Characterization of the role for cadherin 6 in the regulation of human endometrial receptivity

Abstract

Background: The endometrial luminal epithelium is the first point of attachment of embryos during implantation. Failure of embryos to firmly adhere results in implantation failure and infertility. A receptive endometrial luminal epithelium is achieved through the expression of adhesion molecules in the mid-secretory phase and is a requirement for implantation. Cadherin 6 (CDH6) is an adhesion molecule localizing to the endometrial luminal epithelial cell surface in the mid-secretory/receptive phase and knockdown of CDH6 in the Ishikawa cells (receptive endometrial epithelial cell line) compromises cell integrity. However, there are no studies investigating the role of CDH6 on receptivity and infertility. This study aimed to investigate whether CDH6 is dysregulated in the endometrium of women with infertility during the receptive window and the effect of CDH6 on endometrial adhesion and receptivity.

Methods: The expression and the localization of CDH6 in the human endometrium were determined by immunohistochemistry. Ishikawa cells were used to investigate the functional consequences of CDH6 knockdown on endometrial adhesive capacity to HTR8/SVneo (trophoblast cell line) spheroids in vitro. CDH6 knockdown was assessed by qPCR and immunoblotting. After CDH6 knockdown, the expression of type II cadherin family members and CDH6 functional partners were assessed by qPCR. Two-tailed unpaired student's t-test or one-way ANOVA as appropriate were used for statistical analysis with a significance threshold of P < 0.05.

Results: A significant reduction of CDH6 immunolocalization was recorded in the luminal and glandular epithelium of endometrium from women with infertility (P < 0.05) compared to fertile group respective cellular compartments in the mid-secretory phase. Functional analysis using Ishikawa cells demonstrated that knockdown of CDH6 (treated with 50 nM CDH6 siRNA) significantly reduced epithelial adhesive capacity (P < 0.05) to HTR8/SVneo spheroids compared to control and other type II cadherin family members likely failed to compensate for the loss of CDH6. The expression levels of CDH6 functional partners, catenin family members were not changed after CDH6 knockdown in Ishikawa cells.

Conclusion: Together, our data revealed that CDH6 was dysregulated in the endometrium from women with infertility and altered Ishikawa cell adhesive capacity. Our study supports a role for CDH6 in regulating endometrial adhesion and implantation.

Keywords: Adhesive molecules; CDH6; Embryo implantation; Endometrial epithelial cell; Endometrial receptivity; Trophoblast cell.

Fig. 1

Comparison of CDH6 immunolocalization in fertile and infertile mid-secretory phase endometrium. a CDH6 immunolocalized to the luminal epithelium (L), glandular epithelium (G) and stromal cells (S). Higher magnification images are outlined. The specificity of CDH6 labeling was confirmed through the inclusion of an isotype control in which the non-immune antibody of the same isotype was substituted for the CDH6 antibody at the same concertation. Sections were counterstained with hematoxylin to indicate the cell nuclei (blue). b Staining intensity of CDH6 was semi-quantitated by scoring staining in tissues blinded to fertility status. Data were presented as mean ± SEM. (n = 4). *P < 0.05, ns: no significant difference

Fig. 2

Immunocytochemistry detection of CDH6 in the Ishikawa cells and HTR8/SVneo cells. a In Ishikawa cells, CDH6 localization was detected to the plasma membrane with moderate staining appearing in the cytoplasm. b A similar localization was observed in the HTR8/SVneo cells (arrows) with extra staining also found in the cytoplasm. Higher magnification images of CDH6 localization in HTR8/SVneo cells are depicted on the right of panels. The specificity of CDH6 labeling was confirmed through the inclusion of an isotype control as described in Fig. 1. Sections were counterstained with hematoxylin to indicate the cell nuclei (blue)

Fig. 3

Examination of the effect of CDH6 knockdown on Ishikawa cell adhesive capacity. Ishikawa cells were transfected with either scrambled control (50 nM) or CDH6 siRNA (10, 20 or 50 nM) before HTR8/SVneo spheroid adhesion assay. aCDH6 knockdown was determined by qPCR. Expression levels were normalized to 18S (n = 7). b Immunoblotting was also used to determine the CDH6 expression. Blots were co-probed with an anti-GAPDH antibody to confirm equivalent protein loading of each sample. c A siRNA concentration-dependent reduction of the adhesion was observed in Ishikawa cells with the highest concentration of CDH6 siRNA (50 nM) significantly compromised the spheroid adhesion compared to scrambled control. Data were presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Examination of the effect of CDH6 knockdown on the expression of other targets in Ishikawa cells: (a) other Type II cadherin family members and (b) CDH6 functional partners. Expression levels were normalized to 18S (n = 6). Only CDH24 expression was significantly increased after CDH6 knockdown (at 10 nM siRNA treatment) compared to scrambled control. Data were presented as mean ± SEM. *P < 0.05