title: Altered β1,6-GlcNAc and bisecting GlcNAc-branched N-glycan on integrin β1 are associated with early spontaneous miscarriage in humans.
abstract: OBJECTIVE
  Do N-acetylglucosaminyltransferase (GnT-V) and N-acetylglucosaminyltransferase III (GnT-III) play an important role in early spontaneous miscarriage (ESM) in humans.
  
  
  CONCLUSIONS
  The dynamic balance between GnT-V and GnT-III expression in chorionic villi differed between early normal pregnancy and ESM and was associated with altered β1,6-N-acetylglucosamine (β1,6-GlcNAc) and bisecting N-acetylglucosamine (bis-GlcNAc) branched N-glycans on integrin β1.
  
  
  BACKGROUND
  GnT-V contributes to metastasis, while GnT-III is recognized as a metastasis suppressor. It has been reported that GnT-V contributes to placentation in the early phase of pregnancy, possibly regulating trophoblast invasion. However, the expressions of GnT-V and GnT-III in ESM have not been reported.
  
  
  METHODS
  Villous samples from 6 to 9 weeks of gestation were collected in the First Affiliated Hospital of Chongqing Medical University from May 2013 to September 2014 from 60 normal pregnant women undergoing elective termination of pregnancy and from 40 patients with a clinical diagnosis of ESM.
  Quantitative PCR and western blots were used to examine the GnT-V and GnT-III mRNA (Mgat5 and Mgat3) and protein expression, respectively, of chorionic villi in both the ESM group and the normal group from week 6 to week 9. We used immunofluorescence and immunohistochemistry to detect the location of GnT-V and GnT-III. Lectin fluorescence and histochemistry were used to test the location of β1,6-GlcNAc and bis-GlcNAc branching in the normal and ESM groups. To assess the functional capacity of GnT-V and GnT-III in the chorionic villi between the two groups, we used an enzyme-linked immunosorbent assay kit to measure the activity of these enzymes. Using co-precipitated integrin α5β1 followed by phytohaemagglutinin (PHA)-L and PHA-E blotting, we investigated whether GnT-V and GnT-III could modify the N-glycosylation profile in terms of the β1,6-GlcNAc and bis-GlcNAc structures in integrin α5β1 during the first trimester in both groups.
  
  
  RESULTS
  In the normal group expression and activity of GnT-V and the concentration of its product, β1,6-GlcNAc were higher at week 9 than at weeks 6, 7 and 8 (P < 0.05). In contrast, the expression and activity of GnT-III and the concentration of its product, bis-GlcNAc were higher at week 6 than at weeks 7, 8 and 9 (P < 0.05). Compared with the normal group, the ESM group exhibited a lower expression of GnT-V and β1,6-GlcNAc (P < 0.05) and a higher expression of GnT-III and bis-GlcNAc (P < 0.05) with consistent changes in enzymatic activity. Immunofluorescence showed that GnT-V was located mainly in the cytoplasm of syncytiotrophoblasts (STBs) and chorionic villous cytotrophoblasts (CTBs), in both the ESM group and the normal group. β1,6-GlcNAc N-glycan was mainly located outside of the STB and CTB layer in normal villi and was expressed only rarely in the ESM villi. GnT-III was expressed primarily in the cytoplasm of STBs and expressed only very weakly in the CTBs of normal villi, whereas it was highly expressed in both the STBs and CTBs in the ESM group. bis-GlcNAc was primarily located outside of the STBs in the normal villi, whereas it was expressed much more abundantly outside of both the STBs and CTBs in the ESM group at each week of gestation. Moreover, decreased β1,6-GlcNAc-branched N-glycans and increased bis-GlcNAc-branched N-glycans on integrin β1 (P < 0.05) were observed in the ESM group.
  
  
  CONCLUSIONS
  Our findings provide a new insight for studying the mechanism of clinical ESM in humans and it might be valuable for the clinical diagnosis and treatment of ESM.
  
  
  CONCLUSIONS
  The study lacks experiments in vitro to disclose the precise mechanism by which GnT-V and GnT-III regulate ESM. In some cases, degradation of the tissues after the miscarriage event cannot be ruled out.
  
  
  BACKGROUND
  This study was supported by grants from the National Natural Science Foundation of China (31271546). The authors have no competing interests.
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Min
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Meirong
authors.initials: M
authors.lastname: Wang
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Rufei
authors.initials: R
authors.lastname: Gao
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Xueqing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Chen
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Yanqing
authors.initials: Y
authors.lastname: Geng
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Yubin
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China.
authors.forename: Yingxiong
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Laboratory of Reproductive Biology, School of Public Health and Management, Chongqing Medical University, Box 197, No. 1 Yixueyuan Road, Yuzhong District, Chongqing 400016, PR China hejunlin_11@aliyun.com.
authors.forename: Junlin
authors.initials: J
authors.lastname: He
mesh_terms: D000022:Abortion, Spontaneous / Q000201:enzymology / Q000378:metabolism*; D000328:Adult; D002824:Chorionic Villi / Q000201:enzymology / Q000378:metabolism*; D005260:Female; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism*; D010929:Placentation / Q000502:physiology; D011247:Pregnancy; D011261:Pregnancy Trimester, First / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism
chemical_list: D019012:Integrin beta1; D012333:RNA, Messenger; C000601122:Mgat5 protein, human; D017351:N-Acetylglucosaminyltransferases; C054599:beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase
keywords: GnT-III; GnT-V; early spontaneous miscarriage; glycosylation; integrin