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