Embryo implantation is the prerequisite for establishing pregnancy in all eutherian mammals, including humans. It is initiated with the attachment of the embryo to the uterine endometrial epithelium during a limited stage called “Window of implantation” (“WOI”, mid-secretory phase, spanning days 20–24 of the menstrual cycle in humans), followed by the invasion of the embryo into the uterine stromal/decidua tissues 1. Human uterine endometrium is cyclically regulated by the ovarian steroid hormones (17β-estradiol and progesterone), throughout the reproductive life of women 2. In a normal menstrual cycle, if no embryo implantation occurs, the uterine endometrium is shed, followed by rebuilding of the endometrial functional layer within 2 weeks. During the proliferative phase, endometrial glands, stroma, and blood vessels actively grow in response to the estrogens produced by follicles, but the lumen epithelium is non-receptive to the embryo. After ovulation, the corpus luteum produces an increasing concentration of progesterone, which inhibits the proliferative activity of the endometrial epithelial cells, but gradually transforms the endometrial epithelial and stromal/decidua cells into a functional status. At this mid-secretory phase, the receptivity of the uterine endometrium to the embryo reaches the highest 3.According to the investigation by the World Health Organization, female infertility affects more than 48 million women, and implantation failure is thought to be one of the vital reasons 4. Insufficient endometrial receptivity is documented to take responsibility for two-thirds of implantation failure cases and is also considered the primary reason for in-vitro fertilization-embryo transfer (IVF-ET) failure 5. Therefore, understanding the mechanism of embryo attachment, especially the molecular basis of uterine receptivity establishment, is required for improving infertility treatments and exploiting new strategies for contraception.
Protein glycosylation is one of the most common and important post-translational modifications, in which sugar moieties are covalently attached to the specific amino acid sequence, usually yielding N-linked or O-linked glycans6. Generally, the end of the glycans is capped with fucose and sialic acid by fucosyltransferases (FUTs) and sialyltransferases (STs), respectively. Terminal epitopes (fucosylated and/or sialylated oligosaccharide structures) of glycans involve in cell–cell and cell-extracellular matrix interactions, which further affect several cellular biological events such as leukocyte recruitment and cancer metastasis7. During “WOI”, glycoconjugates on human uterine epithelium are abundantly decorated with sialyl Lewis X (sLeX) epitopes, and this terminal tetrasaccharide structure binds with trophoblastic L-selectin to recognize the embryo8. Given the diversity and complexity of glycans, we are interested in whether other glycosylation-dependent molecular mechanisms may exist underlying the blastocyst implantation.
STs are classified into four families: β-galactoside α2,3-STs (ST3GAL1-6); β-galactoside α2,6-STs (ST6GAL1,2), N-acetylgalactosaminide α2,6-STs (ST6GALNAC1-6) and α2,8-polySTs (ST8SIA1-6). ST6GALNAC1 catalyzes the transfer of sialic acid from donor substrate CMP-sialic acid to N-acetylgalactosaminide (GalNAc) with an α2,6-linkage, yielding a truncated O-glycan, which termed sialyl Thomsen-nouvelle (sTn) (Siaα2,6GalNAc-O-Ser/Thr) 9. Notably, sTn is detected by over 80% of human carcinomas and is closely associated with adverse outcomes in cancer patients. The aberrant biosynthesis of sTn has been attributed to the overexpression of ST6GALNAC1, and to loss-of-function in COSMC gene in diverse types of cancer 10. sTn is detected in the upper digestive tract (salivary glands, esophagus, and stomach), urogenital tract (uterine and cervix cells), and testis (interstitial cells) of adult healthy tissues10. Two recent studies revealed that ST6GALNAC1-mediated sTn of mucins in intestinal and ocular goblet cells play crucial roles in maintaining mucus integrity and protecting against allergen particles, respectively11,12. However, very little is known about the physiological roles of ST6GALNAC and sTn in complex biological systems, especially in reproductive processes including embryo implantation.
Sialic acid-binding immunoglobulin-type lectins (Siglecs) are transmembrane proteins, expressed predominantly by immune cells and can be divided into two sub-groups: the conserved Siglecs (Siglec-1, -2, -4, and − 15) and the CD33-related Siglecs (Siglec-3, -5, -6, -7, -8, -9, -10, -11, -14, and − 16). Siglecs belong to the immunoglobulin superfamily (IgSF), which are “immunoglobulin-type” (I-type) lectins that recognize the sialic acid on N-glycans, O-glycans or glycolipids carried by the glycoconjugates13,14. Accumulating evidence reported that Siglecs-sialic acid binding influences cell-cell and cell-pathogen interaction in many pathophysiological processes including immune response, infection, and cancer 15. Siglec-6 is part of the CD33-related Siglecs sub-family, which are rapidly evolving Siglecs, and Siglec-6 is found to express only in primates. Among the primates, only humans express Siglec-6 in their placental trophoblasts, probably explained by mutations in the 5’ untranslated region (5’UTR) of the gene16. Siglec-6 is studied to bind with the sTn17. Interestingly, natural ligands for Siglec-6 are present in the human uterine epithelium by using the recombinant Siglec-6 protein as the probe16. This hints us to raise a presumption that Siglec-6 on embryonic trophectoderm recognizes the sTn-modified glycoproteins on uterine epithelium to facilitate the embryo-endometrium interactions.
In this study, we reported that ST6GALNAC1 was highly expressed by the human uterine endometrium at the mid-secretory phase. Manipulating uterine endometrial sTn by ST6GALNAC1 over- or down-regulation influenced the receptivity of endometrial cells to trophoblastic spheroids in vitro. We also found that adhesion molecule CD44 bears sTn in endometrial cells, and sTn-modified CD44 binds with trophoblastic Siglec-6.