Effects of a gestational level of estradiol on cellular transition, migration, and inflammation in cervical epithelial and stromal cells

Abstract

Problem Estrogen (E2) is one of the main steroid hormones associated with pregnancy and parturition. High levels of E2 increase uterine contractions, promote fetal membrane weakening, and induce degradation of the cervical extracellular matrix (ECM). Current evidence supports the role of E2 in epithelial‐to‐mesenchymal transition (EMT) and inflammation in different cell types; however, its effects on the cellular components of the cervix are still unknown. Method of study In this study, we assessed the effects of gestational levels of E2 in: (a) the cellular transition of endocervical epithelial cells (EEC) and cervical stromal cells (CSC) in vitro using immunocytochemical staining and Western blot analyses for EMT markers (cytokeratin‐18, E‐cadherin, N‐cadherin, SNAIL, and vimentin); (b) cell migration using in vitro scratch assays; (c) inflammatory cytokine (interleukin 1β and TNF‐α) and MMP9 production under untreated and lipopolysaccharide (LPS)‐treated conditions using immunoassays. Results E2 treatment and co‐treatment with LPS as a proxy for infection maintained the metastate of EEC (expression of both cytokeratin and vimentin) and the mesenchymal state of CSC. E2 delayed wound healing, which mimics the tissue remodeling process, in EEC and CSC. E2 led to persistently elevated levels of vimentin throughout the EEC wound healing process. E2 did not affect inflammatory cytokine production by EEC and CSC but increased MMP9 production by EEC. Conclusion Collectively, these results show that third trimester levels of E2 may permit localized inflammation, increase MMP‐9 production, and cause an EMT‐mediated impairment of the remodeling process in the cervix in vitro. These data suggest a potential contribution of E2 in cervical ripening.

Organ‐on‐chip of the cervical epithelial layer: A platform to study normal and pathological cellular remodeling of the cervix

“…The immortalized ectocervical and endocervical epithelial cells used in this study were previously characterized and were shown to have conserved toll‐like receptor (TLR) expression when compared to primary ectocervical and endocervical epithelial cells 25,26 . The cells were immortalized by transduction with PA317⁄LXSN‐16E6E7‐conditioned medium (HPV16 E6/E7 immortalization) and cultured in keratinocyte serum‐free medium (KSFM) (37010022; Thermo Fisher Scientific), a culture medium that is highly selective for epithelial cells, supplemented with bovine pituitary extract (30 μg/mL), epidermal growth factor (0.1 ng/mL), CaCl 2 (0.4 mM), and primocin (0.5 mg/mL) (ant‐pm‐1; Invivogen; San Diego, CA) at 37°C in a 5% CO 2 environment, and grown to 80% confluence.…”

“…Immunocytochemical staining for vimentin (3.7 µL/mL; ab92547; Abcam, Cambridge, MA, USA) and cytokeratin‐18 (CK‐18; 1 µL/mL; ab668; Abcam) was performed after 96 hours, as previously described 26,29 . Manufacturer’s instructions were used to calculate the staining dilutions to ensure uniform staining.…”

“…were shown to have conserved toll-like receptor (TLR) expression when compared to primary ectocervical and endocervical epithelial cells. 25,26 The cells were immortalized by transduction with PA317⁄LXSN-16E6E7-conditioned medium (HPV16 E6/E7 immortalization) and cultured in keratinocyte serum-free medium (KSFM) (37010022; Thermo Fisher Scientific), a culture medium that is highly selective for epithelial cells, supplemented with bovine pituitary extract (30 μg/mL), epidermal growth factor (0.1 ng/ mL), CaCl 2 (0.4 mM), and primocin (0.5 mg/mL) (ant-pm-1; F I G U R E 1 CE-OOC design and cell culture within. The CE-OOC is designed to recapitulate the cervical epithelial layer in vitro by coculturing ectocervical and endocervical epithelial cells separated by type IV collagen-filled microfluidic channels through which cells can migrate to form the transformation zone epithelium of the cervix.…”

Breaking Down the Barrier: The Role of Cervical Infection and Inflammation in Preterm Birth

“…We have also shown that infection and inflammation can promote EMT in cervical epithelial cells ( 81 , 86 , 87 ). EMT was shown to be involved in the cervical changes that happen during parturition.…”

Epithelial to mesenchymal transition (EMT) of feto-maternal reproductive tissues generates inflammation: a detrimental factor for preterm birth

“…Cervix is composed of the epithelial and the stromal layer. The epithelial layer lining the cervical canal is divided into three distinct regions: the ectocervix, the transformation zone, and the endocervix, while the stromal layer is composed mainly of the extracellular matrix (ECM) incorporating fibroblasts, immune, and smooth muscle cells ( 44 , 88 - 91 ). As mentioned above, cervical remodeling is a required process to maintain pregnancy and like fetal membranes, multitudes of cellular matrix remodeling are needed to maintain cervical integrity, maintenance of intact tissue, and natural production of antimicrobial peptides to reduce the influx of vaginal microbiome and provide the barrier functions.…”