy, innate immune modulation is also essential99. This tolerance involves a complex interplay of innate and adaptive immune mechanisms. Beyond its role in preventing excessive inflammatory responses to potential pathogens, TLR4 downregulation may facilitate embryo-maternal communication. One possible mechanism involves extracellular vesicles (EVs) and microRNAs, which are key mediators of embryo–maternal interactions100. Evidence suggests that TLR4 can serve as a target for EVs delivering microRNAs, indicating that TLR4 may play a role in regulating communication between the embryo and the maternal immune system via EV-mediated signaling101. This observation was made in equine species. In bovines, one study indicated that TLR2 expression might be influenced by the embryo culture medium, suggesting that TLR2 expression is neither markedly upregulated nor downregulated in the uterus during embryo implantation (Fig. 3)102. TLR4 appears to have a negative impact on embryo implantation, and TLR2/6 activation has also been associated with decreased embryo implantation and impaired uterine receptivity in mouse models103. Therefore, it seems that TLR2, TLR4, and TLR6 may negatively affect uterine receptivity and embryo implantation (Fig. 3).To investigate the roles of TLRs, particularly TLR2 and TLR4, following embryo implantation and during placental development, studies have revealed distinct effects of their activation in trophoblasts. Activation of TLR4 in these cells has been shown to induce the expression of pro-inflammatory cytokines, while activation of TLR2 can trigger apoptosis104. Trophoblasts express TLR1, TLR2, and TLR4, but reportedly lack TLR6 expression, whereas the mature human placenta expresses a broader repertoire, including TLR1, TLR2, TLR4, and TLR6104,105. Activation of both TLR2 and TLR4 in the placenta has been associated with the release of cytokines and the initiation of local immune responses106.Furthermore, accumulating evidence highlights a critical role for TLR4 in driving excessive uterine inflammation, which can be detrimental to the developing fetus, impair its growth, and ultimately contribute to adverse pregnancy outcomes such as preterm birth (Fig. 3)107. Notably, preclinical studies have demonstrated that blocking TLR4 with specific inhibitors can effectively reduce inflammation and prevent early delivery in certain models107. This highlights the potential for prolonged TLR4-mediated inflammation to negatively impact pregnancy progression. While the effects of TLR2 activation in the context of overall placental development and pregnancy outcomes are less definitively characterized in this section, the apoptotic effect on trophoblasts suggests a potential role in placental remodeling or in pathological conditions. Further research is needed to fully elucidate the contributions of different TLRs to both normal placental development and pregnancy complications.Interplay between TLR2 and TLR4 signalingAs we explained earlier, TLR4 and TLR2 share a common signaling pathway for inducing inflammation. Both utilize MyD88 to activate the NF-κB sub-pathway in response to PAMPs. However, TLR4 can also activate the TRIF-dependent pathway, which TLR2 cannot, resulting in the production of interferons. Since both TLRs rely on MyD88, signaling from one receptor could influence the other, possibly due to competition for the availability of the MyD88 adapter.One mechanism that might downregulate TLR-induced inflammation is the Suppressor of Cytokine Signaling 1 (SOCS-1), which degrades Mal-dependent p65 phosphorylation. It has been reported that activation of TLR2 and TLR4 can lead to the production of SOCS-1108. Therefore, it can be hypothesized that activation of one TLR may induce SOCS-1, which could, in turn, affect the other TLR (Fig. 5)108. Another key regulator of TLR function and cross-talk is A20 (Fig. 5). A20 plays a crucial role in terminating TLR signaling pathways109. It suppresses pro-inflammatory responses by targeting key signaling molecules downstream of TLRs, such as TRAF6 and RIP1, for deubiquitination and degradation110. This mechanism ensures inflammation resolution and prevents immune system overactivation. A20 is particularly important for ligand-specific tolerance. For instance, TLR1/2 activation by PAM3 strongly upregulates A20, contributing to tolerance by inhibiting MAPKs (JNK and p38) and NF-κB pathways upon subsequent PAM3 stimulation109. Similarly, TLR4 stimulation by LPS induces A20, which mediates cross-tolerance by reducing responses to subsequent TLR1/2 activation109. Interestingly, A20 expression is induced not only by TLR ligands but also by pro-inflammatory cytokines like TNF-α and IL-1β, highlighting its broad regulatory role110. This cross-regulatory function suggests that TLR-induced A20 expression could influence other TLR pathways, either by modulating shared downstream signaling components or by creating an anti-inflammatory environment. In the bovine uterus, A20 expression has been observed to reduce inflammation associated with endometritis111. Upregulation of A20 during LPS treatment in the bovine endometrium inhibited LPS-induced inflammation111. These findings suggest that A20 plays a role in inducing tolerance in the endometrium and might mediate cross-talk between TLR2 and TLR4.Fig. 5: Overview of TLR2 and TLR4 signaling pathways and their interplay.Both TLR2 and TLR4 utilize the MyD88-dependent signaling pathway, leading to the production of pro-inflammatory cytokines and inhibitors such as SOCS and A20. These inhibitors modulate MyD88 signaling and contribute to the regulation of the immune response. Additionally, TLR1 and TLR6 influence both the TLR2 and TLR4 systems, playing a role in their interplay and coordination. The figure has been created using BioRender.com software.Full size imageIn addition, other factors can mediate the interplay between TLR2 and TLR4. It has been reported that while TLR4 cannot directly sense gram-positive bacteria, it can amplify the inflammatory response initiated by TLR2112. It has been indicated that the gram-positive bacterium Mycoplasma pneumoniae activates the MyD88 and NF-κB pathways through TLR2/1 and TLR2/6 signaling, and in this model, bacterial clearance was significantly lower in TLR1−/− and TLR6−/− mice compared to wild-type mice, emphasizing the importance of TLR1 and TLR6 in inflammation and bacterial clearance113. Surprisingly, in TLR2−/− mice, accelerated clearance and increased IL-12 production were observed, suggesting that inflammation was heavily dependent on TLR4113. In TLR2−/− mice, MyD88-dependent and -independent pathways were upregulated through TLR4. This evidence underscores the critical role of TLR2-TLR4 interplay in controlling cytokine production, possibly mediated by TLR1 and TLR6. As previously explained, TLR1 and TLR6 elicit different cellular responses47. TLR1 and TLR6 significantly impact TLR2 and TLR4 function, playing key roles in their interplay (Fig. 5). In the bovine uterus, TLR1 is associated with weak, mild, and homeostatic inflammation, while TLR6 generally induces a stronger inflammatory response. Notably, TLR1 expression is higher than TLR6 in the bovine uterus, suggesting that TLR1, possibly via both TLR2/1 and TLR4/1, mediates weak and transient inflammation, promoting homeostasis. Conversely, TLR6 interaction with both TLR2 and TLR4 results in severe inflammation. This suggests that TLR6 may mediate the TLR2-TLR4 interplay, leading to strong inflammatory responses. In conclusion, TLR1 and TLR6 modulate the interplay between TLR2 and TLR4, contributing to either homeostasis or severe inflammation depending on their relative contributions.Future directions and open questionsTLR1 and TLR6 play distinct but critical roles in modulating the functions of TLR2 and TLR4, influencing the inflammatory response and tissue homeostasis. TLR1, when forming heterodimers with TLR2 or TLR4, is associated with recognizing triacylated lipopeptides and promoting weak inflammation and homeostasis, specifically in epithelial cells, potentially through beyond MyD88 and NF-κB or Pi3K/Akt pathways or the preferential recruitment of MyD88 over TRIF adapters114. This limited signaling might lead to reduced NF-κB activation and the induction of anti-inflammatory cytokines like IL-10115. In contrast, TLR6 forms heterodimers with TLR2 or TLR4 to recognize diacylated lipopeptides, triggering severe inflammation and tissue damage. These dimerizations likely recruit both MyD88 and TRIF, amplifying downstream signaling pathways such as NF-κB and IRF3/7, leading to the production of pro-inflammatory cytokines like TNF-α and IL-6, as well as type I interferons. This enhanced response may also involve the generation of ROS and inflammatory mediators that attract neutrophils, exacerbating tissue damage. The difference in signaling strength and outcomes between these dimer pairs may arise from their ligand-binding specificity, interaction with accessory proteins like MD2 and CD14, and recruitment of regulatory molecules like SOCS in TLR1-associated dimers. Future experiments, such as co-immunoprecipitation to study adapter protein interactions, transcriptomics to identify cytokine signatures, and CRISPR-based knockout models for TLR1 or TLR6, could elucidate the precise pathways and mechanisms underlying these distinct dimerization effects.ConclusionThis review highlights the complex interplay between TLR2 and TLR4 in male and female reproductive biology, emphasizing their shared and distinct signaling mechanisms. TLR2 and TLR4 act as a double-edged sword in reproductive processes. On one hand, they are essential for initiating immune responses to defend against pathogens and for critical functions such as sperm activation, sperm-uterine interaction, and uterine tolerance and immunity. On the other hand, excessive activation can lead to severe inflammation and tissue damage, negatively impacting reproductive health and economic outcomes. Balancing immune responses, particularly the collaboration between TLR2 and TLR4, is vital for maintaining cellular homeostasis.Both TLRs rely on the MyD88-dependent pathway to activate NF-κB and drive inflammatory responses, which creates the potential for mutual influence during activation. However, TLR4 uniquely employs the TRIF-dependent pathway to induce interferon production, a mechanism absent in TLR2. Regulatory factors such as SOCS-1 and A20 play critical roles in modulating these pathways, ensuring balanced immune responses and preventing excessive inflammation. Additionally, TLR1 and TLR6 significantly contribute to the TLR2–TLR4 interplay, influencing the balance between transient and severe inflammatory responses depending on their involvement.Importantly, understanding these mechanisms opens new avenues for therapeutic interventions. TLRs may serve as potential drug targets; for example, selective use of TLR agonizts or inhibitors could modulate fertility or mitigate adverse pregnancy outcomes107. However, substantial biological and clinical knowledge gaps remain. For instance, the role of TLRs in innate immune memory within the reproductive tract is not fully understood. Moreover, chronic TLR-mediated activation may have long-term effects, potentially contributing to irregular estrous or menstrual cycles, ovulatory dysfunction, or chronic pelvic pain. Future research addressing these gaps will be essential for developing targeted and safe interventions to support reproductive health in mammalian species.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.ReferencesAkira, S., Takeda, K. & Kaisho, T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. 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W., Smits, A. I. P. M. & Tel, J. Single-cell analysis reveals TLR-induced macrophage heterogeneity and quorum sensing dictate population wide anti-inflammatory feedback in response to LPS. Front. Immunol. 14, 1135223 (2023).Article CAS PubMed PubMed Central Google Scholar Download referencesAcknowledgmentsThe present research work was funded by a Grant-in-Aid for Scientific Research (23H02356 and 24KF0171) of the Japan Society for the Promotion of Science.Author informationAuthors and AffiliationsGlobal AgroMedicine Research Center (GAMRC), Obihiro University of Agriculture and Veterinary Medicine, Obihiro, JapanAlireza Mansouri, Ihshan Akthar & Akio MiyamotoAuthorsAlireza MansouriView author publicationsSearch author on:PubMed Google ScholarIhshan AktharView author publicationsSearch author on:PubMed Google ScholarAkio MiyamotoView author publicationsSearch author on:PubMed Google ScholarContributionsAl.M. and A.M. conceived and planned the manuscript. Al.M. and I.A. drafted the original version. Al.M. prepared the figures. Al.M., I.A., and A.M. reviewed and edited the manuscript.Corresponding authorCorrespondence to Akio Miyamoto.Ethics declarationsCompeting interestsThe authors declare no competing interests.Peer reviewPeer review informationCommunications Biology thanks Hans-Joachim Schuberth and the other, anonymous, reviewer for their contribution to the peer review of this work. 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