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

asgi@hc.msu.edu

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

Women’s Health

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

Women’s Health

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

Women’s Health

• 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:

Women’s Health

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