hCG may favor embryonic implantation and early growth by … · Ali Akoum1, Amélie Bourdiec1,...
1
hCG may favor embryonic implantation and early growth by modulating endometrial cell receptivity to major embryonic signals and targeting different cell types Ali Akoum 1 , Amélie Bourdiec 1 , Mahéra Al-Akoum 1 , Rong Shao 2 , CV Rao 3 1 Obstetrics and Gynecology, Faculty of Medicine, Laval University, Quebec, Qc, Canada; 2 Pioneer Valley Life Sciences Institute, University of Massachusetts Amherst, Springfield, MA, USA; 3 Herbert Wertheim College of Medicine, Reproduction and Development Program, Florida International University, Miami, FL, USA. What is known already Results Conclusions Acknowledgments Endometrial tissue specimens were obtained from normal fertile women with a regular menstrual cycle during the implantation window. Cultures of primary endometrial stromal cells and human microvascular endothelial cells (HMVEC) line were exposed to various doses of hCG, IL1B or a combination of these factors for different time periods. Expression of IL1 receptors (IL1R) and angiogenic factors in hCG and/or IL1-treated cells was determined by real-time PCR, Western blot and ELISA. Cell proliferation and migration was evaluated using BrdU incorporation into cell DNA and wound healing assay. Human Gene 1.0 ST arrays (Affymetrix) was used for microarray analysis. For the first time, a close interaction between hCG and IL1B was found in signaling to uterine stromal cells in vitro. hCG appeared to amplify ESC responsiveness to IL1B and to enhance the expression and release of MCP1, which is shown to stimulate endothelial cell proliferation and migration. Microarray study showed that hCG induces major changes in human ESC phenotype and deeply modulate their responsiveness to a IL1B. Generally via synergistic stimulatory effect, hCG induces significant alterations in the expression of genes known for being involved or having the potential to play an important role in embryonic implantation and remodeling environment around the implanting blastocyst. Together with our previous data using human primary endometrial epithelial cells, our current findings with HMVEC and human endometrial stromal cells, make plausible that the embryo can directly target, interact with and modulate the receptivity of different cell types. Investigations are underway to assess the impact of hCG on human uterine endothelial cells. Our findings are relevant to the physiology of embryo implantation, as IL1B and hCG are both produced by embryos, and broadens the spectrum of hCG’s impact on early embryonic growth and development. The next step in our study is to identify the signaling pathways involved in the angiogenic synergy induced by hCG and IL 1. Our first in vivo data showed a comparable modulation of IL1Rs and MCP1 expression in the endometrium at the embryo implantation sites. During the crucial period of embryo implantation, an intricate network of maternal and embryonic signals coordinates embryo-maternal interaction and leads to timely production of growth,immune- modulating and tissue remodeling factors. Our previous studies provided original evidence that human Chorionic Gonadotropin (hCG) modulates and potentiates endometrial epithelial as well as stromal cell responsiveness to interleukin 1 (IL1), one of the earliest embryonic signals. hCG triggers angiogenesis via the modulation of endometrial stromal cell responsiveness to IL1 IL1R1/ IL1R1 mRNA % of control IL1R2 mRNA % of control IL1RN mRNA % of control sIL1R2 release % of control CCL2 secretion % of control Sample IgG CTRL MAB 279 (anti-MCP1) ESC Supernatant MM 48h CCL2 (MCP1) ESC Supernatant hCG100/IL1 0.1 CCL2 CCL2 CCL2 sIL1RN release % of control sIL1R2 release % of control CCL2 (MCP1) mRNA % of control CCL2 (MCP1) release % of control IL1R2/IL1R1 mRNA 1. Imbalance of IL1Rs and increased responsiveness to IL1 2. Overexpression of IL1R2 in ESC 3. Increased endothelial cell migration and proliferation Transcriptome analysis reveals deep modulation of endometrial stromal cell phenotype by embryo‐derived signals hCG/IL1 5. Validation of main regulated genes Enrichment Score of Biological Processes 4. Hierarchical clustering, Principal Component Analysis (PCA)and Gene Ontology (GO) annotations hCG regulates endothelial cell responsiveness to interleukin 1 6. Proliferation and Migration 7. HMVEC responsiveness to IL1 and IL1R2 overexpression 8. Expression in embryo implantation sites hCG IL1 Blastocyst Epithelium Capillary (endothelial cells) ESC We thank Mrs. Nathalie Bourcier and Monique Longpré for patient recruitment and precious technical assistance. We thank HSFA and CHUL gynecologists for patient evaluation and providing endometrial tissue samples. Supported by an NSERC Discovery Grant to Pr. Ali Akoum, National Researcher, "Fonds de la Recherche du Québec-Santé". Study question To assess whether hCG modulates endometrial cell responsiveness to IL1 by targeting different cell types and gain a broader understanding of the impact of such a mechanism on the acquisition of growth-promoting phenotype. Study design and methods IL1R1 (signaling receptor) IL1R2 IL1RN Inhibitory receptor/antag. Pro-angiogenic effect hCG IL1R2 gene transfer and IL8 secretion IL1Rs in endometrium during gestation in vivo Pregnant mouse uterus d6 Non-pregnant mouse uterus
Transcript of hCG may favor embryonic implantation and early growth by … · Ali Akoum1, Amélie Bourdiec1,...
hCG may favor embryonic implantation and early growth by modulating endometrial cell receptivity to major embryonic
signals and targeting different cell types
Ali Akoum1, Amélie Bourdiec1, Mahéra Al-Akoum1, Rong Shao2, CV Rao31Obstetrics and Gynecology, Faculty of Medicine, Laval University, Quebec, Qc, Canada; 2Pioneer Valley Life Sciences Institute, University of Massachusetts Amherst, Springfield, MA, USA; 3Herbert Wertheim College of Medicine,
Reproduction and Development Program, Florida International University, Miami, FL, USA.
What is known alreadyResults Conclusions
Acknowledgments
Endometrial tissue specimens were obtainedfrom normal fertile women with a regularmenstrual cycle during the implantation window. Cultures of primary endometrial stromal cellsand human microvascular endothelial cells(HMVEC) line were exposed to various doses ofhCG, IL1B or a combination of these factors fordifferent time periods. Expression of IL1 receptors (IL1R) andangiogenic factors in hCG and/or IL1-treatedcells was determined by real-time PCR, Westernblot and ELISA. Cell proliferation and migration was evaluatedusing BrdU incorporation into cell DNA andwound healing assay. Human Gene 1.0 ST arrays (Affymetrix) wasused for microarray analysis.
For the first time, a close interaction between hCG andIL1B was found in signaling to uterine stromal cells invitro. hCG appeared to amplify ESC responsiveness toIL1B and to enhance the expression and release of MCP1,which is shown to stimulate endothelial cell proliferationand migration.
Microarray study showed that hCG induces majorchanges in human ESC phenotype and deeply modulatetheir responsiveness to a IL1B. Generally via synergisticstimulatory effect, hCG induces significant alterations inthe expression of genes known for being involved orhaving the potential to play an important role inembryonic implantation and remodeling environmentaround the implanting blastocyst.
Together with our previous data using human primaryendometrial epithelial cells, our current findings withHMVEC and human endometrial stromal cells, makeplausible that the embryo can directly target, interactwith and modulate the receptivity of different cell types.Investigations are underway to assess the impact of hCGon human uterine endothelial cells.
Our findings are relevant to the physiology of embryoimplantation, as IL1B and hCG are both produced byembryos, and broadens the spectrum of hCG’s impact onearly embryonic growth and development. The next stepin our study is to identify the signaling pathways involvedin the angiogenic synergy induced by hCG and IL 1.Our first in vivo data showed a comparable modulationof IL1Rs and MCP1 expression in the endometrium at theembryo implantation sites.
During the crucial period of embryo implantation,an intricate network of maternal and embryonicsignals coordinates embryo-maternal interactionand leads to timely production of growth, immune-modulating and tissue remodeling factors. Ourprevious studies provided original evidence thathuman Chorionic Gonadotropin (hCG) modulatesand potentiates endometrial epithelial as well asstromal cell responsiveness to interleukin 1 (IL1),one of the earliest embryonic signals.
hCG triggers angiogenesis via the modulation of endometrial stromal cell responsiveness to IL1
IL1R1/
IL1R
1 m
RN
A%
of c
ontro
lIL
1R2
mR
NA
% o
f con
trol
IL1R
N m
RN
A %
of c
ontro
l
sIL1
R2
rele
ase
% o
f con
trol
CC
L2 s
ecre
tion
% o
f con
trol
Sample IgG CTRL MAB 279 (anti-MCP1)
ES
C
Sup
erna
tant
MM
48h
CC
L2(M
CP
1)E
SC
S
uper
nata
nthC
G10
0/IL
1 0.
1
CC
L2C
CL2
CC
L2
sIL1RN release% of control
sIL1R2 release% of control
CC
L2 (M
CP
1) m
RN
A%
of c
ontro
lC
CL2
(MC
P1)
rele
ase
% o
f con
trol
IL1R
2/IL
1R1
mR
NA
1. Imbalance of IL1Rs and increased responsiveness to IL1 2. Overexpression of IL1R2 in ESC 3. Increased endothelial cell migration and proliferation
Transcriptome analysis reveals deep modulation of endometrial stromal cell phenotype by embryo‐derived signals hCG/IL15. Validation of main regulated genes
Enrichment Score of Biological Processes
4. Hierarchical clustering, Principal Component Analysis (PCA)and Gene Ontology (GO) annotations
hCG regulates endothelial cell responsiveness to interleukin 16. Proliferation and Migration 7. HMVEC responsiveness to IL1 and IL1R2 overexpression 8. Expression in embryo implantation sites
hCGIL1
Blastocyst
Epithelium
Capillary(endothelial cells)
ESC
We thank Mrs. Nathalie Bourcier and Monique Longpréfor patient recruitment and precious technicalassistance.
We thank HSFA and CHUL gynecologists for patientevaluation and providing endometrial tissue samples.
Supported by an NSERC Discovery Grant to Pr. AliAkoum, National Researcher, "Fonds de la Recherche duQuébec-Santé".
Study questionTo assess whether hCG modulates endometrialcell responsiveness to IL1 by targeting differentcell types and gain a broader understanding ofthe impact of such a mechanism on the acquisitionof growth-promoting phenotype.
Study design and methods
IL1R1(signalingreceptor) IL1R2
IL1RNInhibitory receptor/antag.
Pro-angiogenic effect
hCG
IL1R2 gene transfer and IL8 secretion
IL1Rs in endometrium during gestation in vivo
Pregnant mouse uterus d6 Non-pregnant mouse uterus
