The expression and activity of Toll-like receptors in the preimplantation human embryo suggest a new role for innate immunity

Abstract

STUDY QUESTION Is the innate immunity system active in early human embryo development? SUMMARY ANSWER The pattern recognition receptors and innate immunity Toll-like receptor (TLR) genes are widely expressed in preimplantation human embryos and the pathway appears to be active in response to TLR ligands. WHAT IS KNOWN ALREADY Early human embryos are highly sensitive to their local environment, however relatively little is known about how embryos detect and respond to specific environmental cues. While the maternal immune response is known to be key to the establishment of pregnancy at implantation, the ability of human embryos to detect and signal the presence of pathogens is unknown. STUDY DESIGN, SIZE, DURATION Expression of TLR family and related genes in human embryos was assessed by analysis of published transcriptome data (n = 40). Day 5 (D-5) human embryos (n = 25) were cultured in the presence of known TLR ligands and gene expression and cytokine production measured compared to controls. PARTICIPANTS/MATERIALS, SETTING, METHODS Human embryos surplus to treatment requirements were donated with informed consent from several ART centres. Embryos were cultured to Day 6 (D-6) in the presence of the TLR3 and TLR5 ligands Poly (I: C) and flagellin, with gene expression measured by quantitative PCR and cytokine release into medium measured using cytometric bead arrays. MAIN RESULTS AND THE ROLE OF CHANCE TLR and related genes, including downstream signalling molecules, were expressed variably at all human embryo developmental stages. Results showed the strongest expression in the blastocyst for TLRs 9 and 5, and throughout development for TLRs 9, 5, 2, 6 and 7. Stimulation of Day 5 blastocysts with TLR3 and TLR5 ligands Poly (I: C) and flagellin produced changes in mRNA expression levels of TLR genes, including the hyaluronan-mediated motility receptor (HMMR), TLR5, TLR7, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and monocyte chemoattractant Protein-1 (MCP-1) (P < 0.05, P < 0.001 compared to unstimulated controls), and release into culture medium of cytokines and chemokines, notably IL8 (P = 0.00005 and 0.01277 for flagellin and Poly (I: C), respectively). LIMITATIONS, REASONS FOR CAUTION This was a descriptive and experimental study which suggests that the TLR system is active in human embryos and capable of function, but does not confirm any particular role. Although we identified embryonic transcripts for a range of TLR genes, the expression patterns were not always consistent across published studies and expression levels of some genes were low, leaving open the possibility that these were expressed from the maternal rather than embryonic genome. WIDER IMPLICATIONS OF THE FINDINGS This is the first report of the expression and activity of a number of components of the innate immunity TLR system in human embryos. Understanding the role of TLRs during preimplantation human development may be important to reveal immunological mechanisms and potential clinical markers of embryo quality and pregnancy initiation during natural conception and in ART. STUDY FUNDING/COMPETING INTEREST(S) This work was funded by the Ministry of Higher Education, The State of Libya, the UK Medical Research Council, and the NIHR Local Comprehensive Research Network and NIHR Manchester Clinical Research Facility and the European Union’s Horizon 2020 Research and Innovation Programmes under the Marie Skłodowska-Curie Grant Agreement No. 812660 (DohART-NET). In accordance with H2020 rules, no new human embryos were sacrificed for research activities performed from the EU funding, which concerned only in silico analyses of recorded time-lapse and transcriptomics datasets. None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER n/a.

Extraction of Innate Immune Genes in Dairy Cattle and the Regulation of Their Expression in Early Embryos

“…Wedad S et al first reported on the expression and activity of many components of the innate immune TLR system in the human embryo. This is important for understanding the role of TLRs in preimplantation human development, which may be important for unraveling immunologic mechanisms and potential clinical markers of embryo quality and pregnancy initiation during natural conception and ART [ 30 ]. According to our analysis, NOD-like receptor signaling pathways and toll-like receptor signaling pathways were significantly enriched in early embryonic development in dairy cows, and the NOD-like receptor signaling pathway was specifically expressed during the 2-cell and morula stage, while the toll-like receptor signaling pathway was specifically expressed only during the morula stage, which suggests a temporal sequence in the construction of innate immunity mechanisms with different periods of early embryonic development, which are concurrently involved in the development of dairy cattle embryos.…”

Toll-like receptors in Immunity and inflammation

“…The preimplantation human embryos also express TLRs [37]. Furthermore, TLR3 in the brain cells of mice in the early embryonic stages and neural stem/progenitor cells (NPCs) control the neurosphere formation.…”

“…This study is critical as ovine and human TLR2, 3, and 4 share 83-88% homology. Human blastocysts highly express TLRs 9 and 5, and TLRs 9, 5, 2, 6, and 7 are expressed throughout embryonic development, and their stimulation in vitro produce different cytokines and chemokines such as IL-8 and monocyte-chemoattractant protein-1 (MCP-1) [37]. Therefore, it will be novel to observe the impact of TLRs in human embryonic development and TLR deficiency on reproduction or reproductive health.…”