Epithelial apical glycosylation changes associated with thin endometrium in women with infertility - a pilot observational study

Abstract

Background: Low endometrial receptivity is one of the major factors affecting successful implantation in assisted reproductive technologies (ART). Infertile patients with thin endometrium have a significantly lower cumulative clinical pregnancy rate than patients with normal endometrium. Molecular pathophysiology of low receptivity of thin endometrium remains understudied. We have investigated composition of glycocalyx of the apical surface of luminal and glandular epithelial cells in thin endometrium of infertile women.

Methods: Thirty-two patients with tubal-peritoneal infertility undergoing in vitro fertilization (IVF) were included in the study. Endometrial samples were obtained in a natural menstrual cycle. Patients were divided into two groups: patients with normal endometrium (≥8 mm) and with thin endometrium (< 8 mm). Histochemical and immunohistochemical analysis of paraffin-embedded endometrial samples was performed using six biotinylated lectins (UEA-I, MAL-II, SNA, VVL, ECL, Con A) and anti-Le^Y and MECA-79 monoclonal antibodies (MAbs).

Results: Complex glycans analysis taking into account the adjusted specificity of glycan-binding MAbs revealed 1.3 times less expression of MECA-79 glycans on the apical surface of the luminal epithelial cells of thin endometrium compared to normal endometrium; this deficiency may adversely affect implantation, since MECA-79 glycans are a ligand of L-selectin and mediate intercellular interactions. The glycans containing a type-2 unit Galβ1-4GlcNAcβ (LacNAc) but lacking sulfo-residues at 6-OH of GlcNAcβ, and binding to MECA-79 MAbs were found; they can be considered as potential markers of endometrium receptivity. Expression of the lectins-stained glycans on the apical surfaces of the luminal and glandular epithelial cells did not differ significantly. Correlation between the expression of difucosylated oligosaccharide Le^Y on the apical surfaces of the luminal and glandular epithelial cells was found in patients with thin endometrium and recurrent implantation failure. A similar relationship was shown for mannose-rich glycans.

Conclusions: Specific features of key glycans expression in epithelial compartments of thin endometrium may be essential for morphogenesis of the endometrial functional layer and explain its low receptivity.

Keywords: Assisted reproductive technologies (ART); Glycans; Glycocalyx; Infertility; Lectin histochemistry; MECA-79; Thin endometrium.

Fig. 1

Glycoconjugates staining in thin endometrium. Staining of the apical surface of the luminal epithelial cells (1) and of the glandular cells (2) with anti-Le^Y MAbs (a) and with Сon A (b). Negative controls were performed without primary antibodies (c) and lectin Con A (d) and with mouse IgG₁ (e). Staining of positive controls: f - placental terminal villi with Con A, g - lung carcinoma tissues with the anti-Le^Y antibody. Magnification: × 400

Fig. 2

Immunolocalization of carbohydrate epitopes MECA-79 on the apical surface of the luminal epithelial cells. a - thin and b - normal endometrium; (1) - luminal cells and (2) - glandular cells. Negative controls were performed without primary antibodies (c) and with isotypic rat IgM (d). Positive control - lymph nodes in breast cancer (e). Magnification: × 400. Histograms demonstrate relative expression of carbohydrate epitopes MECA-79 in thin (n = 14) and normal (n = 18) endometrium. The bar represents optical density as a mean value ± standard deviation. *p < 0.05 compared to normal endometrium (f)

Fig. 3

Top ligands for anti-Le^Y MAbs (a) and MECA-79 MAbs (b). For structures and short names of all ligands of the microarray chip see additional file (Table S1). Fucα1-2Galβ1-4(Fucα1-3) GlcNAcβ- (Le^Y #372). Fucα1-2Galβ1-3(Fucα1-4) GlcNAcβ- (Le^B #371). Fucα1-2Galβ1-3(Fucα1-4) GlcNAcβ1-3Galβ1-4Glcβ- (Le^BLac #496). Galβ1-4GlcNAcβ1-3(GlcNAcβ1-6) Galβ1-4GlcNAcβ (LN3′(GN6′)LN #489). GalNAcβ1-3(Fucα1-2) Galβ1-4(Fucα1-3) GlcNAcβ (ALe^Yb #508). Neu5Acα2-3Galβ1-4(2-O-Su-Fucα1-3) GlcNAcβ (SiaLeX2′′′Su #431). 6-Bn-Galα1-4(6-Bn) GlcNAcβ (Bn2-aLN #126)