Characterization of epidermal growth factor-like domain 7 (EGFL7) expression in normal endometrium and in the endometrium of women with poor reproductive outcomes
Authors: Lacconi V , Massimiani M , Paglione L , Antonaci D , Meneghini C , Klinger F G , Fazleabas A , Stuhlmann H , Rago R , Ticconi C , Campagnolo L 
Published: May 2023

Abstract

STUDY QUESTION
Could epidermal growth factor-like domain 7 (EGFL7) be a factor involved in the preparation of the endometrium for implantation and could its dysregulation be implicated in poor reproductive outcomes?

SUMMARY ANSWER
EGFL7 is highly expressed in the endothelium and glandular epithelium throughout the menstrual cycle; it is upregulated by stromal cells in secretory phase and appears strongly reduced in endometrial biopsies and isolated stromal cells of women with unexplained recurrent pregnancy loss (uRPL) and recurrent implantation failure (RIF).

WHAT IS KNOWN ALREADY
The secreted factor EGFL7, originally identified as a gene primarily expressed in endothelial cells, is also expressed by the mouse blastocyst and by mouse and human trophoblast cells. It regulates trophoblast migration and invasion by activating NOTCH1 signaling. NOTCH1 has been demonstrated to play a fundamental role in endometrial receptivity and its dysregulation may be involved in selected pregnancy complications characterized by altered endometrial receptivity, such as uRPL.

STUDY DESIGN, SIZE, DURATION
This is an exploratory study for which 84 endometrial biopsies were collected from normally fertile women, as well as from women with uRPL and RIF.

PARTICIPANTS/MATERIALS, SETTING, METHODS
Samples were collected from women in both the proliferative and secretory phases of the menstrual cycle and stratified into three sub-groups according to the patient clinical history: 20 fertile women (8 in proliferative and 12 in secretory phase), 41 women with uRPL (6 in proliferative and 35 in secretory phase), and 27 women with RIF (8 in proliferative and 19 in secretory phase). Immunohistochemistry, real-time PCR, and western blot analyses were performed to study the expression of EGFL7 and NOTCH1, as well as the NOTCH target genes.

MAIN RESULTS AND THE ROLE OF CHANCE
Analysis of spatial and temporal distribution of EGFL7 in endometrial biopsies from fertile women revealed higher levels of EGFL7 in samples from the secretory phase compared to proliferative phase. The expected expression of EGFL7 in endothelial cells was shown as well as the novel, not previously reported, expression in endometrial glands and stromal cells. EGFL7 was significantly reduced in the endometrium of women with uRPL and RIF in the secretory phases and this was associated with a downregulation of the NOTCH1 signaling pathway. Human recombinant EGFL7 was able to activate the NOTCH1 signaling pathway in endometrial stromal cells (EndSCs) obtained from fertile women but not in cells from uRPL or RIF patients. EndSCs from fertile women and decidualized in vitro for three days showed an upregulation of EGFL7 expression, whereas cells obtained from women with uRPL and RIF and decidualized in vitro did not.

LIMITATIONS, REASONS FOR CAUTION
This study was conducted with a relatively small number of patient samples. Although results are highly reproducible and consistent, additional observations from multicentric cohorts would strengthen the relevance of the data. Moreover, this is an in vitro study, which might only partially represent the in vivo conditions.

WIDER IMPLICATIONS OF THE FINDINGS
Our results demonstrate for the first time that EGFL7 is new player involved in decidualization and provide new insights into the pathophysiology of selected implantation defects and early pregnancy complications. Our studies have revealed that alterations in EGFL7 expression and the consequent dysregulation of NOTCH signaling are potential underlying causes of RIF and uRPL. Our results might have therapeutic relevance, as the EGFL7/NOTCH pathway may represent a potential target for medical intervention.

STUDY FUNDING/COMPETING INTEREST(S)
This study has been supported by the Grant for Fertility Innovation 2017 (Merck KGaA). There are no competing interests to disclose.

TRIAL REGISTRATION NUMBER
Not applicable.