the endometrial embryo response and use of hcg in ... · endometrial infusion of hcg at the time of...
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Women’s Health
Asgi T. Fazleabas University Distinguished Professor and
Associate Chair for Research Department of Ob, Gyn & Reprod Biol Director, Center for Women’s Health
Research Co-Director, Reproductive & Developmental
Sciences Program Michigan State University
THE ENDOMETRIAL EMBRYO RESPONSE AND USE OF HCG IN IMPROVING IMPLANTATION
Ovarian Club X and CoGEN in Asia Hong Kong, December 16-17, 2017
Women’s Health
OUTLINE
Mechanisms by which hCG modulates uterine receptivity
Consequences of endometriosis on hCG induced changes in uterine receptivity – global and decidualization Effect of hCG on endometrial response following ovarian hyperstimulation
MATERNAL RECOGNITION OF PREGNANCY – PRIMATES
Wilcox AJ, NEJM 10:1796, 1999 Larsen et al., BMC Medicine 11:154, 2013 Macklon F&S 108: July 2107
In 1992 the expression of human chorionic gonadotropin/luteinizing hormone receptors were identified by C.V. Rao in the human endometrium and myometrial blood vessels. (JCEM, 1992)
hCG and the a-subunit induced decidualization of stromal fibroblasts in vitro (JCEM, 1998)
Women’s Health
THE BABOON AS A MODEL
• Not endangered
• Easy monitoring of cycle
• Menstrual cycle/hormonal profile similar to women
• Breeds well in captivity
• Single placenta, pregnancy similar to humans • Access to tissues at time of implantation Phase 1
Phase 2 Phase 3
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MODULATION OF UTERINE RECEPTIVITY – EFFECT OF hCG
Fazleabas et al., PNAS 96:2493, 1999
The blastocyst begins to produce measurable levels of CG Window of time that the blastocyst is free floating in the uterus The corpus luteum is exquisitely responsive to CG
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INHIBITION OF APOPTOSIS BY hCG AND PROGESTERONE
Lovely et al., JCEM 90: 2351, 2005 Women’s Health
Circulating Progesterone
H-Score Quantification of TUNEL Staining
200 mg intravaginal P (D18-26) 10,000 IU hCG (D19)
hCG MEDIATED SIGNALING MECHANISMS IN THE UTERUS
CG
SIGNALING CASCADES
IMPLANTATION
AC cAMP PKA CREB
CG
Nuclear Translocation
MEK
ERK
Raf
Elk1 ERK
PI3K
siRNA against LHCGR
Microarray Analysis
0 min
60 min
10 IU/ml CG
Banerjee et.al., Endo 150:4326, 2009; Srisuparp et.al., BOR 68:457, 2003. Women’s Health
PRE-IMPLANTATION BLASTOCYST
Chorionic Gonadotrophin
RECEPTIVE UTERINE ENDOMETRIUM Activation of MAPK Pathway
Glandular Epithelium Stromal Fibroblasts Uterine Vasculature
Glycodelin
Leukemia Inhibitory Factor
a Smooth Muscle Actin
Notch 1
iNOS & eNOS
VEGF
Modulation of Immune Function
Inhibition of Apoptosis Initiation of Decidualization
Angiogenesis and Vascular Development
Banerjee & Fazleabas, Intl J Dev Biol 54:295,2010; Strug et al., Hum Reprod 31:1552, 2016 Women’s Health
Women’s Health
OUTLINE
Mechanisms by which hCG modulates uterine receptivity
Consequences of endometriosis on hCG induced changes in uterine receptivity – global and decidualization Effect of hCG on endometrial response following ovarian hyperstimulation
EXPERIMENTAL DESIGN INDUCTION OF ENDOMETRIOSIS
Menses Menses
Laparoscopy & Innoculation
with Menstrual Tissue
Control
1 3-4 6-7 9-10 12
Laparotomies
Months
Innoculated Baboons
Harvest Tissues
15
Pelvic cavity prior
to inoculation Pelvic cavity during
to inoculation
Histology of endometrial
tissue in menstrual effluent
ENDOMETRIOSIS SUPPRESSES THE ENDOMETRIAL RESPONSE TO THE PRIMATE EMBRYONIC SIGNAL
Control Endometriotic
a-SMA
Morphology
Glycodelin
Sherwin et al., Endo 151:4982,2010
Induction of endometriosis results in an altered response
to hCG.
• Differential Expression of Endometrial transcripts
• Abnormalities in Decidualization
• Altered immune responses
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• Notch is an evolutionary conserved arbiter of cell fate and regulates diverse cellular functions, including survival, proliferation, and differentiation.
THE NOTCH PATHWAY IN DECIDUALIZATION
Miele L, Clin Cancer Res 2006:
a-SMA
Notch 1
Afshar et al., Endo 2012
NOTCH1 IS CRITICAL FOR IN VITRO DECIDUALIZATION
cont
rol
Lip
o
Vect
or
DN
-Mam
siR
NA
10
0
siR
NA
20
0
d6, cAMP+H
Igfbp1
IL-11
Hey1
Hes5
β-actin
cont
rol
N1
siR
NA
vector
lipo DN-Mam
Afshar et al. Endo 153: 2884, 2012 Women’s Health
ALTERED EXPRESSION OF THE NOTCH SIGNAL TRANSDUCTION CASCADE DURING IN VITRO DECIDUALIZATION
Su et al., JCEM 100:E433, 2015 Women’s Health
MICROARRAY ANALYSIS OF DECIDUALIZATION ASSOCIATED GENES FOLLOWING NOTCH 1 INHIBITION
FOXO1 regulated
genes
Su et al., JCEM 100:E433, 2015 Women’s Health
Decidualization Trigger
Notch 1
aSMA, FOXO1 & FOXO3a
IL11 COX-2 and other FOXO1 regulated genes
Inhibit Apoptosis & Initiate Differentiation
Stromal Fibroblast
Decidual Cell
Progesterone
WORKING HYPOTHESIS
• Notch is an evolutionary conserved arbiter of cell fate and regulates diverse cellular functions, including survival, proliferation, and differentiation.
ERa
Inhibition of Apoptosis
Endometriosis
Afshar et al. Endo 153: 2884, 2012; Su et al., JCEM 100:E433, 2015
Women’s Health
Women’s Health
OUTLINE
Mechanisms by which hCG modulates uterine receptivity
Consequences of endometriosis on hCG induced changes in uterine receptivity – global and decidualization Effect of hCG on endometrial response following ovarian hyperstimulation
43 proteins altered by hCG, including proteins associated with cellular adhesion, extracellular-matrix organization, developmental growth, growth factor regulation, and cell signaling.
These findings indicate that intrauterine administration of hCG-activated autologous PBMC effectively improves the IVF outcomes for RIF patients
Intrauterine injection of 500 IU of hCG before ET statistically significantly improved the implantation and pregnancy rates in IVF/ICSI.
in our study we could not find any evidence for improvement of clinical outcome in blastocyst transfer cycles, neither with top nor with non-top quality morphology.
Intrauterine injection of hCG before ET improves implantation and pregnancy rates and may be considered an adjuvant in IVF/ICSI.
Endometrial infusion of hCG at the time of blastocyst ET does not improve sustained implantation rates.
Cochrane Database of Systematic Reviews 2016.
A definitive large clinical trial with live birth as the primary outcome is recommended. There was no evidence that miscarriage was influenced by intrauterine hCG administration but there were too few events to allow any conclusions to be drawn
STUDY DESIGN FOR HUMAN IN VIVO STUDY
Day of oocyte retrieval = (Day 0)
Stimulated cycle -Approved oocyte donors undergo
oocyte stimulation cycle (which is
independent of this research)
Day 0
Oocyte retrieval
Day 3: Intrauterine infusion of hCG (500IU;N=7)
or vehicle (media;N=8)
Day 5
Obtain Uterine lavage & EMB
COH
Strug et al., Hum Reprod 31:1552, 2016
Women’s Health
Veh hCG0
20
40
60
D-H
SC
OR
E
ERK1/2 (GE)
**
Veh hCG0
10
20
30
40
50
D-H
SC
OR
E
ERK1/2 (Stroma)
*
Veh hCG0
20
40
60
D-H
SC
OR
E
p-ERK1/2 (Glands)
**
Veh hCG0
10
20
30
40
D-H
SC
OR
E
p-ERK1/2 (Stroma)
Veh hCG0
10
20
30
D-H
SC
OR
E
ESR1 (GE)
**
Veh hCG0
1
2
3
4
D-H
SC
OR
E
ESR1 (Stroma)
*
Veh hCG0
10
20
30
40
50
D-H
SC
OR
E
PGR (GE)
*
Veh hCG0
10
20
30
40
D-H
SC
OR
E
PGR (Stroma)
*
Vehicle
hCG
ERK pERK ESR1 PGR
hCG
Veh
0.0
0.5
1.0
1.5
2.0
Re
lativ
e E
xp
res
sio
n/R
PL
17
PGR
*
0.0
0.5
1.0
1.5
2.0
2.5
Re
lativ
e E
xp
res
sio
n/R
PL
17
ESR1
*
hCG INDUCED CHANGES IN ERK AND STEROID HORMONE RECEPTORS
Strug et al., Hum Reprod 31:1552, 2016; Tapia-Pizarro et al., Mol Hum Reprod 23; 393, 2017 Women’s Health
Vehicle
hCG 0.0
0.5
1.0
1.5
2.0
Re
lativ
e E
xp
res
sio
n/R
PL
17
C3
0.0
0.5
1.0
1.5
Re
lativ
e E
xp
res
sio
n/R
PL
17
NOTCH1hCG
Veh
Veh hCG0
50
100
150
D-H
SC
OR
E
NOTCH1 (Stroma)
*
Veh hCG0
60
120
180
D-H
SC
OR
E
NOTCH1 (GE)
**
Veh hCG0
20
40
60
80D
-HS
CO
RE
C3 (GE)
Veh hCG0
25
50
75
100
125
D-H
SC
OR
E
C3 (Stroma)
*
NOTCH 1 COMPLEMENT C3
hCG INDUCED CHANGES IN NOTCH 1 AND COMPLEMENT C3
Strug et al., Hum Reprod 31:1552, 2016 Sherwin et.al., Endo 148:618, 2007; Afshar et al. Endo 153: 2884, 2012; Palomino et al., Repod Sci 20:1103,2013;
Women’s Health
hCG PGR
C3
NOTCH1
α-SMA
ESR1 Stromal Survival and
Decidualization
Immune Priming For Implantation
Stromal Survival and
Transformation for Decidualization
PROPOSED MECHANISM BY WHICH hCG MODULATES UTERINE RECEPTIVITY FOLLOWING COH
Kim et al., Endo 140: 997, 1999; Strug et al., Hum Reprod 31:1552, 2016 Sherwin et.al., Endo 148:618, 2007; Afshar et al. Endo 153: 2884, 2012;
Women’s Health
hCG effectively modulates several metabolic pathways within the endometrium and decidua contributing to endometrial receptivity
Reverses signs of apoptosis apparent by day 26 of the menstrual cycle suggesting a clinical
use for luteal-phase support for treatment of women with infertility and/or recurrent pregnancy loss by restoring synchrony and CL support.
Intrauterine hCG prior to embryo transfer on day 3 in counteracts endometrial
dyssynchrony following ovarian hyper-stimulation and promotes the expression of markers important for immune modulation and stromal survival
Has a clinical impact on reproductive outcomes after endometrial priming with hCG prior to embryo transfer. BUT more studies are needed
IN HUMAN STUDIES:
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ACKNOWLEDGEMENTS Collaborators
Fazleabas Laboratory Mark Olson Alumni Niraj Joshi Yong Song Ren-Wei Su Mike Strug Erin Vegter Ariadna Ochoa Emma Giuliani Danielle Peterse Samantha Bond
Linda Giudice – UCSF Bruce Lessey – Greenville Hospital Steve Young – UNC Rabindranath De La Fuentes – U Georgia Andrew Sharkey – Cambridge Rob Sherwin – Cambridge/Whittenham Lucio Miele – Louisiana State Univ Carlos Simon - IVI Dharani Hapangama – Univ Liverpool Franco DeMayo – NIEHS Jae Wook Jeong – MSU Ripla Arora – MSU Carlo Ticconi – Univ of Rome Tor Vegata Serdar Bulun – Northwestern Carolyn Jones – Univ Manchester Idhaliz Flores – Ponce School of Med Jodi Flaws – Univ of Illinois Paulo Serafini – Univ of San Paolo Sun-Wei Guo – Fudan Univ Lorenzo Sempere - VARI
NIH R01 HD083273, NIH R21 HD083453 NIH R01 HD42280 & NIH F30 HD082951
Women’s Health