Background
Infertility affects a staggering 1 in 10 couples of reproductive age worldwide [
1]. A failure of embryo implantation is a major cause of infertility. Human embryo implantation starts with the initial contact and adhesion between a blastocyst and the endometrial luminal epithelium [
2,
3]. The inadequate adhesive capacity of the endometrial luminal epithelium leads to inadequate embryo attachment and implantation failure. Defective endometrial adhesion is one of the leading causes of implantation failure and infertility [
4,
5] while the mechanisms contributing to this remain poorly defined.
The endometrial luminal epithelium is only receptive to an implanting blastocyst within a narrow window of 2–4 days in the mid-secretory phase [
6,
7]. Within this time frame, the luminal epithelium undergoes a complex series of changes including the altered expression of adhesive molecules such as cadherins to ensure embryos attach and firmly adhere to initiate implantation [
8]. Cadherins comprise a large family of cell adhesion molecules consisting of more than eighty family members [
9] and are key regulators of cell adhesion, sorting and invasion [
10]. Structurally, cadherins contain repeated extracellular domains that interact generally in a homophilic manner to their own family members to mediate cell-cell interactions, whereas their cytoplasmic regions are associated with a wide range of proteins including cytoskeletal regulators, enzymes and transcriptional factors which endow them with diverse downstream functional capabilities [
10].
A previous high-density microarray screen on human endometrium reveals an increase in the expression of cadherins and associated functional partners in mid-secretory phase, compared to the early-secretory phase [
11]. The localization of the three classical cadherin members, E-cadherin, N-cadherin and P-cadherin have been well characterized. The expression of these three cadherins are mainly restricted to the epithelial cell in the secretory phase, although their localisation specifically in the endometrial luminal epithelium is not reported [
12,
13]. Further studies have revealed a potential dual function in facilitating embryo implantation [
14,
15]. As adhesive molecules, the apical surface expression of cadherins can directly contribute to mechanical cell-cell adhesion. An in vitro functional study in a non-receptive endometrial epithelial cell line AN3-CA demonstrated that forced overexpression of E-cadherin in these cells significantly increased their receptivity to trophoblast-like spheroids formed by the BeWo choriocarcinoma cells [
16]. By contrast, downregulation of E-cadherin in the lateral surface of the uterine epithelium in both human (in vitro) and mouse (in vivo) models may serve as a key mechanism to facilitate the loss of apical-basal polarity in the epithelial layer [
17,
18]. Such change is likely to avoid mutual repulsion between the embryo and the polarized endometrial luminal epithelial surface to facilitate embryo attachment and invasion [
19].
Limited studies have investigated the localization and function of type II cadherin family members in the human endometrium. CDH11 is a predominant cadherin subtype in endometrial stromal cells in the secretory phase [
20]. CDH5 is expressed in the late proliferative phase in endometrial explants [
21] and in endometrial mesenchymal stromal cells [
22]. An in vitro study demonstrated that expression of CDH5 in the mouse trophectoderm facilitates embryo implantation [
23]. A previous characterization study revealed that CDH6 expression is decreased in the glandular epithelium and stromal cells in the receptive phase with no information available on the localization and expression in the luminal epithelium [
24]. Another recent study confirmed that CDH6 immunolocalizes to the apical and lateral cell borders in the endometrial luminal epithelium in the mid-secretory phase [
25]. Further assessment using a receptive endometrial epithelial cell line, Ishikawa cells, indicates that knockdown of
CDH6 in Ishikawa cells at high siRNA concentrations (50 and 100 nM) impact the integrity of Ishikawa cell monolayers compared to low siRNA concentrations (10 and 20 nM) or control siRNA [
25].
To the best of our knowledge, there is no research exploring whether CDH6 plays a role in regulating endometrial epithelial cell adhesive capacity and receptivity and whether it is dysregulated in the endometrium of women with infertility during the receptive window. We examined the clinical relevance of CDH6 on receptivity by determining CDH6 immunostaining levels in mid-secretory phase endometrium from fertile and infertile patients. We used the Ishikawa cells as an in vitro model of endometrial epithelial cells to determine whether siRNA knockdown of
CDH6 compromised their adhesive capacity to HTR8/SVneo trophoblast spheroids. It has been previously identified that in neurons, other cadherins can compensate for the loss of CDH6 to maintain the correct positioning of neurons in the mouse model [
26]. We thus also investigated the effect of
CDH6 knockdown on the expression of other type II cadherin family members and CDH6 functional partners in Ishikawa cells.
Discussion
Adhesive proteins in the endometrial luminal epithelium fulfill essential roles in facilitating embryo attachment. In this study, we demonstrated for the first time that CDH6 reduction was associated with infertile endometrium during the receptive window. We used Ishikawa cells as an in vitro model to explore the functional consequences of CDH6 knockdown on their adhesive capacity to HTR8/SVneo spheroids. We demonstrated that knockdown of CDH6 significantly reduced the spheroid adhesion compared to scrambled control under 50 nM condition and did not affect the expression of other type II cadherin family members or CDH6 functional partners, indicating CDH6 has a non-redundant role in regulating endometrial receptivity.
CDH6 belongs to type II classical cadherin and it regulates cell adhesion via a homophilic binding to its own family members. In order to regulate embryo adhesion, cadherin family members are required to be expressed in both endometrial epithelial cells and blastocyst trophectoderm. qPCR analysis from our study revealed that all type II cadherin family members were expressed in the Ishikawa cells. Gene expression analysis of trophectoderm isolated from preimplantation human embryos demonstrates all type II cadherin family members are expressed in the trophectoderm [
35]. This indicates that all type II classical cadherins are able to directly regulate embryo adhesion onto the endometrial luminal epithelium. Our data showed that although lower concentrations of
CDH6 siRNA treatment (10 and 20 nM) in the Ishikawa cells did not compromise spheroid adhesion, 50 nM
CDH6 siRNA treatment significantly reduced their adhesive capacity to HTR8/SVneo spheroids suggesting that either the level of CDH6 was critical for adhesion or the existence of other type II classical cadherins were not able to compensate for the severe loss of CDH6. It is not unusual that cadherin family members play non-redundant roles in cell adhesion and invasion. A previous study in mouse embryos reveals a unique role of E-cadherin in regulating the formation of polarized functional trophectoderm by demonstrating that the replacement of E-cadherin by N-cadherin is not sufficient to form an intact trophectoderm [
36]. After embryo attachment, the trophectoderm differentiates into trophoblast and starts to invade into the uterus. It has been revealed that in the invasive front of extravillous cytotrophoblasts, CDH6 is the predominant cadherin subtype and its expression promotes the cell invasive capacity [
20,
37].
This leads to an intriguing notion of cadherin expression that has been recorded during epithelial to mesenchymal transition in embryonic development, namely the cadherin switch [
38]. During this process, the expression of the epithelial cell marker E-cadherin is reduced whereas the expression of another cadherin family member N-cadherin is increased. Such an expression switch coincides with a morphological and phenotypic transition of the pre-migrating cells [
39]. A similar change in cadherin expression has also been recorded in tumor cells. Loss of E-cadherin and increased expression of N-cadherin and CDH11 in the human prostate cancer cells is able to change the invasive capacity and metastasis of the cells [
40]. In the mid-secretory phase human endometrium, in contrast to the localization of CDH6 in the apical and lateral surface of endometrial luminal epithelium, E-cadherin protein expression is reduced and shows minimal levels [
41]. Such an expression reduction has been proven essential for embryo implantation and invasion in mice [
17]. It is likely that selective changes in expression of E-cadherin and CDH6, along with other notable cadherin family members that have been reported in the human endometrium [
12,
13,
20] contribute to a functional transition that is required for successful embryo implantation.
A particular curiosity of this hypothesis is how the differential expression of the same cadherin family members lead to a functional transition in endometrial epithelial cells. One possible explanation is that different family members may have different isoforms. The isoform differences enable even the same cadherin family member to have different functional capacities. It has recently been demonstrated that CDH6 has two isoforms that are inherently different: CDH6-long isoform and CDH6-short isoform [
37]. The difference between these two isoforms is that the CDH6-short isoform does not have an intracellular domain to interact with its functional partners [
37] and it may lose the ability to activate downstream signaling pathways. It is likely that this isoform only functions as an adhesive molecule during cell-cell interactions. The antibody we used in this study binds to the extracellular domain thus recognizes both isoforms. We remain uncertain if these two isoforms co-exist in the endometrial luminal epithelium to regulate different functional aspects of the implantation process. Different isoforms of cadherin family members may fulfill specific functional requirements within a specific cell type or development stage.
Previous studies have also proposed a cellular ‘redistribution’ theory that may contribute to the functional complexity of the cadherins. In the human endometrium, the lateral surface expression of CDH6 is essential to form the adherens junctions to maintain the integrity of the endometrial luminal epithelium before implantation. Once the embryo contacts the endometrial luminal epithelium, the lateral adherens junction proteins such as CDH6 may redistribute to the apical surface to facilitate the attachment of the embryo and such redistribution may break down the adherens junctions to allow or facilitate embryo invasion [
25,
42]. The redistribution of CDH6 also releases the cadherin functional partners such as catenin proteins that are required to form adherens junctions. Another role of catenin proteins is to participate in Wnt/β-catenin (CTNNB1) signaling that is essential for implantation in vivo in mice [
43]. Wnt/β-catenin (CTNNB1) signaling activation similarly requires the local stimulus of the embryo [
43] which supports the ‘redistribution’ theory. Our immunohistochemistry data revealed that CDH6 was downregulated in the apical and lateral surfaces of the endometrial luminal epithelium in the infertile endometrium during the mid-secretory phase suggesting CDH6 affects both adhesion and integrity of the endometrial luminal epithelium.
Several mechanisms can reduce CDH6 expression in the infertile endometrium. microRNA downregulates gene expression [
44] and our previous studies reveal that specific microRNAs are increased in the infertile human endometrium and affect endometrial adhesive capacity and receptivity by downregulating essential gene targets [
30,
31].
CDH6 is a direct gene target of miR-223-3p and forced overexpression of miR-223-3p in cultured osteosarcoma cells reduces
CDH6 expression and inhibits cell invasion and migration [
45]. miR-223-3p also suppresses the expression of leukemia inhibitory factor (
LIF) during the implantation window in mouse uterus [
46], a target that is essential for embryo implantation in both humans and mice [
47,
48]. A previous study confirms the upregulation of miR-223-3p in human endometrium with a compromised receptivity phenotype and its interaction with
LIF [
49]. In support, in our recent study we reveal that miR-223-3p is detected at low levels in the primary fertile human endometrial epithelial cells [
50]. However, whether
CDH6 is directly regulated by miR-223-3p in the infertile human endometrium is unknown. Similarly,
CDH6 may be downregulated in infertile endometrium epigenetically by DNA methylation. An in vitro study on human endometrial epithelial cell line AN3-CA has confirmed that E-cadherin gene expression is negatively controlled by DNA methyltransferase enzymes. Inhibition of these enzymes increases the expression of E-cadherin and switch the non-receptive endometrial epithelial cells to become receptive to trophoblast-like spheroids formed by the BeWo choriocarcinoma cells [
16]. In support, an in vivo mouse model has revealed that increased DNA methylation on the promoter of homeobox A10 (
Hoxa10) reduces the expression of
Hoxa10 and directly affects endometrial receptivity [
51]. There are no studies that have investigated whether the downregulation of
CDH6 is related to increased DNA methylation in any biological system. Whether this is the case in the infertile endometrium warrants further investigation.
Ishikawa cells transfected with 50 nM of
CDH6 siRNA did not compromise cell integrity which is in contrast to a previous report [
25]. This may have been due to methodological differences between the two studies. One notable difference in our study is that we transfected the Ishikawa cells at 70–80% confluency while in the previous report the cells were transfected at 40–50% confluency. After transfection we cultured the Ishikawa cells for 48 h while in the previous study, the Ishikawa cells were cultured for up to 120 h which may have severely impacted the cell integrity.
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