Elsevier

Placenta

Volume 36, Issue 12, December 2015, Pages 1405-1411
Placenta

Fibulin-5 is upregulated in decidualized human endometrial stromal cells and promotes primary human extravillous trophoblast outgrowth

https://doi.org/10.1016/j.placenta.2015.10.008Get rights and content

Highlights

  • Fibulin-5 is produced by first trimester decidua and extravillous trophoblast.

  • Fibulin-5 does not promote decidualization.

  • Fibulin-5 does enhance primary human first trimester trophoblast invasion in vitro.

Abstract

Interactions between the highly invasive trophoblasts and the maternal uterine decidual extracellular matrix (ECM) are crucial in the determination of a successful pregnancy. Fibulin-5 (FBLN5) is a member of the fibulin family that alters cell adhesive and invasive properties and is expressed in human villous cytotrophoblasts.

We aimed to determine the expression and immunolocalization of FBLN5 in human first trimester decidua and examine the effect of FBLN5 in trophoblast invasion in vitro using a first trimester placental villous outgrowth assay. We demonstrated that FBLN5 mRNA expression is upregulated in response to cAMP-mediated decidualization of primary human endometrial stromal cells, although FBLN5 itself does not enhance decidualization. We reported for the first time, FBLN5 protein production in first trimester decidual cells and also co-localization to HLAG-positive EVTs in first trimester decidua. Consequently, we investigated the effects of exogenous FBLN5 on placental villous outgrowth in vitro and demonstrated that FBLN5 promotes EVT migration/invasion. This is the first study to identify FBLN5 in decidualized human endometrial stromal cells, first trimester decidua and EVT and determine a functional role for FBLN5 in human EVTs, suggesting that decidual and or EVT-derived FBLN5 regulates EVT invasion and placentation in women.

Introduction

During the establishment of pregnancy, cytotrophoblasts derived from the embryonic trophectoderm proliferate and differentiate into syncytiotrophoblast and highly invasive extravillous trophoblast (EVT) [1]. EVTs migrate into the maternal decidua and myometrium to penetrate the lumen of the spiral arterioles, replacing endothelial cells in a process of endovascular invasion known as ‘spiral artery remodeling’. This creates low-resistance, large-diameter vessels that facilitate the increased blood supply required to sustain fetal growth and development [2]. Initiation of proper placental development therefore depends largely on the proliferative, invasive and/or migratory capability of EVTs [2]. Conversely, abnormal trophoblast function is associated with placental insufficiency and adverse pregnancy outcomes, such as the pregnancy disorders preeclampsia and intrauterine growth restriction (IUGR) [3]. Understanding the critical regulators of trophoblast function could provide much needed insight into the etiology of these disorders.

Interactions between the highly invasive trophoblasts and the maternal uterine decidual environment are crucial in the determination of a successful pregnancy [2], [4]. Prior to embryo implantation, stromal cells of the human uterine endometrium differentiate in the process of ‘decidualization’ during the late secretory phase of the menstrual cycle. Decidualization describes the dramatic morphological transformation of endometrial stromal cells into decidual cells, which become rounded with altered secretory and ECM expression [5]. If implantation occurs, decidualization intensifies and continues to form the decidua of pregnancy [6].

Fibulin-5 (FBLN5) also known as DANCE (developmental arteries and neural crest EGF-Like) or EVEC (embryonic vascular EGF-like repeat containing) is a member of the fibulin family, characterized by calcium-binding epidermal growth factor (EGF)-like repeats and a globular carboxyl-terminal fibulin type structure [7]. The fibulin family comprises six family members (1–6), which are commonly expressed extracellular matrix (ECM) proteins localized to the basement membrane, stroma, and ECM fibers [8]. FBLN5−/− mice do not have any primary fertility defects, although they do exhibit dramatic changes in elastic fiber homeostasis in the uterus and vagina during pregnancy and parturition, resulting in pelvic organ prolapse [9]. Since the placental morphology in these mice has not been examined, this does not provide insight into its role in early pregnancy or placental development. Moreover, we aimed to investigate its role in human placental development, which has numerous differences when compared with the mouse.

FBLN5 was previously identified in first trimester human placenta at the transcript level [10] and protein has since been immunolocalized to the villous cytotrophoblasts and cytotrophoblasts of the proximal cell column [11]. Expression of FBLN5 protein in the non-pregnant human endometrium or uterine decidua during early pregnancy has not been reported. Functionally, FBLN5 regulates cell-to-cell and cell-to-matrix communication and alters ECM structure [7], [12], which are crucial events in the initiation of pregnancy during trophoblast invasion and remodeling of the uterine decidual environment.

In this study, we aimed to determine the expression of FBLN5 in human first trimester decidua and investigate whether FBLN5 promotes primary human endometrial stromal cell decidualization and placental outgrowth in vitro.

Section snippets

Endometrial tissue collection ethics statement

Written informed consent was obtained from each patient and the study was approved by the Southern Health Research and Ethics Committee (#09317B) at Monash Medical Centre Melbourne, Australia.

Endometrial tissue collection

Endometrial biopsies were collected by curettage from women with regular menstrual cycles throughout the menstrual cycle. Biopsies were obtained from women with known normal fertility in late secretory phases of the menstrual cycle (n = 4). All women had regular menstrual cycles of 26–32 days, were not

Statistical analysis

All data are presented as mean ± SEM. Statistical analysis was carried out using GraphPad Prism (GraphPad Software 6.0). Data was assessed by Student's t-test when comparing two groups. Multiple groups were compared using 1-way ANOVA, with Tukey's post-hoc test. Results of p < 0.05 were considered statistically significant.

Fibulin-5 expression is increased in response to cAMP induced decidualization

FBLN5 mRNA was significantly increased in the E + P + cAMP and cAMP treatment groups at day 7 (p < 0.05) (Fig. 1A) and by day 14, FBLN5 mRNA was significantly elevated in the cAMP treatment group, suggesting that FBLN5 expression is cAMP dependant (p < 0.05) (Fig. 1B). Decidualization was confirmed by increased prolactin (PRL) protein secretion levels present at day 7 in the CM in the E + P + cAMP and cAMP treatment groups compared to E + P or control (p < 0.05) (Fig. 1B). At day 14,

Discussion

This is the first study to identify and determine a functional role for fibulin-5 (FBLN5) in human EVTs. We demonstrated that FBLN5 gene expression is upregulated in response to decidualization of primary human endometrial stromal cells, although FBLN5 itself does not enhance decidualization. We reported FBLN5 protein production in first trimester decidual cells and showed that it also co-localized to HLAG-positive EVTs in first trimester decidua. Consequently, we investigated the effects of

Acknowledgments

This work was supported by the Victorian Government's Operational Infrastructure Support Program. ED was supported by NHMRC (Australia) Senior Research Fellowship (#550905). AW and CC were supported by Australian post-graduate Awards. The authors would like to acknowledge the contribution of Sister Judy Hocking for the consenting of patients and collection of tissues. The authors are grateful to Dr. Ellen Menkhorst and Dr. Michelle Van Sinderen for their technical assistance.

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