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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013.

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The Role of Maspin in Human Placental Development

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The role of maspin in the human reproductive tract is unknown. The human placenta is a physiological site of invasion where cytotrophoblasts invade into maternal tissues during gestation. This process is spatially and temporally regulated, but the role of tumor suppressor genes in this invasive model has not been studied in detail. We have examined the expression of maspin in human placenta during all three trimesters of pregnancy and hypothesize a potential role for maspin in regulating invasion at this unique site.

The human placenta is hemochorial and displays highly regulated invasive activity and exponential growth potential. The stem cell cytotrophoblasts undergo differentiation along two pathways: they fuse to form multinucleate syncytiotrophoblasts or they detach from the basement membrane to form mononuclear cell columns and anchoring villi that invade into the endometrium, myometrium, and spiral arteries. Thus, cytotrophoblast invasion physically anchors the fetus to the mother. The depth of placental invasion is precisely controlled and temporally regulated, and abnormalities in invasion can have clinically relevant consequences. For example, shallow invasion of cytotrophoblasts has been reported in conditions such as preeclampsia and intrauterine growth retardation.1,2 Increased invasion is associated with conditions such as placenta accreta and placenta increta, which are both associated with postpartum hemorrhage. Despite the extensive spatial invasion, the risk of developing an invasive mole or choriocarcinoma remains low (1 in 20,000–40,000 pregnancies).

Regulation of Cytotrophoblasts Invasion

The dynamic process of invasion demonstrated by extravillous cytotrophoblasts appears to be regulated by similar mechanisms as have been described in cancer cells: extracellular matrix (ECM) degrading enzymes, matrix metalloproteases (MMPs), adhesion molecules (cadherins), ECM receptors (integrins), and cytokines. The invasive capacity of cytotrophoblasts, as assessed in vitro, decreases from the first to the third trimester, mimicking the in vivo situation.3 The stem cell cytotrophoblast populations express high levels of E-cadherin and α6β4 integrins.4 During the process of invasion into the maternal surface, the extravillous cytotrophoblasts down-regulate their expression of E-cadherin and up-regulate α1β1 integrins (laminin receptor). Further, invading cytotrophoblasts upregulate the expression of MMPs, especially MMP-9.5 More importantly, during the first trimester both inhibitors of MMPs, TIMP and TIMP-2, have been shown to decrease cytotrophoblast invasion in vitro. MMPs and their inhibitors are also regulated in the first trimester placenta by the cytokines IL-1β,6 TGFβ1 and TGFβ27 and EGF.3

Tumor Suppressor Genes in the Human Placenta

A few tumor suppressor genes have been detected in the human placenta including p53,8 WT19 and maspin.10 In the placenta, maspin is localized to cytotrophoblasts in chorionic villi.10 However, there is no information in the literature regarding the putative biological role of any of the above tumor suppressor genes in the human placenta or the female reproductive tract. Maspin (mammary serine protease inhibitor) is a tumor suppressor gene which has been shown to have inhibitory actions on motility, invasion, metastasis, and angiogenesis in human breast and prostate cancer cells.11,12 Loss of maspin expression in these cancers correlates with tumor invasiveness and breast cancer recurrence. Further, treatment of human breast and prostate cancer cells with recombinant maspin (rMaspin) inhibited cell motility.13 These observations suggest that maspin expression plays an important role in regulating tumor cell invasion and metastasis.

Maspin in the Human Placenta

We were interested in examining the role of maspin in regulating cytotrophoblast invasion and therefore determined the expression of maspin in human placenta obtained after first- and second-trimester terminations (7–20 weeks) and after delivery by cesarean section at term (37–40 weeks). Maspin mRNA expression was maximally detected in third-trimester placentae by semi-quantitative RT-PCR, as compared to first- and second-trimester placental tissues (Fig. 1A). Similar results were obtained when cytotrophoblasts isolated by sequential enzymatic digestion (first- and second-trimester isolation,14 third trimester15) were examined. In contrast, expression of maspin was not detected in the invasive extravillous cytotrophoblast cell line derived from a first-trimester placenta (HTR-8/SVneo, kind gift from Dr. Charles Graham, Canada) nor in the choriocarcinoma cell lines (JEG, JAR) (Fig. 1A).

Figure 1. (A) Maspin mRNA expression in human placenta.

Figure 1

(A) Maspin mRNA expression in human placenta. Total RNA from human placentae from first (T1), second (T2), and third (T3) trimesters, HTR-8/SVneo (SV), JEG (J1) and JAR (J2) and MDA-MB-231 (invasive breast cancer cell line used as a negative control) (more...)

Consistent with maspin mRNA expression, maspin protein was also maximally detected by Western blot in cell lysates from cytotrophoblasts isolated from term placentae (Fig. 1B). The level of maspin protein was 2-fold higher in the second trimester compared to the first trimester, and 4.5-fold higher in the third trimester compared to the first trimester, as measured by densitometric analysis. In addition, immunohistochemistry was used to localize maspin in placental tissues using a monoclonal mouse anti-human maspin antibody (Pharmingen, San Diego, CA). In the first trimester, maspin staining was patchy and restricted to the cytotrophoblast layer with no staining detected in the syncytiotrophoblast layer (Fig. 2A). There was more uniform staining in the syncytiocytotrophoblast layers later in gestation with maximum staining in the third trimester (Fig. 2B).

Figure 2. Immunohistochemistry of human placentae with mouse anti-human maspin monoclonal antibody.

Figure 2

Immunohistochemistry of human placentae with mouse anti-human maspin monoclonal antibody. (A) First-trimester placenta shows faint positive staining for the cytotrophoblast layer (Cy) and occasional cells in the mesenchymal villous core (V). (B) Third (more...)

The invasive ability of cytotrophoblasts from all three trimesters was assessed using an in vitro invasion assay, which measured the ability of the cells to traverse a defined human basement membrane matrix over 60 hours using membrane invasion culture system (MICS) chambers.16 Cytotrophoblasts isolated from the first (mean±SE: 695±37/filter) and second (677±50/filter) trimesters demonstrated a significantly higher invasive ability as compared to those isolated from term placentae (298±77, p<0.02). The invasive ability of both HTR-8/SVneo (16264±1117, p<0.004) and the choriocarcinoma cell line, JEG (2943±634, p<0.03), was significantly higher when compared to first-trimester cytotrophoblasts.

Next, we examined the direct effects of rMaspin (Arriva Pharmaceuticals Inc, Alameda, CA) on cytotrophoblast invasion using the same in vitro invasion assay system. There was a dose-dependent decrease in the invasive ability of cytotrophoblasts in all three trimesters receiving rMaspin treatment for 24 hours (Table 1). Upon the addition of 60 μg/ml rMaspin to the test wells, the first- and second-trimester cytotrophoblasts showed a 50% decrease and the third-trimester cytotrophoblasts showed a 40% decrease in invasive ability when compared to the untreated controls.

Table 1. Effects of recombinant maspin on cytotrophoblast invasion in vitro.

Table 1

Effects of recombinant maspin on cytotrophoblast invasion in vitro.


Our data demonstrate for the first time that maspin, a tumor suppressor gene, is differentially expressed in the human placenta and maximally expressed in the third trimester. Furthermore, the in vitro invasion experiments demonstrate that cytotrophoblasts isolated from the third trimester have the least invasive ability when compared to cytotrophoblasts isolated from first and second trimesters. These results indicate that the decreased expression of maspin in the first trimester corresponds to the period of maximum cytotrophoblast invasion as shown in vitro. This hypothesis is further supported by the absence of maspin expression in the invasive cell lines, HTR-8/SVneo and JEG. In addition, we detected a 40–50% decrease in invasive ability with the addition of rMaspin to cytotrophoblasts, as compared to controls. These data suggest that maspin may play a role in the regulation of cell invasion at a physiological site, the human placenta.

Studies from our laboratory have previously shown that treatment of highly invasive breast cancer cells with recombinant maspin causes increased surface expression of the α5 integrin and down-regulation of MMPs, which converts these cells to a more benign epithelial phenotype.17 Interestingly, first-trimester cytotrophoblasts cultured in vitro rapidly change their integrin repertoire to mimic invasive (α1β1) rather than stem cell cytotrophoblasts (α6β4).4 However, the authors reported that this ability of the cells to differentiate along the invasive pathway was lost in cytotrophoblasts isolated from term placentae. Hence it is possible that increased expression of maspin at term may regulate cytotrophoblast invasion by altering the surface integrin expression of these cells. The hierarchical role of the above-described factors including tumor suppressor genes, cytokines, MMPs, and integrins in human placental development in vivo remains to be determined. The down-regulation of maspin expression may be critical at the time of implantation and early placental development, whereas up-regulation of maspin may provide a signal for cytotrophoblasts to decrease invasive activity at the end of gestation.


This work was supported by a grant to Anuja Dokras from the Reproductive Scientist Development Program through NIH grant #5K12HD00849 and the ASRM.


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