Endometrial cell migration and endometriosis - · PDF fileadenomyosis, mullerianosis,...

IRCAD Barretos 1-8-16

Philippe R. Koninckx, Anastasia Ussia

Gruppo Italo Belga Prof em KU leuven Belgium, Univ Oxford UK, Univ Sacro Cuore, Italy, Honorary Consultant UK, Hon Prof Moscow Univ Gruppo Italo Belga, Leuven –Rome, Belgium Italy.

Endometrial cell migration and endometriosis

What is endometriosis ? The definition 1 disease ?

Progressive and recurrent ?

Hereditary – cancer risk ?

Only in women and primates The endometrium – placentation - JZ

P koninckx A Ussia, Gruppo italo belga

The Endometrium

• Glands & stroma

• during menstrual cyle = dating

• Pregnancy-> decidualisation

• Functionalis and basalis

• Different hormonal control

• Junctional zone & spiral arteries



The Endometrial function

• The most regenerative tissue

• Hormonal sensitivity

• Peristalsis

• Pregnancy

• Invasion

• immunology

Brosens 68

Leyendecker 85

Archimetra

Endometriosis : Facts 1

different presentations – prevalences - histology

100% Retrograde menstruation

80% Subtle with remodeling

15% typical 10% cystic 1% deep

adenomyosis, mullerianosis, stromatosis, pockets,

active no fibrosis

Inactive with fibrosis


Pathophysiology : Sampson Theory

Sampson : retrograde menstruation Viable cells in menstruation

Retrograde menstruation

Viable cells in PF

Implantation potential

Keebiel WC, Stein RJ. Am J Obstet Gynecol 1951; 61:440-442.

Koninckx PR et al. J.Reprod.Med. 1980; 24:257-260.

In humans, in primates, in nude mice, in vitro

Metaplasia Subtle lesions Retrograde

menstruation,

Microscopical

Remodeling P koninckx A Ussia, Gruppo italo belga


Prevalence of subtle lesions

Koninckx et al 1991

0

20

40

60

80

Inf Pain Inf+pain

n= 1297 918 267


• In normal peritoneum • 10-15%

• In lymph nodes

• 15% in deep endo

• In bowel deep endo

Microscopical endometriosis

P koninckx A Ussia, Gruppo italo belga Fertil steril, 2016, 105,305-6


Conclusion 1: subtle - microscopic

Glands and stroma outside the uterine cavity

• Are not always pathology

• We need another definition


Sampson’s speculation

Sampson Viable cells in menstruation

Retrograde menstruation

Viable cells in PF

Implantation potential

Keebiel WC, Stein RJ. Am J Obstet Gynecol 1951; 61:440-442.

Koninckx PR et al. J.Reprod.Med. 1980; 24:257-260.

In humans, in primates, in nude mice, in vitro

We see We Imagine

Abdominal wall growth stops fibrosis


Evidence for progression ? • Subtle to typical ?

• Typical to severe cystic ?

deep ?

• At diagnosis most lesions are no longer progressive

No evidence

in the human

in primates

Circomstantial evidence of the

contrary

historical data in Leuven :

typical in 1980 ; deep in the 90’s

in primates : no evolution


A recurrent disease ?

• Subtle 100% • Typical 20% • Cystic 5% • Deep 1%


Clonality in endometriosis

Monoclonal neoplasm

Genetic damage to single

progenitor cell Clonal

expansion

Cystic ovarian endometriosis Yes

• Jimbo et al (1997) Am J Pathol 150, 1173 ;21 samples from 11

endometriomas ; Marker = X-linked HUMARA gene

21/21 samples monoclonal

• Tamura et al (1998) Lab Invest 78, 213 ;25 epithelial cells from 25

archival endometriomas ; Controls = 25 matched ovarian stroma

tissue ; Marker = X-linked PGK gene

10/25 samples informative (all 10 monoclonal)

Deep endometriosis


Germline predisposition • Familial clustering Humans: Kennedy et al (1995) J Assist Reprod Genet 12,

Rhesus: Hadfield et al (1997) Hum Reprod Update 3, 109 • Twin studies : MZ >> DZ Moen (1994) Acta Obstet Gynecol Scand 73, 59

Hadfield et al (1997) Fertil Steril 68, 941

Treloar et al (1999) Fertil Steril 71, 701 • Heredity symptom onset age in non-twin sisters

Kennedy et al (1996) Hum Reprod 11, 101

6-9x increased prevalence in 1st degree relatives

Simpson et al (1980) Am J Obstet Gynecol 137, 327

Coxhead & Thomas (1993) J Obstet Gynaecol 13, 42

Moen & Magnus (1993) Acta Obstet Gynecol Scand 72, 560

15% prevalence in 1st degree relatives (using MRI)

Kennedy et al (1998) Lancet 352, 1440


Typical-Cystic-Deep

• Progression or end stage ?

• Cystic and deep are clonal

• Wise to consider separately

• Associated with (typical) • Pain and infertility

• Low grade inflammation in peritoneal fluid

• Heriditary, age

• ........food, ......fat

• Cancer, nerve cells in endometrium

• For surgeons : association is not causal

Philippe R. Koninckx

Prof em KU leuven Belgium, Univ Oxford UK, Univ Sacro Cuore,

I taly, Honorary Consultant UK, Hon Prof Moscow Univ

Gruppo Italo Belga, Rome

www.gynsurgery.org

Disclosure : CEO EndoSAT www.endosat.com

Pathophysiology of

TYPICAL, CYSTIC, DEEP endometriosis

The theories

Sampson’s theory is speculation and incomplete

does not explain typical, cystic, deep

…….forgets angiogenic-lymphogenic spread

…….forgets metaplasia

What is missing : Endometriotic disease theory

The modulators

peritoneal fluid

immunology

genetics

http://www.gynsurgery.org/

http://www.endosat.com/


Sampson is historical speculation

Whether considered as retrograde menstruation, as stem cells, as pale cells……..

• Does not explain metaplasia

• Does not explain progression to typical, cystic or deep endometriosis

• Cannot explain hereditary aspect

• Is incompatible with clonal aspect

• Does not explain effect of dioxin, radiation etc;

Something is missing


The Endometriotic Disease Theory

Something

Causes a cell to

change behavour

Koninckx P.R., Kennedy S., Barlow D.,

Gyn Obstet Invest 1999,47,1-10

Genetic mutation

Epigenetics

Environment


Implantation Metaplasia Progression

Endometriotic

disease Endometriosis

Deep

Cystic Ovarian

Adhesions

Typical

Subtle lesions

Retrograde

menstruation,

Remodeling,

(epi) Genetic

mutation favorised by

heredity

immunology

volume

environment

Sampson - Metaplasia The Endometriotic Disease Theory Koninckx P.R., Kennedy S., Barlow D., Gyn Obstet Invest 1999,47,1-10


Conclusion 1: subtle is not pathology

Glands and stroma outside the uterine cavity

• Are not always pathology

• We need another definition


rr

Conclusion II Typical – cystic - deep

1. A different cell

coming from endometrium ? basalis or functionalis ?

stem cells ? Pale cells ? Bone marrow ?

genetic –epigenetic-microenvironment

altered immunology

endometrium

peritoneal cavity

systemic

Environment

blood

peritoneal fluid

JZ ‘blocks’ invasion

rr 2. Growth


Epigenetics • Epigenetics are stable heritable traits (or "phenotypes") that cannot be

explained by changes in DNA sequence…may result from external or environmental factors .phenotype changes without genotype changes.

https://en.wikipedia.org/wiki/Phenotype

https://en.wikipedia.org/wiki/DNA_sequence

https://en.wikipedia.org/wiki/Environment_(biophysical)


Which cell ?

• Endometrium – basalis

• Stem cells

• Bone marrow

• Pale cells

• Looks like trigger ?

• More retrograde menstruation

• In endometrium endometrium

• In peritoneal cavity = trigger

• Epithelial-mesenchymal transformation

• In endometrium bone marrow

• In endometriosis cells


• CD68

• CD45


Why does a cell change behaviour

A benign tumour

• an insult

• genetic predisposition

Mutation Epigenetics

Endometrium Stem cell Mesothelial cells


Pollution and Endometriosis

• Dioxins and PCB’s

• Is Endometriosis increasing in the human ?

• Is endometriosis linked to pollution ?

• Animal models

• Human


0

1

2

3

4

5

6

7

C 5 25

IV

III

II

I

0

Endometriosis in Rhesus Monkeys chronically exposed to dioxin

Rier SE, Martin DC, Bowman RE, Dmowski, WP, Becker JL Fundam. Appl. Toxicol,

1991,111,422-431

Dioxin dose (ppt)

n

rAFS


Endometriosis in Rhesus Monkeys

• Spontaneous :

• clinical endo in autopsy 30% after 13 years

• Following

• dioxin : increased and dose dependent (Rier 1991)

• PCB : increased ( Campbell , unpublished)

• single energy & mixed energy proton irradiation, X rays (Wood 83, Fanton 91, Wood 91)

THE SHORTEST TIME

BETWEEN IRRADIATION AND

ENDOMETRIOSIS IS 6 YEARS


Dioxin Concentrations in women with endometriosis

Mayani A, Barel S, Soback S, Almagor M. Human Repr 1997, 12, 373-375

0

5

10

15

20

25

No I+II III+IV

% of women with detectable dioxin in blood

Stage of Endometriosis

1/35

3/24

5/20

Dioxin concentration (part per trillion)

0 I-II III-IV0.00

0.25

0.50

0.75

1.00

1.25

Stage of Endometriosis


Plasma Dioxin concentrations & risk

• Rhesus monkeys 25ppt 4 years (Bowman, 1989)

• T1/2 : 180-780 days

• adipose tissue concentration : 250-810 ppt

• Humans (Mocarelli, 1991)

• T1/2 : 7 years

• Seweso subjects : 2.000-35.000 ppt

• Area under time-concentration curve for 14 years • rhesus monkeys 388-1.400 (*1000)

• humans Seweso 5.500-112.000 (*1000) AUC T1/2=7 years

Bois FY, Eskenazi B, Environ. Health Perspect. 1994, 102,476


Dioxin and Radiation

• Dioxin binds to the DNA through specific receptor • Pseudo steroid

• Direct DNA effect

• transmissible effect eg sperm up to third generation

• Radiation has a direct DNA effect

Mutagenic factors


Genetic predisposition

• Loss of heterozygosity • Germ cell predisposition


Loss of Heterozygocity

X

X

X

Germline

Mutation

=Heredity

Somatic

Mutation

1st Hit Deletion or

Translocation

X X X

Duplication

X X

Point

Mutation

First

Hit

Second

Hit

Where should we look for a first hit ?

in the endometrium of women

with & without endometriosis


• loss of heterozygosity on p16(Ink4), GALT, and p53, as well as on APOA2, a region

frequently lost in ovarian cancer, occurs in endometriosis Goumenou AG, Arvanitis DA, Matalliotakis IM, Koumantakis EE, Spandidos DA. Microsatellite DNA assays reveal an allelic imbalance in p16(Ink4), GALT, p53, and APOA2 loci in patients with endometriosis. Fertil Steril 2001; 75(1):160-165.

• endometrium from women with endometriosis releases more ICAM-1, which can inhibit NK activity Somigliana E, Vigano P, Gaffuri B, Guarneri D, Busacca M, and Vignali M. (1996) Human endometrial stromal cells as a source of soluble intercellular adhesion molecule (ICAM)-1 molecules. Hum. Reprod. 11, 1190-1194

• P450aromatase transcripts & IL-6 and IL-11 transcripts in endometriosis women Noble LS, Simpson ER, Johns A, and Bulun SE. (1996) Aromatase expression in endometriosis. J. Clin. Endocrinol. Metab. 81, 174-179

• . Increased expression of heat shock protein 27 Ota H, Igarashi S, Hatazawa J, and Tanaka T. (1997) Distribution of heat shock proteins in eutopic and ectopic endometrium in endometriosis and adenomyosis. FERT. STERIL. 68, 23-28

Endometrium in Endometriosis


Genetics of endometriosis

Which Gene ?

Oxegene project

Stephen Kennedy

University of Oxford

Specific genes

Non hypothesis driven

Linkage analysis

S. Kennedy , 2005

Molecular genetic evidence


Linkage analysis 2006

• Extremely expensive

• Probably 2 chromosomes identified

• Highly secretive for patent reasons

• Clinically irrelevant for the next 10 years

• Potentially very important

Philippe R. Koninckx Anastasia Ussia

New Frontiers in Endometriosis

Ara Pacis Museum, Rome 28-10-2011

www.mondoginecologico.it Gruppo Italo Belga

Sub;itted

Gynaecological surgery, 2016

Ackowledged : Jacquez Donnez, Camran Nezhat, Charles Koh, Antonio Setubal

DEEP is increasing in number and severity

Deep Endometriosis surgeons > 20 Years




Why does a cell change behaviour

Growth

• blood stream

• peritoneal cavity

Cellular Micro

Environment


• Surgery : more in women than in men

• increases the cancer risk Hysterectomy increases the risk of renal cancer,10,11

and total joint arthroplasty elevate the risk of prostate cancer.12

• promotes cancer metastasis.

• activates adrenergic signaling,

• which suppresses cell-mediated immunity

• promotes angiogenesis

• and metastasis

• and accelerates the growth of endometriotic

lesions in mice, - abrogated by b-blockade

Long, Q., Liu, X., and Guo, S. W. Surgery accelerates the development of endometriosis in mice. Am.J.Obstet.Gynecol. 215(3), 320. 2016.


Long, Q., Liu, X., and Guo, S. W. Surgery accelerates the development of endometriosis in mice. Am.J.Obstet.Gynecol. 215(3), 320. 2016.


Haematologic and lymphogenic spread

• Haematologic

• Lung endometriosis

Lymphatic

•Deep endo

•Umbilical ? •


• Mesothelial cells are very sensitive • React by retraction and bulging thus exposing intracellular spaces

• An acute inflammation of the entire exposed area : fast within hours

• With as consequences pain, adhesion formation, fluid resorbtion, ....

• Full- conditioning = prevention of this acute inflammation

• Surgery • = access to the cavity = air, CO2, fuids

• = manipulation, surgical trauma and irrigation

The mesothelial cell


Peritoneal fluid

• An ovarian exudate

• Volume

// with ovarian activity

(Koninckx PR, Renaer M, and Brosens IA. (1980) Origin

of peritoneal fluid in women: An ovarian exudation

product. Br. J. Obstet. Gynaecol. 87, 177-183).


Peritoneal fluid

• An ovarian exudate

• Volume

// with ovarian activity

• steroid hormone concentrations

always higher than in plasma

• protein concentrations :

60% of plasma

lower for larger molecules

(Koninckx PR, Renaer M, and Brosens IA. (1980) Origin of peritoneal fluid in women: An ovarian

exudation product. Br. J. Obstet. Gynaecol. 87, 177-183).

Koninckx PR, Heyns W, Verhoeven G, Van Baelen H, Lissens W, and De Moor P. (1980) Biochemical

characterisation of peritoneal fluid in women during the menstrual cycle. J. Clin. Endocrinol. Metab. 51,

1239-1244


LUF as a cofactor for Endometriosis

• Since

• steroid hormone concentrations are low Koninckx PR, De Moor P, and

Brosens IA. (1980) Diagnosis of the luteinized unruptured follicle syndrome by steroid hormone assays on peritoneal fluid. Br. J.

Obstet. Gynaecol. 87, 929-934

• association with endometriosis Brosens IA, Koninckx PR, and Corveleyn PA. (1978)

A study of plasma progesterone, oestradiol-17b, prolactin and LH levels, and of the luteal phase appearance of the ovaries in

patients with endometriosis and infertility. Br. J. Obstet. Gynaecol. 85, 246-250

• A cofactor in endometriosis Koninckx PR, Ide P, Vandenbroucke W, and

Brosens IA. (1980b) New aspects of the pathophysiology of endometriosis and associated infertility. J Reprod

Med, 24, 257-260


Peritoneal fluid in Endometriosis

• Low grade inflammation

with more and activated macrophages • increase in chemotactic activity by 20kD protein Weil SJ, Wang SY, Perez MC, and Lyttle CR.

(1997) Chemotaxis of macrophages by a peritoneal fluid protein in women with endometriosis. FERT. STERIL. 67, 865-869

• decreased by medical therapy Leiva MC, Hasty LA, Pfeifer S, Mastroianni L, Jr., and Lyttle CR. (1993) Increased

chemotactic activity of peritoneal fluid in patients with endometriosis. Am J Obstet. Gynecol. 168, 592-598

• in vitro secretion of a monocyte chemoattractant Akoum A, Lemay A, Brunet C, and

Hebert J. (1995) Cytokine-induced secretion of monocyte chemotactic protein-1 by human endometriotic cells in culture. the groupe d'investigation en

gynecologie. Am. J. Obstet. Gynecol. 172, 594-600

• RANTES Hornung D, Ryan IP, Chao VA, Vigne JL, Schriock ED, and Taylor RN. (1997) Immunolocalization and regulation of the chemokine RANTES in

hunan endothelial and endometriosis tissues and cells. JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM, 82, 1621-1628



• More and activated Macrophages = secretion products

• Bax+ macrophages McLaren J, Prentice A, Charnock-Jones DS, Sharkey AM, and Smith SK. (1997) Immunolocalization

of the apoptosis regulating proteins Bcl-2 and Bax in human endometrium and isolated peritoneal fluid macrophages in endometriosis.

HUMAN REPRODUCTION, 12, 146-152

• angiogenic activity

in vivo Oosterlynck D. (1993a) Angiogenic activity of peritoneal fluid from women with endometriosis. Fertil. Steril. 59, 778-782.

TGF Oosterlynck DJ, Meuleman C, Waer M, and Koninckx PR. (1994) Transforming Growth Factor-beta Activity Is

Increased in Peritoneal Fluid from Women with Endometriosis. Obstet. Gynecol. 83, 287-292 VEGF McLaren J, Prentice A, Charnock Jones DS, and Smith SK. (1996) Vascular endothelial growth factor (VEGF)

concentrations are elevated in peritoneal fluid of women with endometriosis. Hum. Reprod. 11, 220-223.

McLaren J, Prentice A, Charnock Jones DS et al. (1996) Vascular endothelial growth

factor is produced by peritoneal fluid macrophages in endometriosis and is regulated by ovarian steroids. J. Clin. Invest.

98, 482-489

• Cytokines Koninckx PR, Kennedy SH, Barlow DH.(1999) Pathogenesis of endometriosis: the role of

peritoneal fluid. Gynecol Obstet Invest 47 Suppl 1:23-33.



• Others • The IGF system

• Platelet activating factor and altered fibrinolytic system

• Prostaglandins

• decreased NK cell activity Oosterlynck DJ, Meuleman C, Waer M, Vandeputte M, and

Koninckx PR. (1992) The natural killer activity of peritoneal fluid lymphocytes is decreased in women with endometriosis. Fertil. Steril. 58, 290-295

• inhibition of activity Oosterlynck D. (1993b) Immunosuppressive activity of peritoneal fluid in

women with endometriosis. Obstet. Gynecol. 82, 206-212

• increased shedding of ICAM-1 by endometrial cells (Somigliana, Vigano, et al. 1996 )

• high local concentrations of glycodelins


Decreased NK cell activity

• Decreased activity

• In plasma & peritoneal fluid

• More in more severe endo

• Local shielding and glycodelins ?

• The chicken or the egg

• 4 mths after excision of deep endo

CA125 decreased

NK and endometrium resistance unchanged

Before

Oosterlynck,D.J., Meuleman,C., Waer,M. Koninckx PR CO2-laser excision of endometriosis does

not improve the decreased natural killer activity. Acta Obstet.Gynecol.Scand.1994, 73, 333-337.

after


Bolton AE, Pockley AG, Clough KJ, Mowles EA, Stoker RJ,

Westwood OM et al. Identification of placental protein 14 as an

immunosuppressive factor in human reproduction. Lancet

1987; 1(8533):593-595

Okamoto N, Uchida A, Takakura K, Kariya Y, Kanzaki H,

Riittinen L, et al. Suppression by human placental protein 14

of natural killer cell activity. Am J Reprod Immunol 1991;

26:137-142

PP14 Inhibit NK cells

requires 18 h of contact

min -max dose : 5-50µgr/ml

potent suppressive activity

in mixed lymphocyte cultures


Koninckx P.R., Kennedy S., Barlow

D., Gyn Obstet Invest 1999,47,1-10

Angiogenetic factors

• Bioassay • VEGF • TGF

Like most benign tumors

Chicken allantoic membrane

Oosterlynck, Waer, Koninckx 1994


For the surgeon : Cancer Stem cells

Cut the head and the rest will die : do not be too agressive in deep endo


Conclusions

• Retrograde menstruation + blood+ lymph • Implantation and mesothelial trauma

• Subtle is not (yet) pathology

• Endometrium-pale-stem-mesothelial cell-bone marrow

• Something more is needed • Genetic incident / epigenetics / predisposition

• Incident :genetics epigenetics

• Typical, cystic, and deep : different endpoints

• Growth

• Angiogenesis- hormonal dependent- stress

Endometrial cell migration and endometriosis - · PDF fileadenomyosis, mullerianosis,...

Documents

Transcript of Endometrial cell migration and endometriosis - · PDF fileadenomyosis, mullerianosis,...