Divergent endometrial inflammatory cytokine expression at peri-implantation period and after the stimulation by copper intrauterine device

Chia-Hung Chou, Shee-Uan Chen, Chia-Tung Shun, Po-Nien Tsao, Yu-Shih Yang, Jehn-Hsiahn Yang, Chia-Hung Chou, Shee-Uan Chen, Chia-Tung Shun, Po-Nien Tsao, Yu-Shih Yang, Jehn-Hsiahn Yang

Abstract

Endometrial inflammation has contradictory effects. The one occurring at peri-implantation period is favourable for embryo implantation, whereas the other occurring after the stimulation by copper intrauterine device (Cu-IUD) prevents from embryo implantation. In this study, 8 week female ICR mice were used to investigate the endometrial inflammation, in which they were at proestrus stage (Group 1), at peri-implantation period (Group 2), and had a copper wire implanted into right uterine horn (Group 3). Cytokine array revealed that two cytokines were highly expressed in Group 2 and Group 3 as compared with Group 1, and seven cytokines, including tumour necrosis factor α (TNF-α), had selectively strong expression in Group 3. Immunohistochemistry demonstrated prominent TNF-α staining on the endometrium after Cu-IUD stimulation, and in vitro culture of human endometrial glandular cells with Cu induced TNF-α secretion. The increased TNF-α concentration enhanced in vitro THP-1 cells chemotaxis, and reduced embryo implantation rates. These results suggest that inflammatory cytokine profiles of endometrium are different between those at peri-implantation period and after Cu-IUD stimulation, and TNF-α is the one with selectively strong expression in the latter. It might account for the contradictory biological effects of endometrial inflammation.

Figures

Figure 1
Figure 1
(A) Brief protocols of the three groups are demonstrated. (B) Midline laparotomy is performed and a copper wire with the length of 5 mm and a diameter of 0.3 mm is implanted into the right uterine horn (arrow) in a 8-week-old ICR female mouse for 21 days. (C) The location of copper wire is confirmed with computed tomography (arrowhead). (D) The right uterine horn is dissected and cut open (E), and the endometrium is obtained by curettage.
Figure 2
Figure 2
(A) Signals of cytokine antibody array are shown. (B) Relative expression ratio of Group 2 (at peri-implantation period) and Group 3 (after Cu-IUD stimulation) is calculated using Group 1 (control group) as the benchmark. Using the cutoff value of 1.8, IL-1α and thymus and activation regulated chemokine (TARC) are more strongly expressed both in Group 2 and Group 3 as compared with those in Group 1. By contrast, TNF-α, IFN-γ, IL-1β, monokine induced by gamma interferon (MIG), macrophage inflammatory protein 1β (MIP-1β), MIP-2, and triggering receptor expressed on myeloid cells 1 (TREM-1) are more strongly expressed after the stimulation by Cu-IUD than those in the other two groups.
Figure 3
Figure 3
Haematoxylin and eosin (H & E) stain demonstrates subepithelial lymphocytic infiltration in the endometrium at peri-implantation period (Group 2) and after the stimulation by Cu-IUD (Group 3), but not evident in the control group (Group 1). The lymphocytic infiltration is more prominent in Group 3 than that in Group 2. Immunohistochemisty shows more obvious F4/80 (macrophage) staining (n = 10, p 

Figure 4

( A ) In vitro…

Figure 4

( A ) In vitro treatment of human endometrial glandular cells (hEGCs) with…

Figure 4
(A) In vitro treatment of human endometrial glandular cells (hEGCs) with CuSO4 achieves a dose-dependent release of TNF-α (n = 3). The higher concentrations of CuSO4, the more TNF-α is produced by hEGCs (p < 0.001). (B) Immunohistochemisty reveals prominent TNF-α expression in the endometrium after Cu-IUD stimulation (Group 3), but not in the other two groups.

Figure 5

( A , B )…

Figure 5

( A , B ) In vitro chemotaxis assay (n = 4) shows…

Figure 5
(A,B) In vitro chemotaxis assay (n = 4) shows that conditioned medium (CM) obtained from Cu-treated human endometrial glandular cells (hEGCs) significantly enhances chemotaxis of THP-1 cells, as compared with that obtained from hEGCs without Cu treatment (p < 0.001), and the addition of TNF-α neutralising antibody reduces chemotactic effect (p < 0.001).

Figure 6

BeWo spheroids formed spontaneously by…

Figure 6

BeWo spheroids formed spontaneously by cell aggregation after 24 hours of culture (…

Figure 6
BeWo spheroids formed spontaneously by cell aggregation after 24 hours of culture (B). In vitro BeWo spheroid- human endometrial glandular cells (hEGCs) implantation assay (n = 3) demonstrates that embryo implantation potentials decrease gradually as hEGCs are treated with higher TNF-α concentrations. When hEGCs are treated with 50 (p = 0.003 and 0.011 respectively), 100 (p < 0.001), and 200 (p < 0.001) pg/mL TNF-α, the embryo implantation rates are much lower than those with 0, and 10 pg/mL TNF-α treatment (A,C).

Figure 7

Schematic effects of Cu-IUD reveals…

Figure 7

Schematic effects of Cu-IUD reveals that it stimulates endometrial cells to secrete TNF-α.…

Figure 7
Schematic effects of Cu-IUD reveals that it stimulates endometrial cells to secrete TNF-α. The increased TNF-α concentration results in immune cells aggregation in the endometrium and provokes intense endometrial inflammation, which accordingly interferes with embryo implantation.
All figures (7)
Figure 4
Figure 4
(A) In vitro treatment of human endometrial glandular cells (hEGCs) with CuSO4 achieves a dose-dependent release of TNF-α (n = 3). The higher concentrations of CuSO4, the more TNF-α is produced by hEGCs (p < 0.001). (B) Immunohistochemisty reveals prominent TNF-α expression in the endometrium after Cu-IUD stimulation (Group 3), but not in the other two groups.
Figure 5
Figure 5
(A,B) In vitro chemotaxis assay (n = 4) shows that conditioned medium (CM) obtained from Cu-treated human endometrial glandular cells (hEGCs) significantly enhances chemotaxis of THP-1 cells, as compared with that obtained from hEGCs without Cu treatment (p < 0.001), and the addition of TNF-α neutralising antibody reduces chemotactic effect (p < 0.001).
Figure 6
Figure 6
BeWo spheroids formed spontaneously by cell aggregation after 24 hours of culture (B). In vitro BeWo spheroid- human endometrial glandular cells (hEGCs) implantation assay (n = 3) demonstrates that embryo implantation potentials decrease gradually as hEGCs are treated with higher TNF-α concentrations. When hEGCs are treated with 50 (p = 0.003 and 0.011 respectively), 100 (p < 0.001), and 200 (p < 0.001) pg/mL TNF-α, the embryo implantation rates are much lower than those with 0, and 10 pg/mL TNF-α treatment (A,C).
Figure 7
Figure 7
Schematic effects of Cu-IUD reveals that it stimulates endometrial cells to secrete TNF-α. The increased TNF-α concentration results in immune cells aggregation in the endometrium and provokes intense endometrial inflammation, which accordingly interferes with embryo implantation.

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