Expression of ST6GalNAc1 and sialyl-Tn antigen enhances endometrial receptivity

Expression of ST6GalNAc1 and sialyl-Tn antigen enhances endometrial receptivity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Expression of ST6GalNAc1 and sialyl-Tn antigen enhances endometrial receptivity Hee-Jung Choi, Tae-Wook Chung, Jihyun Kim, Bo-Sung Kim, Mi-Ju Park, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.22965/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background To characterize molecular mechanism underlying the regulation of sialylated glycan expression and its roles for endometrial receptivity and embryo implantation. Here, we characterized the role of a truncated form of sialylated O -glycan, sialyl-Tn, for endometrial receptivity. Methods The transcriptomes of human endometrium at mid-secretory phase were analyzed by Bioinformatics. Changes in gene expression, protein, and signal pathway were measured using RT-PCR and Western blot. The cell adhesion assay was visualized using a fluorescent microscope. In peri-implantation phase of mice, the expression of leukemia inhibitory factor (LIF) and sialyl-Tn were confirmed using immunohistochemistry and immnofluorescence analysis. The effect of sialy-Tn expression on embryo implantation was estimated by in vitro fertilization and embryo transfer using mice. Results In in silico analysis, expression of O -glycosylation genes, especially ST6GalNAc1 , was significantly increased in the human uterus of mid-secretory phase. Overexpression of the ST6GalNAc1 gene in non-receptive human endometrial AN3CA cells enhances the attachment of trophoblastic JAr cells. In an animal study, the results clearly indicated that sialyl-Tn was expressed on the surface of the mid-secretory uterus. In addition, blockade of the receptor using free sialyl-Tn epitope diminished the implantation rates of intrauterine transferred murine embryos. Conclusion From these results, here we suggest that sialyl-Tn expression might be a novel factor regulating the endometrial receptivity for successful embryo implantation. Endocrinology & Metabolism Glycosylation Sialyl-Tn ST6GalNAc1 Embryo implantation Endometrial Receptivity Figures Figure 1 Figure 2 Figure 3 Background Embryo implantation, a key process for a successful pregnancy, is one of the remaining unmet needs to improve the success rate of assisted reproduction [ 1 ]. The endometrium is receptive to embryo only during the implantation window, a short period of time about 6–10 days after ovulation [ 1 , 2 ]. A large number of molecular mediators including adhesive proteins, hormones, cytokines, and growth factors, which are involved in implantation have been identified. Among these molecules, several glycoproteins such as mucin-1 (MUC-1) and human chorionic gonadotrophin (hCG) have been focused on its possible function in relation to embryo implantation [ 3 , 4 ]. However, the molecular mechanisms regulating endometrial receptivity are remained poorly understood [ 5 ]. Protein glycosylation, a highly elaborative process of post-translational modification, is tightly controlled by enzymatic regulation [ 6 ]. The expression of terminal fucose residue generated by the action of fucosyltransferases, such as an increase of sialyl Lewis X antigen synthesized by fucosyltransferase VII, was reported as a positive factor of embryo implantation [ 7 , 8 ]. In spite of given importance of the terminal sialic acid residues on the cell surface for cell-cell interaction owing to its negative charge [ 9 ], the function of the sialic acid epitopes in the embryo implantation is still not elucidated. Sialyl-Tn antigen, a type of truncated O -glycosylation, has been reported as a key player in cancer progression including proliferation, invasion, and metastasis [ 10 – 12 ]. Although the expressions of sialyl-Tn were previously examined in the normal human reproductive organs including uterus, testis, and amniotic fluid [ 13 , 14 ], there are no previous report on the expression of sialyl-Tn nor their function in receptive endometrium. In this study, we firstly demonstrate that the expression of the sialyl-Tn antigen is increased in receptive endometrium is essential for the process of embryo implantation in both in vitro human cell lines and in vivo mouse experiment. Materials And Methods Materials Anti-sialyl-Tn antibody [STn 219] (FITC) was supplied from Abcam (Cambridge, UK). The free sialyl-Tn epitope was supplied from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-β-actin antibody, β-estradiol, and progesterone were purchased from Sigma-Aldrich (St. Louis, MO). Cell Tracker™ Green CMFDA (5-chloromethylfluorescein diacetate) fluorescence dye was provided from Thermo Fisher Scientific (Waltham, MA). Bioinformatic analyses Gene Set Enrichment Analysis (GSEA; www.broadinstitute.org/gsea ) was performed as described in previous studies [ 15 , 16 ] using the transcriptomes of human proliferative and mid-secretory endometrium (NCBI GEO dataset, GSE4888) [ 17 ]. Correlation analyses were performed also using the transcriptomes of human proliferative and secretory endometrium (NCBI GEO dataset, GSE4888) as described in previous studies [ 18 , 19 ]. Correlogram was built using corrgram package in RStudio (R Consortium Inc, Boston, MA), with positive and negative correlations represented in blue and red edges, respectively. Only correlations with Spearman's Rho > 0.5 or < − 0.5 (p < 0.05) are displayed in the interaction network. Cell culture Human uterine endometrial Ishikawa cell line, derived from a uterine adenocarcinoma [ 20 ], was kindly provided by Dr. Jacques Simard (CHUL Research Center, Quebec, Canada). Non-receptive uterine endometrial AN3CA cell lines [ 21 ] were purchased from American Type Culture Collection (ATCC; Manassas, VA). The cells were maintained at 37 °C in an atmosphere containing 5% CO 2 /air in Dulbecco’s Modified Eagle’s Medium (DMEM; Welgene, Daegu, Korea) and Roswell Park Memorial Institute medium (RPMI-1640; Welgene) containing 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin/streptomycin (PSA; Thermo Fisher Scientific), respectively. The choriocarcinoma JAr cell was obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI1640 (Welgene) containing 10% FBS and 1% PSA. Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was isolated from cells using RiboEx™ (GeneAll, Seoul, Korea) and then subjected to reverse transcription with oligo-dT primers using M-MLV reverse transcriptase (Thermo Fisher Scientific). The cDNA was amplified by PCR using AccuPower®PCR PreMix (Bioneer Co., Daejeon, Korea). The primers used in this study were as follows: ST6GalNAc1 , forward 5’-GTCCAGTGGTCCTTGCTTCTGGCTG-3’ and reverse 5’-ATTTCCTTGGGTCGTCTTTGTGTCC-3’; β-actin , forward 5’-CAAGAGATGGCCACGGCTGCT-3’ and reverse 5’-TCCTTCTGCATCCTGTCGGCA-3’. Expression of ST6GalNAc1 gene AN3CA cells were transfected with the ST6GalNAc1 gene in the mammalian expression vector, pcDNA3.1, which was kindly gifted from Dr. Philippe Delannoy (University of Lille, France). Flow cytometric analysis AN3CA cells were fixed with 3.7% formaldehyde and incubated with FITC-conjugated anti-sialyl-Tn antibody in Carbo-free blocking solution for 2 h. The cells were evaluated by flow cytometry (BD FACS CANTO II; BD Biosciences, San Jose, CA). Adhesion assay AN3CA cells were seeded into 6 well plates and cultured to a confluent monolayer. After the pretreatment of hLA, the cells were incubated with leukemia inhibitory factor (LIF) treatment for 48 h. The JAr cells were labeled with Cell Tracker™ Green CMFDA (Invitrogen, Carlsbad, CA) and gently added onto a monolayer of AN3CA cells. After gently shaking at 40 rpm for 30 min at 37 °C, the cells were vigorously washed to remove non-binding cells. The attached JAr cells were visualized by using a fluorescent microscope (Axio Imager M1, Carl-Zeiss, Oberkochen, Germany). Five fields of each sample were chosen randomly and the number of adhered cells was averaged after quantifying with ImageJ software (NIH; Bethesda, MD) Animals Female C57BL/6 mice (7–8 weeks old, weight 20–22 g) were purchased from Orient Bio, Co. (Seongnam, Korea). Mice were ovariectomized (OVEX) and recuperated 14 days later. Animals were fed on a standard diet with ad libitum access to water, and kept on a 12 h light: 12 h dark cycle. All experimental procedures were examined and approved by the Animal Research Ethics Committee at the Pusan University of Korea (no. PNU-2016-1212). Induction of peri-implantation phase and immunochemistry Twenty-eight female mice were randomly divided into two groups: control and PE. In the prime phase, mice were injected subcutaneously (s.c.) with 100 ng β-estradiol (in 100 µl corn oil) or vehicle for 3 days. In interphase, no hormone was treated for the next two days. In the peri-implantation phase, mice were daily injected s.c. with 6.7 ng β-estradiol and 1 mg progesterone (in 100 µl corn oil) for 6 more days. Mice were respectively sacrificed on day 6, 8 and 10, and both uterine horns were excised to determine the expression of LIF and sialyl-Tn using immunohistochemistry and immunofluorescence analysis, respectively. The uterine tissues of mice were fixed in 3.7% formalin and embedded in paraffin and were then cut into 4 µm serial sections. The sections were immunostained with LIF or sialyl-Tn (FITC) antibodies, visualized with the Dako Envision kit (Dako, Jena, Germany) and fluorescent microscope (Axio Imager M1) respectively. Animals and embryo collection and Embryo transfer in utero Inbred ICR female mice (6–8 weeks old) were induced to superovulate by intraperitoneal injection of 5 IU serum gonadotropin from a pregnant mare (Sigma-Aldrich) followed by injection with 5 IU human chorionic gonadotropin (hCG; Sigma-Aldrich) 46 later. Superovulated female mice were mated with fertile male mice and euthanized by cervical dislocation 46 h after hCG injection. The day of vaginal plugging was designated as 0.5 days post coitum (dpc). Mouse 2-cell embryos were collected from oviducts on 1.5 dpc and cultured in pre-warmed the Quinn’s Advantage Blastocyst Medium (SAGE/Origio, Malov, Denmark) containing 10% serum protein substitute in an incubator at 37 and 5% CO 2 . Implantation potential of embryos from the control and sialyl-Tn epitope groups was examined via embryo transfer as described previously [ 22 , 23 ]. In the sialyl-Tn epitope group, embryos were transferred with a 50 ng/ml sialyl-Tn epitope. Five to six embryos from two different groups were transferred on 3.5 dpc to the contralateral uterine horn of pseudopregnant recipients provided by mating with a vasectomized male. Two days after transfer, implantation sites in uterine horns of pregnancy were detected by intravenous injection of Chicago blue dye. Clear blue bands in utero were considered implantation sites. Statistical analysis Statistical analysis of results was performed by a Student’s t -test (for in vitro adhesion assay) or Chi-square test (for in vivo embryo transfer) using GraphPad Prism (GraphPad Software, San Diego, CA). Values are expressed as the mean ± standard deviation (SD) and the minimum significance level was set at a P value of 0.05. All experiments except for animal studies were independently performed at least 3 times. Results Bioinformatic analysis To identify the functions of protein glycosylation in human embryo implantation, we applied a bioinformatic approach with the NCBI GEO database (GSE4888) [ 17 ]. The results from GSEA using the transcriptomes of human proliferative and mid-secretory endometrium indicated that glycosylation, especially O -linked glycosylation is the most prominent and significant gene set (Fig. 1 A and B). Among the genes involved in O -glycosylation, the expression of sialyltransferases, such as ST6GalNAc1 , ST6Gal1 , ST3Gal6 , are significantly increased in the uterus of mid-secretory phase, which is corresponding to implantation window (Fig. 1 C). To identify putative targets of LIF, a major cytokine regulating endometrial receptivity [ 24 ], we performed correlation analyses using the same GEO dataset. Interestingly, a highly significant positive correlation between LIF/gp130/STAT3 signaling pathway and ST6GalNAc1 was observed (Fig. 1 D and E). Other cytokines and growth factors related to endometrial receptivity including IL-1α, IL-6, IL-8, IL-11, IL-15, and HB-EGF [ 25 ] showed a lower positive correlation with the expression of ST6GalNAc1 compared with LIF. ST6GalNAc1 expression mediated adhesion of trophoblast to endometrial cells Among the sialic acid-binding immunoglobulin-type lectins (Siglecs), Siglec-6 is a receptor for the sialyl-Tn epitope and expressed on the surface of trophoblast [ 26 ]. As ST6GalNAc1 is responsible for the expression of sialyl-Tn antigen and termination of O -glycan (Fig. 2 A), we confirmed the expression of the ST6GalNAc1 gene in the receptive endometrial Ishikawa and non-receptive AN3CA cells [ 21 , 27 ]. The results shown in Fig. 2 B demonstrated that non-receptive AN3CA cells did not express ST6GalNAc1 at the presence of LIF or not. Whereas, receptive Ishikawa cells expressed ST6GalNAc1 even at the absence of LIF. Thus, we exogenously overexpressed the ST6GalNAc1 gene in non-receptive endometrial AN3CA cells. The overexpressed ST6GalNAc1 gene successfully induced both sialyl-Tn expression and receptivity to JAr cells (Fig. 2 C-E). These results clearly demonstrated that the expression of ST6GalNAc1 and sialyl-Tn were sufficient for mediating the cell-cell interaction between trophoblast and endometrial cells. Sialyl-Tn expressed at implantation window was crucial for endometrial receptivity To examine the expression of sialyl-Tn at the period of implantation window in mice endometrium, estradiol and progesterone were serially treated to ovariectomized female mice for mimicking normal hormonal cycle (Fig. 3 A), according to previous studies [ 28 , 29 ]. The expression of LIF appeared in the uterus of estradiol and progesterone treated-mice (PE group) from day 8 and prominent at day 10 (Fig. 3 B). The expression of sialyl-Tn was elevated from day 6 and day 8 in the uterus of the PE group than the control group. However, the expressions are fuzzily distributed in the stoma region of the uterus. On day 10, the sialyl-Tn was expressed highest and prominent in the luminal region of the endometrium (Fig. 3 C). To confirm the function of ST6GalNAc1 expression in embryo implantation in vivo , the genetic abrogation of its expression is required. However, there are no available knockout mice in previous research papers or mouse phenotyping centers. It is possible that knockdown of ST6GalNAc1 may be embryonic lethal. Thus, to overcome the absence of knockout mice, we confirm the in vivo effect of sialyl-Tn on endometrial receptivity by blocking its receptor using free sialyl-Tn epitopes in murine IVF model. The result clearly demonstrates that the pre-incubation of the murine embryo with free sialyl-Tn for blocking its receptor can reduce the implantation rate of the transferred murine embryo (Table 1 ). These results collectively suggested that the sialyl-Tn antigen expressed on the endometrium induced by LIF in the period of implantation window had a key role in the adhesion between trophoblast and endometrium. Table 1. Effect of sTn-Ser on embryos implantation rate in mice. Recipients No. of embryos transferred Implanted embryos Implantation rate * Average Con #1 6 5 83.3 64.2 ± 9.4 a (29/44) #2 6 3 50.0 #3 5 2 40.0 #4 6 6 100.0 #5 5 2 40.0 #6 6 6 100.0 #7 5 2 40.0 #8 5 3 60.0 Free sialyl-Tn #1 6 4 66.7 32.1 ± 8.0 b (14/44) #2 6 0 0.0 #3 5 1 20.0 #4 6 1 16.7 #5 5 3 60.0 #6 6 2 33.3 #7 5 1 20.0 #8 5 2 40.0 * Implantation rate = Implanted embryos / Transferred embryos ab Different letters indicate statistically significance by Chi-square test (p < 0.001) Discussion Previously, several sialic acid-containing glycan epitopes, such as α2,3, α2,6, and α2,8 sialic acids linked to glycosphingolipids or glycoproteins, are expressed on the surface of uterine endometrium [ 30 , 31 ]. The sialic acid epitopes expressed on the surface of the uterine endometrium was affected by hormonal regulation and aging [ 31 – 35 ]. In addition, injection of LIF could modulate glycoconjugates on the apical surface of the murine uterine epithelium including terminal sialic acid [ 36 ]. However, the expression and roles of sialic acid epitopes in the embryo implantation site are still controversial [ 34 , 35 , 37 ]. In this study, we firstly found that the expression of a unique α2,6 sialic acid epitopes, sialyl-Tn antigen on the surface of endometrial cells might play a crucial role in receptivity toward trophoblast during the implantation window period. The sialyl-Tn, which is a truncated form of O -glycan synthesized by ST6GalNAc1, is related to the development of various epithelial tumors including gastric, colon, breast, lung, prostate, cervical, endometrial cancer, and endometriosis [ 38 – 40 ]. Its receptor, Siglec-6 was reported that highly expressed on the immune cells and placental trophoblast [ 41 ]. The roles of Siglec-6 in the leptin-related regulation of gestational trophoblast disease [ 42 ] and preterm preeclampsia [ 43 ] were suggested, its function in embryo implantation are still not reported. In this study, we blocked the receptor-ligand binding by using free sialyl-Tn epitopes, and they successfully abrogated the adhesion of trophoblast to endometrial cells in murine IVF model. However, the precise mechanism underlying the LIF/sialyl-Tn/Siglec-6 axis should be illuminated by further extensive experiments. From our recent study, the expression of integrin αVβ3 and αVβ5 induced by LIF treatment are required for endometrial receptivity [ 44 ]. However, the pattern of integrin expression in AN3CA is very similar to receptive endometrial cells, such as HEC-1A and Ishikawa [ 45 ]. To explain the phenomena, an element added to the integrins are required. Previous studies reported that several integrins including α2, α2b, α5, β1, and β3, are O -glycosylated proteins [ 46 , 47 ]. In addition, other adhesive molecules such as CD44 and trophinin are also highly glycosylated proteins [ 48 ]. Thus, the precise proteins, which added by sialyl-Tn and mediating embryo implantation, should be elucidated by further extensive studies. In this study, we shed a light on the understanding of the role of protein sialylation in endometrial receptivity. Conclusions LIF increases the cell surface sialyl-Tn antigen in uterine endometrial cells through the induction of ST6GalNAc1 expression. LIF-stimulated sialyl-Tn expression enhances the endometrial receptivity by increasing cell-cell interaction between trophoblast and endometrial cells by means of in vitro and in vivo model. From these results, we suggest that sialyl-Tn expression might be a key factor regulating the endometrial receptivity for successful embryo implantation. Abbreviations MUC-1: mucin-1 hCG: human chorionic gonadotrophin LIF: leukemia inhibitory factor GSEA: Gene Set Enrichment Analysis RT-PCR: Reverse transcription-polymerase chain reaction OVEX: Mice were ovariectomized s.c: subcutaneously dpc: days post coitum Siglecs: sialic acid-binding immunoglobulin-type lectins Declarations Ethics approval and consent to participate All experimental procedures were examined and approved by the Animal Research Ethics Committee at the Pusan University of Korea (no. PNU-2016-1212). Consent for publication Not applicable. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This study was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, of the Korean Government (Grant no. NRF-2014R1A5A20009936). Authors’ contributions HJC and TWC designed the experiments and performed the major molecular experiment. JK and JHJ performed in vitro fertilization and embryo transfer. BSK and MJP assisted the molecular experiments. KJK perfoermed the immunohistochemistry and immunoflurescent staining. JKJ and CHK analyzed the data. DR performed the in silico analysis. DR and KTH prepared manuscript. KTH designed the experiments and supervised the study. All authors have gone through and approved the manuscript. All authors have agreed both to be personally accountable for the author’s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work. Acknowledrements Not Applicable. References Salamonsen, L.A., et al., The Microenvironment of Human Implantation: Determinant of Reproductive Success. Am J Reprod Immunol, 2016. 75 (3): p. 218-25. Achache, H. and A. 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Chung, T.W., et al., Integrin alphaVbeta3 and alphaVbeta5 are required for leukemia inhibitory factor-mediated the adhesion of trophoblast cells to the endometrial cells. Biochem Biophys Res Commun, 2016. 469 (4): p. 936-40. Prifti, S., et al., Role of integrins in invasion of endometrial cancer cell lines. Gynecol Oncol, 2002. 84 (1): p. 12-20. King, S.L., et al., Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells. Blood Adv, 2017. 1 (7): p. 429-442. Liu, C.H., et al., C1GALT1 promotes invasive phenotypes of hepatocellular carcinoma cells by modulating integrin beta1 glycosylation and activity. PLoS One, 2014. 9 (8): p. e94995. Aplin, J.D. and P.T. Ruane, Embryo-epithelium interactions during implantation at a glance. J Cell Sci, 2017. 130 (1): p. 15-22. 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(A-C) Bubble plot, enrichment plot, and heat map summarizing the results of Gene Set Enrichment Analysis (GSEA) using the transcriptomes of human normal endometrium at distinct phases of the menstrual cycle (NCBI GEO dataset, GSE4888). (A) Bubble plot showing nominal p-value (x-axis), family-wise error rate (FWER) p-value (y-axis), and false discovery rate (FDR) q-value (size of the bubbles, -log10) of GSEA. (B) GSEA enrichment plot showing the enrichment scores (ES) of the GO_Glycosylation gene set. (C) Corresponding heat map displaying the expression values for the top genes of GO_Glycosylation gene set in human proliferative and secretory endometrium. (D, E) Correlogram and interaction network showing correlations among ST6GALNAC1, cytokines and their down-streams in using the transcriptomes of human normal endometrium (NCBI GEO dataset, GSE4888). (D) The depth of the shading at the correlogram displays the magnitude of the correlation (Spearman’s Rho). Positive and negative correlations are represented in blue and red, respectively. (E) Interaction network showing correlations of indicated genes. Positive and negative Spearman’s Rank correlation coefficients are represented by blue or red edges (Rho = |0.5-1.0|, p \u003c 0.05), respectively.","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/22452e1d-88fa-48b5-bb72-47282f222da8/v1/1.png"},{"id":471877,"identity":"e1e67654-c56f-4227-8717-bff9a071a731","added_by":"auto","created_at":"2020-02-10 16:17:10","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":978472,"visible":true,"origin":"","legend":"The adhesion of JAr cells to non-receptive AN3CA cells was reversed by ST6GalNAc1 expression. (A) Schematic diagram of O-glycan synthesis. (B) AN3CA cells were treated with LIF for 24 h. The expression of ST6GalNAc1 was determined by RT-PCR. The expression of ST6GalNAc1 in Ishikawa cells was used for positive control. (C) AN3CA cells were transfected with the ST6GalNAc1 gene in the mammalian expression vector pcDNA3.1. ST6GalNAc1 mRNA levels in total RNA obtained from each cell were detected by RT-PCR (D). In pcDNA3.1- or ST6GalNAc1-transfected AN3CA cells, the sialyl-Tn expression and the number of adherent JAr cells were measured by FACS analysis with FITC-labeled sialyl-Tn antibody. (E) The fluorescent labeled-JAr cells attached to pcDNA3.1 or ST6GalNAc1-transfected AN3CA cells were pictured and calculated. Five different sites per well were pictured and the number of adherent cells was calculated as mean ± SD of three independent experiments. **P \u003c 0.01 and ***P \u003c 0.001 compared to each group.","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/22452e1d-88fa-48b5-bb72-47282f222da8/v1/2.png"},{"id":471878,"identity":"dd729178-d0fe-4848-ae77-0635051c5423","added_by":"auto","created_at":"2020-02-10 16:17:10","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2745910,"visible":true,"origin":"","legend":"The expression of LIF and sialyl-Tn in the implantation window. (A) Schematic diagram of induction of peri-implantation phase. The prime phase was induced on day 0. Control and PE groups were injected s.c. with 100 ng of β-estradiol or vehicle for 3 days. The interphase was not treated on day 4. In the peri-implantation phase, mice were daily injected s.c. with 6.7 ng of β-estradiol and 1 mg of progesterone for 6 more days. Mice were sacrificed on days 6, 8 and 10, and both uterine horns were excised. The uterine tissues of mice were embedded in paraffin. The serial sections were immunostained with LIF (B) and FITC-labeled sialyl-Tn antibodies (C). The expression of LIF and sialyl-Tn were indicated by arrows.","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/22452e1d-88fa-48b5-bb72-47282f222da8/v1/3.png"},{"id":15665953,"identity":"6233761e-2123-4453-8412-ecbb17dab35d","added_by":"auto","created_at":"2021-11-18 13:34:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2425165,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-13537/v1/bd7059bf-f4dc-4028-9eb6-0bed3dd300be.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eExpression of \u003cem\u003eST6GalNAc1\u003c/em\u003e and sialyl-Tn antigen enhances endometrial receptivity\u003c/p\u003e","fulltext":[{"header":"Background","content":" \u003cp\u003eEmbryo implantation, a key process for a successful pregnancy, is one of the remaining unmet needs to improve the success rate of assisted reproduction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. The endometrium is receptive to embryo only during the implantation window, a short period of time about 6\u0026ndash;10 days after ovulation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A large number of molecular mediators including adhesive proteins, hormones, cytokines, and growth factors, which are involved in implantation have been identified. Among these molecules, several glycoproteins such as mucin-1 (MUC-1) and human chorionic gonadotrophin (hCG) have been focused on its possible function in relation to embryo implantation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, the molecular mechanisms regulating endometrial receptivity are remained poorly understood [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProtein glycosylation, a highly elaborative process of post-translational modification, is tightly controlled by enzymatic regulation [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The expression of terminal fucose residue generated by the action of fucosyltransferases, such as an increase of sialyl Lewis X antigen synthesized by fucosyltransferase VII, was reported as a positive factor of embryo implantation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In spite of given importance of the terminal sialic acid residues on the cell surface for cell-cell interaction owing to its negative charge [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], the function of the sialic acid epitopes in the embryo implantation is still not elucidated. Sialyl-Tn antigen, a type of truncated \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eO\u003c/span\u003e-glycosylation, has been reported as a key player in cancer progression including proliferation, invasion, and metastasis [\u003cspan additionalcitationids=\"CR11\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Although the expressions of sialyl-Tn were previously examined in the normal human reproductive organs including uterus, testis, and amniotic fluid [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], there are no previous report on the expression of sialyl-Tn nor their function in receptive endometrium.\u003c/p\u003e \u003cp\u003eIn this study, we firstly demonstrate that the expression of the sialyl-Tn antigen is increased in receptive endometrium is essential for the process of embryo implantation in both \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e human cell lines and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e mouse experiment.\u003c/p\u003e "},{"header":"Materials And Methods","content":" \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAnti-sialyl-Tn antibody [STn 219] (FITC) was supplied from Abcam (Cambridge, UK). The free sialyl-Tn epitope was supplied from Santa Cruz Biotechnology (Santa Cruz, CA). An anti-β-actin antibody, β-estradiol, and progesterone were purchased from Sigma-Aldrich (St. Louis, MO). Cell Tracker\u0026trade; Green CMFDA (5-chloromethylfluorescein diacetate) fluorescence dye was provided from Thermo Fisher Scientific (Waltham, MA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic analyses\u003c/h2\u003e \u003cp\u003eGene Set Enrichment Analysis (GSEA; \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.broadinstitute.org/gsea\u003c/span\u003e\u003c/span\u003e) was performed as described in previous studies [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] using the transcriptomes of human proliferative and mid-secretory endometrium (NCBI GEO dataset, GSE4888) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Correlation analyses were performed also using the transcriptomes of human proliferative and secretory endometrium (NCBI GEO dataset, GSE4888) as described in previous studies [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Correlogram was built using \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ecorrgram\u003c/span\u003e package in RStudio (R Consortium Inc, Boston, MA), with positive and negative correlations represented in blue and red edges, respectively. Only correlations with Spearman's Rho\u0026thinsp;\u0026gt;\u0026thinsp;0.5 or \u0026lt;\u0026thinsp;\u0026minus;\u0026thinsp;0.5 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) are displayed in the interaction network.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman uterine endometrial Ishikawa cell line, derived from a uterine adenocarcinoma [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], was kindly provided by Dr. Jacques Simard (CHUL Research Center, Quebec, Canada). Non-receptive uterine endometrial AN3CA cell lines [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] were purchased from American Type Culture Collection (ATCC; Manassas, VA). The cells were maintained at 37\u0026nbsp;\u0026deg;C in an atmosphere containing 5% CO\u003csub\u003e2\u003c/sub\u003e/air in Dulbecco\u0026rsquo;s Modified Eagle\u0026rsquo;s Medium (DMEM; Welgene, Daegu, Korea) and Roswell Park Memorial Institute medium (RPMI-1640; Welgene) containing 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific) and 1% penicillin/streptomycin (PSA; Thermo Fisher Scientific), respectively. The choriocarcinoma JAr cell was obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI1640 (Welgene) containing 10% FBS and 1% PSA.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eReverse transcription-polymerase chain reaction (RT-PCR)\u003c/h2\u003e \u003cp\u003eTotal RNA was isolated from cells using RiboEx\u0026trade; (GeneAll, Seoul, Korea) and then subjected to reverse transcription with oligo-dT primers using M-MLV reverse transcriptase (Thermo Fisher Scientific). The cDNA was amplified by PCR using AccuPower\u0026reg;PCR PreMix (Bioneer Co., Daejeon, Korea). The primers used in this study were as follows: \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eST6GalNAc1\u003c/span\u003e, forward 5\u0026rsquo;-GTCCAGTGGTCCTTGCTTCTGGCTG-3\u0026rsquo; and reverse 5\u0026rsquo;-ATTTCCTTGGGTCGTCTTTGTGTCC-3\u0026rsquo;; \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eβ-actin\u003c/span\u003e, forward 5\u0026rsquo;-CAAGAGATGGCCACGGCTGCT-3\u0026rsquo; and reverse 5\u0026rsquo;-TCCTTCTGCATCCTGTCGGCA-3\u0026rsquo;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eExpression of ST6GalNAc1 gene\u003c/h2\u003e \u003cp\u003eAN3CA cells were transfected with the \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eST6GalNAc1\u003c/span\u003e gene in the mammalian expression vector, pcDNA3.1, which was kindly gifted from Dr. Philippe Delannoy (University of Lille, France).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometric analysis\u003c/h2\u003e \u003cp\u003eAN3CA cells were fixed with 3.7% formaldehyde and incubated with FITC-conjugated anti-sialyl-Tn antibody in Carbo-free blocking solution for 2\u0026nbsp;h. The cells were evaluated by flow cytometry (BD FACS CANTO II; BD Biosciences, San Jose, CA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAdhesion assay\u003c/h2\u003e \u003cp\u003eAN3CA cells were seeded into 6 well plates and cultured to a confluent monolayer. After the pretreatment of hLA, the cells were incubated with leukemia inhibitory factor (LIF) treatment for 48\u0026nbsp;h. The JAr cells were labeled with Cell Tracker\u0026trade; Green CMFDA (Invitrogen, Carlsbad, CA) and gently added onto a monolayer of AN3CA cells. After gently shaking at 40\u0026nbsp;rpm for 30\u0026nbsp;min at 37\u0026nbsp;\u0026deg;C, the cells were vigorously washed to remove non-binding cells. The attached JAr cells were visualized by using a fluorescent microscope (Axio Imager M1, Carl-Zeiss, Oberkochen, Germany). Five fields of each sample were chosen randomly and the number of adhered cells was averaged after quantifying with ImageJ software (NIH; Bethesda, MD)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eFemale C57BL/6 mice (7\u0026ndash;8 weeks old, weight 20\u0026ndash;22\u0026nbsp;g) were purchased from Orient Bio, Co. (Seongnam, Korea). Mice were ovariectomized (OVEX) and recuperated 14 days later. Animals were fed on a standard diet with \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ead libitum\u003c/span\u003e access to water, and kept on a 12\u0026nbsp;h light: 12\u0026nbsp;h dark cycle. All experimental procedures were examined and approved by the Animal Research Ethics Committee at the Pusan University of Korea (no. PNU-2016-1212).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInduction of peri-implantation phase and immunochemistry\u003c/h2\u003e \u003cp\u003eTwenty-eight female mice were randomly divided into two groups: control and PE. In the prime phase, mice were injected subcutaneously (s.c.) with 100\u0026nbsp;ng β-estradiol (in 100\u0026nbsp;\u0026micro;l corn oil) or vehicle for 3 days. In interphase, no hormone was treated for the next two days. In the peri-implantation phase, mice were daily injected s.c. with 6.7\u0026nbsp;ng β-estradiol and 1\u0026nbsp;mg progesterone (in 100\u0026nbsp;\u0026micro;l corn oil) for 6 more days. Mice were respectively sacrificed on day 6, 8 and 10, and both uterine horns were excised to determine the expression of LIF and sialyl-Tn using immunohistochemistry and immunofluorescence analysis, respectively. The uterine tissues of mice were fixed in 3.7% formalin and embedded in paraffin and were then cut into 4\u0026nbsp;\u0026micro;m serial sections. The sections were immunostained with LIF or sialyl-Tn (FITC) antibodies, visualized with the Dako Envision kit (Dako, Jena, Germany) and fluorescent microscope (Axio Imager M1) respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and embryo collection and Embryo transfer in utero\u003c/h2\u003e \u003cp\u003eInbred ICR female mice (6\u0026ndash;8 weeks old) were induced to superovulate by intraperitoneal injection of 5\u0026nbsp;IU serum gonadotropin from a pregnant mare (Sigma-Aldrich) followed by injection with 5\u0026nbsp;IU human chorionic gonadotropin (hCG; Sigma-Aldrich) 46 later. Superovulated female mice were mated with fertile male mice and euthanized by cervical dislocation 46\u0026nbsp;h after hCG injection. The day of vaginal plugging was designated as 0.5 days post coitum (dpc). Mouse 2-cell embryos were collected from oviducts on 1.5 dpc and cultured in pre-warmed the Quinn\u0026rsquo;s Advantage Blastocyst Medium (SAGE/Origio, Malov, Denmark) containing 10% serum protein substitute in an incubator at 37 and 5% CO\u003csub\u003e2\u003c/sub\u003e. Implantation potential of embryos from the control and sialyl-Tn epitope groups was examined via embryo transfer as described previously [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In the sialyl-Tn epitope group, embryos were transferred with a 50\u0026nbsp;ng/ml sialyl-Tn epitope. Five to six embryos from two different groups were transferred on 3.5 dpc to the contralateral uterine horn of pseudopregnant recipients provided by mating with a vasectomized male. Two days after transfer, implantation sites in uterine horns of pregnancy were detected by intravenous injection of Chicago blue dye. Clear blue bands in utero were considered implantation sites.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of results was performed by a Student\u0026rsquo;s \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003et\u003c/span\u003e-test (for \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e adhesion assay) or Chi-square test (for \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e embryo transfer) using GraphPad Prism (GraphPad Software, San Diego, CA). Values are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and the minimum significance level was set at a \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eP\u003c/span\u003e value of 0.05. All experiments except for animal studies were independently performed at least 3 times.\u003c/p\u003e \u003c/div\u003e "},{"header":"Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eBioinformatic analysis\u003c/h2\u003e\n\u003cp\u003eTo identify the functions of protein glycosylation in human embryo implantation, we applied a bioinformatic approach with the NCBI GEO database (GSE4888) [\u003cspan class=\"CitationRef\"\u003e17\u003c/span\u003e]. The results from GSEA using the transcriptomes of human proliferative and mid-secretory endometrium indicated that glycosylation, especially \u003cspan class=\"Italic\"\u003eO\u003c/span\u003e-linked glycosylation is the most prominent and significant gene set (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). Among the genes involved in \u003cspan class=\"Italic\"\u003eO\u003c/span\u003e-glycosylation, the expression of sialyltransferases, such as \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e, \u003cspan class=\"Italic\"\u003eST6Gal1\u003c/span\u003e, \u003cspan class=\"Italic\"\u003eST3Gal6\u003c/span\u003e, are significantly increased in the uterus of mid-secretory phase, which is corresponding to implantation window (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC). To identify putative targets of LIF, a major cytokine regulating endometrial receptivity [\u003cspan class=\"CitationRef\"\u003e24\u003c/span\u003e], we performed correlation analyses using the same GEO dataset. Interestingly, a highly significant positive correlation between LIF/gp130/STAT3 signaling pathway and \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e was observed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD and E). Other cytokines and growth factors related to endometrial receptivity including IL-1\u0026alpha;, IL-6, IL-8, IL-11, IL-15, and HB-EGF [\u003cspan class=\"CitationRef\"\u003e25\u003c/span\u003e] showed a lower positive correlation with the expression of \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e compared with LIF.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eST6GalNAc1 expression mediated adhesion of trophoblast to endometrial cells\u003c/h2\u003e\n\u003cp\u003eAmong the sialic acid-binding immunoglobulin-type lectins (Siglecs), Siglec-6 is a receptor for the sialyl-Tn epitope and expressed on the surface of trophoblast [\u003cspan class=\"CitationRef\"\u003e26\u003c/span\u003e]. As ST6GalNAc1 is responsible for the expression of sialyl-Tn antigen and termination of \u003cspan class=\"Italic\"\u003eO\u003c/span\u003e-glycan (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA), we confirmed the expression of the \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e gene in the receptive endometrial Ishikawa and non-receptive AN3CA cells [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e27\u003c/span\u003e]. The results shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB demonstrated that non-receptive AN3CA cells did not express \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e at the presence of LIF or not. Whereas, receptive Ishikawa cells expressed ST6GalNAc1 even at the absence of LIF. Thus, we exogenously overexpressed the \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e gene in non-receptive endometrial AN3CA cells. The overexpressed \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e gene successfully induced both sialyl-Tn expression and receptivity to JAr cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). These results clearly demonstrated that the expression of \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e and sialyl-Tn were sufficient for mediating the cell-cell interaction between trophoblast and endometrial cells.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eSialyl-Tn expressed at implantation window was crucial for endometrial receptivity\u003c/h2\u003e\n\u003cp\u003eTo examine the expression of sialyl-Tn at the period of implantation window in mice endometrium, estradiol and progesterone were serially treated to ovariectomized female mice for mimicking normal hormonal cycle (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), according to previous studies [\u003cspan class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e]. The expression of LIF appeared in the uterus of estradiol and progesterone treated-mice (PE group) from day 8 and prominent at day 10 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). The expression of sialyl-Tn was elevated from day 6 and day 8 in the uterus of the PE group than the control group. However, the expressions are fuzzily distributed in the stoma region of the uterus. On day 10, the sialyl-Tn was expressed highest and prominent in the luminal region of the endometrium (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). To confirm the function of \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e expression in embryo implantation \u003cspan class=\"Italic\"\u003ein vivo\u003c/span\u003e, the genetic abrogation of its expression is required. However, there are no available knockout mice in previous research papers or mouse phenotyping centers. It is possible that knockdown of \u003cspan class=\"Italic\"\u003eST6GalNAc1\u003c/span\u003e may be embryonic lethal. Thus, to overcome the absence of knockout mice, we confirm the \u003cspan class=\"Italic\"\u003ein vivo\u003c/span\u003e effect of sialyl-Tn on endometrial receptivity by blocking its receptor using free sialyl-Tn epitopes in murine IVF model. The result clearly demonstrates that the pre-incubation of the murine embryo with free sialyl-Tn for blocking its receptor can reduce the implantation rate of the transferred murine embryo (Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). These results collectively suggested that the sialyl-Tn antigen expressed on the endometrium induced by LIF in the period of implantation window had a key role in the adhesion between trophoblast and endometrium.\u003c/p\u003e\n\u003cdiv class=\"gridtable\"\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1. Effect of sTn-Ser on embryos implantation rate in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable style=\"width: 446.55pt; border-collapse: collapse;\" border=\"0\" width=\"595\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n\u003ctbody\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 51.15pt; border: solid black 1.0pt; border-left: none; padding: .75pt .75pt 0in .75pt; height: 33.75pt;\" width=\"11.428571428571429%\"\u003e\u0026nbsp;\u003c/td\u003e\n\u003ctd style=\"width: 62.15pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 33.75pt;\" width=\"13.949579831932773%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eRecipients\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 33.75pt;\" width=\"17.478991596638654%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eNo. of embryos\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003etransferred\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 33.75pt;\" width=\"17.478991596638654%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eImplanted \u003cbr /\u003e\u0026nbsp;embryos \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: solid black 1.0pt; border-left: none; padding: .75pt .75pt 0in .75pt; height: 33.75pt;\" width=\"18.991596638655462%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eImplantation \u003cbr /\u003e rate\u003csup\u003e*\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border-top: solid black 1.0pt; border-left: none; border-bottom: solid black 1.0pt; border-right: none; padding: .75pt .75pt 0in .75pt; height: 33.75pt;\" width=\"20.672268907563026%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eAverage\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 51.15pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" rowspan=\"8\" width=\"11.428571428571429%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eCon\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"13.949579831932773%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"17.478991596638654%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"17.478991596638654%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; 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padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: none; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e100.0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"20.544554455445546%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e2\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: none; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e40.0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"20.544554455445546%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: none; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e100.0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"20.544554455445546%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#7\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e2\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: none; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e40.0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 62.15pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.25pt;\" width=\"20.544554455445546%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#8\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.25pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.25pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.25pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e60.0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 51.15pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" rowspan=\"8\" width=\"11.428571428571429%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003eFree sialyl-Tn\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"13.949579831932773%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"17.478991596638654%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"17.478991596638654%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e4\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: none; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"18.991596638655462%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e66.7\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 92.15pt; border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" rowspan=\"8\" width=\"20.672268907563026%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e32.1 \u0026plusmn; 8.0\u003csup\u003eb \u003cbr /\u003e\u0026nbsp; \u003c/sup\u003e(14/44)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"20.544554455445546%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#2\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: none; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e0.0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"20.544554455445546%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#3\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: none; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e20.0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"20.544554455445546%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#4\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e6\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e1\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border: none; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e16.7\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr\u003e\n\u003ctd style=\"width: 62.15pt; border: none; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"20.544554455445546%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e#5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 78.1pt; padding: .75pt .75pt 0in .75pt; height: 16.5pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e5\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 77.95pt; 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border: none; border-bottom: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.25pt;\" width=\"25.742574257425744%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e2\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 85.05pt; border-top: none; border-left: none; border-bottom: solid black 1.0pt; border-right: solid black 1.0pt; padding: .75pt .75pt 0in .75pt; height: 17.25pt;\" width=\"27.97029702970297%\"\u003e\n\u003cp style=\"text-align: center; line-height: 200%; font-size: 13px; font-family: 'Malgun Gothic',sans-serif; margin: 0in 0in .0001pt 0in;\"\u003e\u003cspan style=\"line-height: 200%; font-family: 'Times New Roman',serif;\"\u003e40.0\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"padding-left: 30px;\"\u003e\u003csup\u003e* \u003c/sup\u003eImplantation rate = Implanted embryos / Transferred embryos\u003c/p\u003e\n\u003cp style=\"padding-left: 30px;\"\u003e\u003csup\u003eab\u003c/sup\u003e Different letters indicate statistically significance by Chi-square test (p \u0026lt; 0.001)\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":" \u003cp\u003ePreviously, several sialic acid-containing glycan epitopes, such as α2,3, α2,6, and α2,8 sialic acids linked to glycosphingolipids or glycoproteins, are expressed on the surface of uterine endometrium [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The sialic acid epitopes expressed on the surface of the uterine endometrium was affected by hormonal regulation and aging [\u003cspan additionalcitationids=\"CR32 CR33 CR34\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In addition, injection of LIF could modulate glycoconjugates on the apical surface of the murine uterine epithelium including terminal sialic acid [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, the expression and roles of sialic acid epitopes in the embryo implantation site are still controversial [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In this study, we firstly found that the expression of a unique α2,6 sialic acid epitopes, sialyl-Tn antigen on the surface of endometrial cells might play a crucial role in receptivity toward trophoblast during the implantation window period.\u003c/p\u003e \u003cp\u003eThe sialyl-Tn, which is a truncated form of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eO\u003c/span\u003e-glycan synthesized by ST6GalNAc1, is related to the development of various epithelial tumors including gastric, colon, breast, lung, prostate, cervical, endometrial cancer, and endometriosis [\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Its receptor, Siglec-6 was reported that highly expressed on the immune cells and placental trophoblast [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The roles of Siglec-6 in the leptin-related regulation of gestational trophoblast disease [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] and preterm preeclampsia [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e] were suggested, its function in embryo implantation are still not reported. In this study, we blocked the receptor-ligand binding by using free sialyl-Tn epitopes, and they successfully abrogated the adhesion of trophoblast to endometrial cells in murine IVF model. However, the precise mechanism underlying the LIF/sialyl-Tn/Siglec-6 axis should be illuminated by further extensive experiments.\u003c/p\u003e \u003cp\u003eFrom our recent study, the expression of integrin αVβ3 and αVβ5 induced by LIF treatment are required for endometrial receptivity [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, the pattern of integrin expression in AN3CA is very similar to receptive endometrial cells, such as HEC-1A and Ishikawa [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. To explain the phenomena, an element added to the integrins are required. Previous studies reported that several integrins including α2, α2b, α5, β1, and β3, are \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eO\u003c/span\u003e-glycosylated proteins [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. In addition, other adhesive molecules such as CD44 and trophinin are also highly glycosylated proteins [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Thus, the precise proteins, which added by sialyl-Tn and mediating embryo implantation, should be elucidated by further extensive studies. In this study, we shed a light on the understanding of the role of protein sialylation in endometrial receptivity.\u003c/p\u003e "},{"header":"Conclusions","content":" \u003cp\u003eLIF increases the cell surface sialyl-Tn antigen in uterine endometrial cells through the induction of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003eST6GalNAc1\u003c/span\u003e expression. LIF-stimulated sialyl-Tn expression enhances the endometrial receptivity by increasing cell-cell interaction between trophoblast and endometrial cells by means of \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vitro\u003c/span\u003e and \u003cspan type=\"Italic\" class=\"Italic\" name=\"Emphasis\"\u003ein vivo\u003c/span\u003e model. From these results, we suggest that sialyl-Tn expression might be a key factor regulating the endometrial receptivity for successful embryo implantation.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eMUC-1: mucin-1\u003c/p\u003e\n\u003cp\u003ehCG: human chorionic gonadotrophin\u003c/p\u003e\n\u003cp\u003eLIF: leukemia inhibitory factor\u003c/p\u003e\n\u003cp\u003eGSEA: Gene Set Enrichment Analysis\u003c/p\u003e\n\u003cp\u003eRT-PCR: Reverse transcription-polymerase chain reaction\u003c/p\u003e\n\u003cp\u003eOVEX: Mice were ovariectomized\u003c/p\u003e\n\u003cp\u003es.c: subcutaneously\u003c/p\u003e\n\u003cp\u003edpc: days post coitum\u003c/p\u003e\n\u003cp\u003eSiglecs: sialic acid-binding immunoglobulin-type lectins\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were examined and approved by the Animal Research Ethics Committee at the Pusan University of Korea (no. PNU-2016-1212).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr /\u003e Consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eCompeting interests \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by a grant from the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT, of the Korean Government (Grant no. NRF-2014R1A5A20009936).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHJC and TWC designed the experiments and performed the major molecular experiment. JK and JHJ performed \u003cem\u003ein vitro\u003c/em\u003e fertilization and embryo transfer. BSK and MJP assisted the molecular experiments. KJK perfoermed the immunohistochemistry and immunoflurescent staining. JKJ and CHK analyzed the data. DR performed the \u003cem\u003ein silico\u003c/em\u003e analysis. DR and KTH prepared manuscript. KTH designed the experiments and supervised the study. All authors have gone through and approved the manuscript. All authors have agreed both to be personally accountable for the author\u0026rsquo;s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledrements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSalamonsen, L.A., et al., \u003cem\u003eThe Microenvironment of Human Implantation: Determinant of Reproductive Success.\u003c/em\u003e Am J Reprod Immunol, 2016. \u003cstrong\u003e75\u003c/strong\u003e(3): p. 218-25.\u003c/li\u003e\n\u003cli\u003eAchache, H. and A. 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Ruane, \u003cem\u003eEmbryo-epithelium interactions during implantation at a glance.\u003c/em\u003e J Cell Sci, 2017. \u003cstrong\u003e130\u003c/strong\u003e(1): p. 15-22.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Glycosylation, Sialyl-Tn, ST6GalNAc1, Embryo implantation, Endometrial Receptivity","lastPublishedDoi":"10.21203/rs.2.22965/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.22965/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eTo characterize molecular mechanism underlying the regulation of sialylated glycan expression and its roles for endometrial receptivity and embryo implantation. Here, we characterized the role of a truncated form of sialylated \u003cem\u003eO\u003c/em\u003e-glycan, sialyl-Tn, for endometrial receptivity.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eThe transcriptomes of human endometrium at mid-secretory phase were analyzed by Bioinformatics. Changes in gene expression, protein, and signal pathway were measured using RT-PCR and Western blot. The cell adhesion assay was visualized using a fluorescent microscope. In peri-implantation phase of mice, the expression of leukemia inhibitory factor (LIF) and sialyl-Tn were confirmed using immunohistochemistry and immnofluorescence analysis. The effect of sialy-Tn expression on embryo implantation was estimated by \u003cem\u003ein vitro\u003c/em\u003e fertilization and embryo transfer using mice.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eIn\u003cem\u003e in silico\u003c/em\u003e analysis, expression of \u003cem\u003eO\u003c/em\u003e-glycosylation genes, especially \u003cem\u003eST6GalNAc1\u003c/em\u003e, was significantly increased in the human uterus of mid-secretory phase. Overexpression of the \u003cem\u003eST6GalNAc1\u003c/em\u003e gene in non-receptive human endometrial AN3CA cells enhances the attachment of trophoblastic JAr cells. In an animal study, the results clearly indicated that sialyl-Tn was expressed on the surface of the mid-secretory uterus. In addition, blockade of the receptor using free sialyl-Tn epitope diminished the implantation rates of intrauterine transferred murine embryos.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\u003cp\u003eFrom these results, here we suggest that sialyl-Tn expression might be a novel factor regulating the endometrial receptivity for successful embryo implantation.\u003c/p\u003e","manuscriptTitle":"Expression of ST6GalNAc1 and sialyl-Tn antigen enhances endometrial receptivity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2020-02-10 16:17:09","doi":"10.21203/rs.2.22965/v1","editorialEvents":[{"type":"communityComments","content":1}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"65bb0882-9887-442b-8747-82f97a0bb4ac","owner":[],"postedDate":"February 10th, 2020","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56949,"name":"Endocrinology \u0026 Metabolism"}],"tags":[],"updatedAt":"","versionOfRecord":[],"versionCreatedAt":"2020-02-10 16:17:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-13537","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"identity":"rs-13537","version":["v1"]},"buildId":"wLkW0s4AflPzk-lpfg-fK","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}