Matrix metalloproteinase 15 plays a pivotal role in human first trimester cytotrophoblast invasion and is not altered by maternal obesity

Abstract

Adequate anchoring of the placenta in the uterus through invasion of first trimester cytotrophoblasts (CTB) is required for a successful pregnancy. This process is mediated by matrix metalloproteinases (MMPs) and regulated by the maternal environment. Obesity is known to alter the intrauterine milieu and has been related to impaired invasion. We hypothesized that placental MMP15, a novel membrane-type MMP, is involved in CTB invasion and regulated by maternal obesity in early pregnancy. Thus, in this study MMP15 was immunolocalized to invasive extravillous and interstitial CTB. MMP15 silencing in chorionic villous explants using two different siRNAs reduced trophoblast outgrowth length (-35%, P ≤ .001 and -26%, P < .05) and area (-43%, P ≤ .001 and -36%, P ≤ .01) without altering trophoblast proliferation or apoptosis. Short-term treatment of primary first trimester trophoblasts with IL-6 (10 ng/mL), interleukin 10 (IL-10) (50 ng/mL), and tumor necrosis factor α (TNF-α) (10 ng/mL) did not affect MMP15 protein levels. Likewise, MMP15 mRNA and protein levels were unaltered between human first trimester placentas from control pregnancies vs those complicated with maternal obesity. Overall, our results suggest that the role of MMP15 in placental development and function in early pregnancy is limited to CTB invasion without being affected by short- and long-term inflammation.

Keywords: early pregnancy; inflammation; placenta; trophoblasts.

FIGURE 1

Extravillous (eCTB) and interstitial cytotrophoblasts (iCTB) are the main source of MMP15 in human early pregnancy. MMP15 immunofluorescence staining was performed in serial sections of human placental tissue (GW 6‐10) and decidua basalis (GW 7‐10). MMP15 localized to eCTB in the cell columns (CC, A and D) as well as to iCTB invading through the decidua (G). Villous cytotrophoblasts (vCTB) and syncytiotrophoblast (STB) were identified by cytokeratin 7 (K7) immunostaining (B and C). eCTB and iCTB were further identified by positive immunostaining for HLA‐G (E and H). Image overlay showed placental and decidual MMP15 and HLA‐G co‐localization (F and I). Nuclei were counterstained with DAPI. Scale bar: 100 µm (A‐F) and 50 µm (G‐I)

FIGURE 2

MMP15 silencing decreases trophoblast outgrowth in first trimester chorionic villous explants. Dissected chorionic villi from human first trimester placentas (GW 6‐10) were incubated with non‐targeting (NT)‐siRNA (control, A) or with two different MMP15‐specific siRNA (si5‐ and si6‐siRNA, C and E) for 24 h. Trophoblast outgrowth was monitored for 72 h, and quantified as the length between the villous tip and the front of the outgrowing sheet (dotted lines, B, D, and F), or as the outgrowth area (solid line, B, D, and F). Results were calculated relative to NT‐siRNA (G and H), arbitrarily set to 1, and expressed as mean ± SEM. Knockdown efficiency was validated by MMP15 (I) and MMP14 (J) RT‐qPCR, with HLA‐G and β‐actin as housekeeping genes, respectively. n = 4 different placentas, 3‐11 villi for each condition. *P < .05; **P ≤ .01; ***P ≤ .001 vs NT‐siRNA

FIGURE 3

MMP15 silencing does not affect trophoblast proliferation and apoptosis in first trimester chorionic villous explants. Dissected chorionic villi from human first trimester placentas (GW 6‐10) were incubated for 24 h with non‐targeting (NT)‐siRNA (control) or with two different MMP15‐specific siRNA (si5‐ and si6‐siRNA). After 72 h, trophoblast proliferation and apoptosis were determined by Ki67 (A‐C) and caspase‐cleaved cytokeratin 18 (cleaved K18, E‐G) immunofluorescence staining, respectively. Results were calculated as percentage of Ki67 and cleaved K18 positive cells (arrows) relative to the number of cytokeratin 7 (K7) positive cells (D and H). Data are expressed as mean ± SEM. n = 4 different placentas, 2‐3 cell columns for each condition

FIGURE 4

MMP15 is unaffected by short‐term exposure to obesity‐associated pro‐inflammatory cytokines. Primary first trimester trophoblasts (GW 7‐9) were incubated in the absence (control) or the presence of IL‐6 (10 ng/mL), IL‐10 (50 ng/mL), or TNF‐α (10 ng/mL). MMP15 protein levels (pro‐MMP15, active (act)‐MMP15, and total‐MMP15: pro + act‐MMP15) were determined by Western blotting (A). Results were normalized to HLA‐G protein levels and expressed as mean ± SEM (B). n = 5 different placentas, each treatment was assayed in duplicates

FIGURE 5

MMP15 is unaffected by long‐term exposure to obesity‐associated intrauterine environment. MMP15 expression and protein levels (pro‐MMP15, active (act)‐MMP15, and total‐MMP15: pro + act‐MMP15) were determined in first trimester placental tissue from lean (GW 5‐11, n = 24) and obese (GW 5‐10, n = 18) women by RT‐qPCR (A) and Western blotting (B), respectively. Results were normalized to HLA‐G expression (−ΔCt) and protein levels and expressed as mean ± SEM. GA, gestational age (days)