IRCAD Barretos 1-8-16

Pathophysiology of endometriosis

Philippe R. Koninckx

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.

Disclosure : shareholder EndoSAT

Pathophysiology for surgeons

• What is endometriosis ?

• Different presentations : 1 disease ?

• Progressive and recurrent ?

• What do we know ?

• What is surgically useful ?

R S

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

Symptoms vary with lesion Prevalence Pain Infertility

• Subtle : 80% no no

• Typical : 25% in 50% + ?

• Cystic : 10% in 80% +++ ++++

• Deep : 2-3% in 95% ++++ ???

• Adenomyosis

• Peritoneal pockets – Müllerianosis - Choristoma

• Stromatosis

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

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

c

5 cm

Bowel resection: 10-20% positive margins

• 40% Remorgida V, Ragni N, Ferrero S, Anserini P, Torelli P, Fulcheri E. How complete is full thickness disc resection of bowel endometriotic lesions? A prospective surgical and histological study. Hum Reprod 2005;20: 2317–20.

• 9% Anaf V, El Nakadi I, Simon P, Van de Stadt J, Fayt I, Simonart T, Noel JC. Preferential infiltration of large bowel endometriosis along the nerves of the colon. Hum Reprod 2004; 19: 996-1002

• 16% Nirgianakis K, McKinnon B, Imboden S, Knabben L, Gloor B, Mueller MD. Laparoscopic management of bowel endometriosis: resection margins as a predictor of recurrences. Acta Obstet Gynecol Scand 2014; 93: 1262-7.

• 19 % Mabrouk M, Spagnolo E, Raimondo D, D‟Errico A, Caprara G, Malvi D, Catena F, Ferrini G, Paradisi R, Seracchioli R. Hum Reprod 2012; 27: 1314-9.

• 27% Roman Horace fertil Steril 2016

microscopic nests

Fertil steril, 2016, 105,305-6

Fertil steril, 2016, 105,305-6

What is endometriosis ?

Glands and stroma outside the uterine cavity

• Always pathology ? • No

• We need another definition

Subtle lesions ? Microscopic ?

• White & red vesicles, flame like • More tetrograde menstruation ?

• Progressive : the large majority not

• Microscopic : no known associated pathology

• Lymph nodes

• Bowel resection

• Pain & infertility

• For the surgeon :

• not too aggressive

• No wide perineum ablation

Pathophysiology : Theories

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 ?

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%

Prevalences and age

• nn

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

Facts ?

• 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 endometriosis

The theories

Sampson

Angiogenic-lymphogenic spread

Endometriotic disease theory

The modulators

peritoneal fluid

immunology

genetics

Evidence for progression ? • Subtle to typical ?

• Typical to severe ?

No evidence

in the human

in primates

Why progression in some women only ?

Endometriosis is a genetic disease • Hereditary

• Deep and cystic are clonal in origin

Total body radiation, Dioxin

The Endometriotic Disease Theory

Genetic mutation

cause a cell to

become tumorous

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

Gyn Obstet Invest 1999,47,1-10

Implantation Metaplasia Progression

Endometriotic

disease Endometriosis

Deep

Cystic Ovarian

Adhesions

Typical

Subtle lesions

Retrograde

menstruation,

Remodeling,

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

rr

Conclusion I which cell ?

endometriosis versus endometrium ?

stem cells ?

basalis or functionalis ?

“cancer like” invasion & metastasis ?

altered immunology

endometrium

peritoneal cavity

systemic

Environment

blood

peritoneal fluid

JZ ‘blocks’ invasion

Haematologic and lymphogenic spread

• Haematologic

• Lung endometriosis

Lymphatic

•Deep endo

•Umbilical ? •

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

Peritoneal fluid in Endometriosis

• 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.

Peritoneal fluid in Endometriosis

• 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

Endometriosis as a benign tumour

A benign tumour means

genetic predisposition

an insult

age radiation dioxin

progression

environment, immunology

A benign tumor

• loss in E-cadherin receptors in some foci of

endometriosis Gaetje R, Kotzian S, Herrmann G, Baumann R, and

Starzinski-Powitz A. (1997) Nonmalignant epithelial cells, potentially invasive in human

endometriosis, lack the tumor suppressor molecule E-cadherin. AMER. J. PATHOL. 150,

461-467

• suppressing metalloproteinase secretion in

vitro with progesterone or with a natural

inhibitor, inhibits endometriosis formation Bruner KL, Matrisian LM, Rodgers WH, Gorstein F, and Osteen KG. (1997) Suppression of

matrix metalloproteinases inhibits establishment of ectopic lesions by human

endometrium in nude mice. JOURNAL OF CLINICAL INVESTIGATION, 99, 2851-2857

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

For the surgeon : Cancer Stem cells –

progression - endometriosis ??

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, in press

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

Moscow 7-11/6/2016

Deep Endometriosis surgeons > 20 Years

Conclusions

• Typical, cystic, and deep : different endpoints

• Impression that deep is progressing

Endometriosis 2015

Tissue proliferative

Invasive Immunologic

Genomic incident

Peritoneal cavity 3 or more diseases

rarely proliferative Non recurrent

Definition has to be changed No animal model -as for placentation

Koninckx PR Ussia A Moscow 2015

For the surgeon ?

• excise

For the surgeon ?

• If no adhesions :Cystic corpus luteum

• Excise / coagulate small

• Pathology

• Glands and stroma are

always found

For the surgeon ?

• Every day more arguments not to be too agressive

• Leave a rim of fibrosis

• Small resections

• Some are different

For the surgeon : the literature

• Research : Significance

Increases with N

Does not permit a conclusion of the population

Length of women men

• Clinically useful

• Outcome

• Accidents

• Sensitivity/specificity

for population

170 180 cm

170 180 cm