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.

Tripeptidyl peptidase I promotes human endometrial epithelial cell adhesive capacity implying a role in receptivity

“…Immunohistochemistry for TPP1 was performed on formalin-fixed endometrial tissue as described [ 12 ]. Fixed 4 μm sections on tissue slides were rehydrated and then antigen retrieval performed by microwave heating in 10 mM sodium citrate buffer for 5 min.…”

“…Staining intensity scores were determined by two individual scorers blinded to the patient characteristic and cycle stage of each section, as previously described [ 12 ]. Briefly, scores were given to each tissue type (luminal epithelium, glandular epithelium, stroma) on a scale of 0–3 (0: no stain, 3: strong stain).…”

“…Ishikawa endometrial epithelial cells were provided by Dr. M. Nishida (Tsukuba University, Tochigi, Japan) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal calf serum (FCS; Gibco). Ishikawa cells are receptive endometrial epithelial cells possessing apical adhesiveness, as such are commonly used to study endometrial receptivity [ 12 , 22 ] as it has similar characteristics to endometrial luminal and glandular epithelium, including the expression of steroid hormone receptors and possess apical cell adhesiveness [ 23 ]. The HTR8/SVneo trophoblast cell line (CRL-3271) exhibits features of invasive trophoblast cells, such as human leukocyte antigen-G (extravillous trophoblast marker) and cytokeratin-7 expression [ 23 ].…”

“…Spheroid number per well was counted and recorded using a light microscope before incubation at 37 °C for 2 h. Following incubation, wells were gently washed with serum-free DMEM/F12 to remove non-adherent spheroids. The remaining spheroids were counted, and a percentage of adhered spheroids calculated [ 12 , 24 ]. For qPCR analysis, Ishikawa cells were washed to remove spheroids then collected in TRI reagent for RNA extraction.…”

“…Additionally, endometrial stromal cells isolated from women with infertility do not decidualize as effectively in vitro as stromal cells isolated from women with infertility [ 7 ]. Several cytokines, chemokines, growth factors, matrix metalloproteases and adhesion molecules all have important contributions to the implantation process as well as processes preceding it [ 8 – 12 ]. Information on lesser known proteases such as tripeptidyl peptidase I (TPP1) are lacking.…”