Habitual implantation Habitual implantation failure /RM,RIF/failure /RM,RIF/

S. ŠkrablinS. Škrablin

Successful Successful implantationimplantation

Materno-fetal dialogueMaterno-fetal dialogue-Competent blastocystCompetent blastocyst

- receptive endometriumreceptive endometrium

““Window of implantation”Window of implantation”

19-23 cycle day (6. day after 19-23 cycle day (6. day after ovulation!!!) ovulation!!!)

Stromal cells transformation into decidual Stromal cells transformation into decidual cells: secretory granules, pinopodes, cells: secretory granules, pinopodes, disappearance of mycrovilli…… disappearance of mycrovilli……

Cytokines, chemokines, ligands…….Cytokines, chemokines, ligands……. dNK, macrophages, uDCsdNK, macrophages, uDCs

Regulatory role in trophoblast invasion

Angiogenesis, decidualisationAPC

“antigen presenting cells”

Th1 phenomenon

LIF LIF andand p53 p53 (Stewart LC, Nature 2007; (Stewart LC, Nature 2007; 450:619, Hu W et al. Nature 2007; 450:721)450:619, Hu W et al. Nature 2007; 450:721)

LIF – receptor interractionendometrial receptivity

HCG LIF

Acquisition of adhaesion ligands and loss of inhibitory molecules

HOX gensHOX gens

HOX genHOX genss – endometrial development – endometrial development - steroid hormones function- steroid hormones function - elevated expression during secretory phase- elevated expression during secretory phase - with successful implantation increased - with successful implantation increased

decidual expression decidual expression - regulation of morphological parameters of - regulation of morphological parameters of

implantation windowimplantation window: pinopodes, beta 3 integrin, : pinopodes, beta 3 integrin, IGFBP1IGFBP1

- essential for implantation – low - essential for implantation – low HOX HOX genes expression during the secretory phase - genes expression during the secretory phase - infertilityinfertility /Taylor et al.1999, Rackow and Taylor 2010//Taylor et al.1999, Rackow and Taylor 2010/

““unexplained infertility” unexplained infertility” /Brosens et al., 2009//Brosens et al., 2009/

Segmental body identity

HOX expression manipulationHOX expression manipulation- increased implantation with EAI - increased implantation with EAI

/in vitro //in vitro / /Bagot et al., 2000//Bagot et al., 2000/

Implantation Implantation failurefailure

/Cakmak H, Taylor /Cakmak H, Taylor HS. 2011/HS. 2011/

LeiomyomaLeiomyoma- deformitydeformity- occlusionocclusion- Low Low HOXA10 and BTEB1HOXA10 and BTEB1

exp.exp.

■■ PCOSPCOS- Decreased Decreased αvβ3 integrinαvβ3 integrin, ,

HOXA-10 and IGFBP-1 HOXA-10 and IGFBP-1 - ““OverexpressionOverexpression” of ” of

androgene receptorsandrogene receptors- Absence of Absence of estrogen estrogen

receptor-α downregulatreceptor-α downregulation ion during implantation windowduring implantation window

- OverexpressionOverexpression of of steroidsteroid receptorreceptor coactivators - coactivators - AIB1 AIB1 and TIF2and TIF2

EndometriEndometrialal pol polyypp- Gamete transportGamete transport- Decreased Decreased IGFBP-1 IGFBP-1 ii

osteopontin osteopontin - Low pLow progesteronrogesteron

receptorsreceptors

Implantation failureImplantation failure /Cakmak H, Taylor /Cakmak H, Taylor

HS. 2011/HS. 2011/

EndometrioEndometriosissis: : - Decreased - Decreased αvβ3 αvβ3

integrinintegrins and s and LIF LIF - Low- Low IL-11 IL-11 and and IL-11R IL-11R - Absence of - Absence of HOXA10 HOXA10

andand HOXA11 HOXA11 increaseincrease - Elevated- Elevated EMX2 EMX2 - P- Progesteronrogesteron resistence resistence - Disregulation of - Disregulation of PR-A PR-A / /

PR-B PR-B - HOX 10 promotor - HOX 10 promotor

hypermetilationhypermetilation

HydrosalpinxHydrosalpinx- Toxic effect of tubal secretionsToxic effect of tubal secretions- Decrease of Decrease of avβ3 integrin avβ3 integrin andand

LIFLIF-a-a - Low HOXLow HOXA10 A10 expression expression

■■ Septum, arcuatusSeptum, arcuatus■■ Sy AshermanSy Asherman■■ Thin endometriumThin endometrium■ ■ Ovarian stimulationOvarian stimulation■ ■ AdiposityAdiposity■ ■ TrombophiliaTrombophilian Impaired endometrial Impaired endometrial

functionfunctionn Immunologic factorsImmunologic factors

ENDOMETRIOSISENDOMETRIOSIS

Hox10/HOX10 geni –Hox10/HOX10 geni – ECTOPIC AND EUTOPIC ECTOPIC AND EUTOPIC

ENDOMETRIUMENDOMETRIUM /Zanatta et. /Zanatta et. al., 2010/al., 2010/

Embrional remnants – regrowth Embrional remnants – regrowth during and after pubertyduring and after puberty

““De novo” creation of eutopic or De novo” creation of eutopic or ectopic endometrium – when, why, ectopic endometrium – when, why, what is the initial stimulus ????what is the initial stimulus ????

“Mulleriosis”11% female fetuses !!!!!!11% female fetuses !!!!!!

Hoxa 9-tubesHoxa 11-cervixHoxa 13-vagina

HOX10-epithelial, stromal and myometrial embriogenesis Expression is regulated by cyclical E/P variation

Endometrial receptivity and nidation window

EAIEAI /Zanatta et. Al., /Zanatta et. Al., 2010/2010/

- Poor oocyte quality- Poor oocyte quality

- Implantation failure- Implantation failure

Ectopic towards eutopic endometrium - communication:?

Could radical surgery of ectopic endometrium assure normal eutopic edometrium function

- Low endometrial alfa, beta integrin -No IL -11 i IL 11R i LIF-a

-After GNRH analogues or ablation increased HOX expression and

integrins /Lessey i Young 1997, Daftary et al., 2007/

Low FC, abnormal follicular maturation,Low oocyte quality/Toya et al. 2000/

In 50% no alfa 5 beta 3 integrin

- Infertile with mild endometriosis/M. Donaghay, B.A. Lessey

2007/

Molecular differences Molecular differences

Ectopic: Ectopic: 17beta-hydroxsteroid d., 17beta-hydroxsteroid d.,

aromatase, P rec., ERbeta 140x aromatase, P rec., ERbeta 140x P rezistenceP rezistence apoA-I – plasma, endometrium, apoA-I – plasma, endometrium,

HCG resistant /beta HCG can HCG resistant /beta HCG can not inhibit apo A-Inot inhibit apo A-I/Brosens et al., 2009//Brosens et al., 2009/

Medical od radical surgical silencing of extrauterine genes could PREVENT e. Radical surgery of e.foci DOUBLED success in ART cycles /Bianchi et al., 2009/

“hallmark” of e.

HYDROSALPINXHYDROSALPINXENDOMETRITIS CHRENDOMETRITIS CHR

PID , hydrosalpinx PID , hydrosalpinx /Chukwuemeka et /Chukwuemeka et

al, 2002/al, 2002/

Hydrosalpinx:Hydrosalpinx:

►►Flushing effect, pinopod Flushing effect, pinopod malfunction, malfunction malfunction, malfunction of endometrial epitelial of endometrial epitelial cells….cells….

►►Embriotoxic /animal Embriotoxic /animal studies/studies/

►►Out of phase Out of phase endometrium low endometrium low integrins , LIF, HOXintegrins , LIF, HOX……

Chl. inf. , PID:Chl. inf. , PID:- endometritis endometritis - HSP- scar tissueHSP- scar tissue

- anti Chl HSP IgA – - anti Chl HSP IgA – marker for unsuccessful marker for unsuccessful IVFIVF

- HSP 10– tubal infertility- HSP 10– tubal infertility

- HSP 60- early - HSP 60- early misccariagemisccariage

IVF success after salpingectomy / Strandel et al., 2001, Cochrane dtb2002 /

FIBROIDSFIBROIDS

IVF/ICSIIVF/ICSI – meta analysis – meta analysis /Bajekal , Li 2000//Bajekal , Li 2000/

0

10

20

30

40

50

60

70

80

90

SM Healthy IM Healthy SS Healthy

PRLBMR

P <0,05

Compared to infertileCompared to infertileSM – decreased fertilitySM – decreased fertility

/RR PR 0,30//RR PR 0,30//Somigliana et al. 2011//Somigliana et al. 2011/

Similar results 3 META ANALYSES:

Pritts et al, 2009Sunkara et al, 2010

Metwally et al, 2011.

The effect of submucous fibroidsThe effect of submucous fibroids /Rackow and Taylor, 2010//Rackow and Taylor, 2010/

Decreased HOXA10, HOXA11, LIF, BTEB1Decreased HOXA10, HOXA11, LIF, BTEB1

MRNA HOXA 10 i HOXA 11MRNA HOXA 10 i HOXA 11

compared to intramuralcompared to intramural Global – entire endometrium, not Global – entire endometrium, not

only beneath myomaonly beneath myoma Size of myoma not important!!!!!Size of myoma not important!!!!!

Glycodelin, IL-10 – only with SM /Ben-Nagi J et al., 2010/

Inflammatory reaction with Inflammatory reaction with fibroidsfibroids

/Miura S et al., 2006//Miura S et al., 2006/

Monocyte chemotactic protein – (MCP-1), Monocyte chemotactic protein – (MCP-1), macrophage infiltration, PGF2alfa - SM macrophage infiltration, PGF2alfa - SM and IM and IM sigg. elevatedsigg. elevated compared to SSM, compared to SSM, healthy myometrium or endometriumhealthy myometrium or endometrium

Size of myoma not important!!!!!Size of myoma not important!!!!!

The outcome of pregnancy with fibroidsThe outcome of pregnancy with fibroids

Univ. Med. School ZagrebUniv. Med. School Zagreb 2008-2012, 2008-2012, N=135N=135

mean age 35,4 y.mean age 35,4 y.

17 previous 17 previous myomectomy:17,6%myomectomy:17,6%

35 previous delivery: 25, 9%35 previous delivery: 25, 9% 84 first pregnancy: 62, 2%84 first pregnancy: 62, 2% 22 previous misccariage: 22 previous misccariage:

16, 2%16, 2% IVF: 9 (6,6%)IVF: 9 (6,6%)

0

20

40

60

80

100

>3732-3628-31<28

>37 87,4

32-36 5,9

28-31 2,9

<28 3,7

%

Vaginal delivery: 39 (28, 8%)Complications: 6 (4, 4%)

Ruptura uteri completa 1Hysterectomia abd.: 5 N newborns >22 tj = 134 (1 triplets , 1 twins)

Perinatal mortality 1/134 = 7,4%o23 weeks, Apgar 1/0

Skrablin et al.Eur J Obstet Gynecol Reprod Biol.

2005;118(1):115-6.

Ap.1.:9,1Ap.2.:9,4*pH 7,26

The ooutcome of pregnancy after The ooutcome of pregnancy after myomectomy myomectomy

Univ. Med. school ZagrebUniv. Med. school Zagreb 2008-2012, 2008-2012, N=50N=50

mean age 35,9 y.mean age 35,9 y.

15 previous birth: 30,0%15 previous birth: 30,0% 27 first pregnancy: 54,0%27 first pregnancy: 54,0% 9 previous miscarriage: 9 previous miscarriage:

18,0%18,0% 3 IVF: 6%3 IVF: 6%

0

20

40

60

80

100

>3732-3628-31<28

>37 88

32-36 2

28-31 6

<28 2

%

Vaginal birth: 4 %Complications: 1 (2, 0%)

Ruptura uteri completa 1 (31 tj., mors fetus in grav)

Technique: 18 lap: 36,0 %

26 LPSC: 52,0%2 Hys: 4%1 sc: 2%

1 conversion lpsc to lap : 2%2?

N = 52 newborns(3 twins)

Perinatal mortality:3/52= 57,6%o

(26 gemin31 ruptura32 IUGR)

Skrablin et al.. Successful pregnancy

after spontaneous rupture of scarred uterus following

fundal myomectomy. Eur J Obstet Gynecol Reprod Biol.

2005;121(2):251-2

Ap.1.:8,7Ap.2:9,1*pH: 7,16

The effect of myomectomy

PCT – spontaneous pregnancies- 15% increase in fertility, outcome of

pregnancy unknown /Casini ML et al., 2006/

- 10 reports – risk of complications after myomectomy can not be determined with certainty

/Viswanathan et al., 2007/

SM

The effect of myomectomy randomized “matched control”studyrandomized “matched control”study

/Shokeir T, et al. 2010//Shokeir T, et al. 2010/

Hys miomectomyN = 101

Hys – biopsyN=103

63,4% 28,2%pregnancy

Success with op. with type O and I, but NOT with myoma type II

SM

The effect of myomectomy /Pritts EA, /Pritts EA,

Parker WH, Olive DL, 2009/Parker WH, Olive DL, 2009/

IM IM N RR pN RR p

(studies)(studies)

CPR CPR 2 3,76 2 3,76 n.s.n.s.

LBR LBR 1 1,67 1 1,67 n.s.n.s.

SASA 1 0,76 1 0,76 n.s.n.s.

“..as yet no data

to support myomectomy in the treatment of IM myomas to improve fertility outcome”

IM

Tulandi, Barbieri, Falk, Uptodate Tulandi, Barbieri, Falk, Uptodate 20112011

AAsymptomatic leiomyomassymptomatic leiomyomas

- Suggestion - Suggestion not not to to postpone pregnancypostpone pregnancy, since leiomyomas, , since leiomyomas, combined with advanced maternal age, may impair fertility and adversely combined with advanced maternal age, may impair fertility and adversely impact pregnancy (impact pregnancy (Grade 2CGrade 2C))

- - Suggestion nSuggestion not ot to to perform prophylactic perform prophylactic myomectomy to myomectomy to prevent pregnancy prevent pregnancy complicationscomplications ( (Grade 2CGrade 2C))

IInfertile or a history of recurrent pregnancy nfertile or a history of recurrent pregnancy loss: loss:

- S- Submucosal or an intracavitary componentubmucosal or an intracavitary component - - myomectomy ( myomectomy (Grade 2CGrade 2C).). - S- Subserosalubserosal - - against myomectomy ( against myomectomy (Grade 2CGrade 2C).). - I- Intramural fibroids that do not distort the uterine cavity, other sources of ntramural fibroids that do not distort the uterine cavity, other sources of

infertility should be infertility should be addressed. The decision to perform a myomectomy addressed. The decision to perform a myomectomy should be made based on patient preference and clinical factors (eg, should be made based on patient preference and clinical factors (eg, obstructing of a fallopian tube or the cervical canal or failure of other obstructing of a fallopian tube or the cervical canal or failure of other infertility treatments). infertility treatments).

IVF Submucosal fibroid or an intramural fibroid that deforms the uterine cavity

should be removed (Grade 2C).

ENDOMETRIAL POLYPENDOMETRIAL POLYP

Implantation with Implantation with endometrial polypendometrial polyp

Low IGFBP1 and osteopontin Low IGFBP1 and osteopontin Low P receptor expression – P Low P receptor expression – P

resistencyresistency After polypectomy elevation of After polypectomy elevation of

IGFBP1, osteopontin levels IGFBP1, osteopontin levels /Ben-Nagy et /Ben-Nagy et al., 2009/al., 2009/

The effect of polypectomy in The effect of polypectomy in subfertile womensubfertile women

0

10

20

30

40

50

60

70

S C S C S C

Implantation ratePregnancy rate %Miscarriage %Clinical pregnancy %Live birth %

Perez-Medina et al.2005IUI

randomizirana

Lass et al.1999 Isikoglu et al.2006

S- IVF/ICSI/IUI Polipectomy

C-IVF/ICSI/IUI

–Without polipectomy

The only randomized

Polyps- prior to IVF

should be removed-during the course of COH-

- management individualized/Afifi K, et al., 2010/

Septum uteriSeptum uteri /Revel, 2012//Revel, 2012/

Blood flow dearrangedBlood flow dearranged UTERUS ARCUATUS - less than 1 cmUTERUS ARCUATUS - less than 1 cm, , - -

probably without ill effectprobably without ill effect Hys -Hys - simple simple Still dubious whether to perform op. before Still dubious whether to perform op. before

conceptionconception PPeer opinion polleer opinion poll – – in support of in support of

prophylactic operation less than 50% of prophylactic operation less than 50% of experts!!!!experts!!!! //M.J. Cohen, T.S. Rosenzweig, A. RevelM.J. Cohen, T.S. Rosenzweig, A. Revel, 2007/, 2007/

Asherman SyAsherman Sy

Sperm transport or implantation compromisedSperm transport or implantation compromised InfertilitInfertility iny in 802 802 / 21/ 2151 (43%)51 (43%)-- depending on severitydepending on severity PR – no operation PR – no operation 51% (540 of 1052), 51% (540 of 1052),

- no therapy 46% - no therapy 46% (133 of 292) (133 of 292) - Hys 74%- Hys 74% (468 of 632) (468 of 632) //J.G. Schenker, E.J. Margalioth J.G. Schenker, E.J. Margalioth 1982/1982/

After surgery : IUD ili baloonsAfter surgery : IUD ili baloons Bone marrow stem cells - regenerationBone marrow stem cells - regeneration //H.S. Taylor H.S. Taylor 2004/ 2004/

Thin endometriumThin endometrium

Problem : elevated oxygen in Problem : elevated oxygen in endometriumendometrium //R.F. Casper R.F. Casper 2011/2011/

But, implantation could be successful even But, implantation could be successful even with very thin endometriumwith very thin endometrium //3.7 mm3.7 mm//J.H. J.H. Check, R. Cohen Check, R. Cohen 2011/2011/

3D – endometrial volum compared to 2D 3D – endometrial volum compared to 2D endometrial thickness - not better for IVF endometrial thickness - not better for IVF success estimation success estimation //J. Alcazar J. Alcazar 2006/2006/

Ovarian stimulationOvarian stimulation

E elevation – endometrial E elevation – endometrial receptivivity decreasedreceptivivity decreased

CPR per transfer – sigg. better CPR per transfer – sigg. better after transfer of frozen embria after transfer of frozen embria //B.S. Shapiro B.S. Shapiro et al., 2011/et al., 2011/

PCOSPCOS

PCOSPCOS

OligoovulationOligoovulation P low and its regulatory function P low and its regulatory function

dearranged, constant unopposed E2 dearranged, constant unopposed E2 Low alfa and beta integrin, HOX-10 i Low alfa and beta integrin, HOX-10 i

IGFBP1 during secretory phase even in IGFBP1 during secretory phase even in those with ovulationsthose with ovulations

Elevated androgens - decreased HOX 10 Elevated androgens - decreased HOX 10 expression expression

Elevated A receptors, no downregulatiom Elevated A receptors, no downregulatiom Ealfa receptors Ealfa receptors

Low receptivity and steroid receptor disregulationThe consequence of low P or insulin/androgen imbalance?

/Cakmak H, Taylor HS, 2011/

ENDOMETRAL EVALUATIONENDOMETRAL EVALUATION ClinicalClinical

US– thicknes and texture - 6mmUS– thicknes and texture - 6mm- No correlation with hystologyNo correlation with hystology- ““Proliferative phase defect” –Proliferative phase defect” –- Subendometrial blood flow – no Subendometrial blood flow – no

correlation with implantation correlation with implantation success or failuresuccess or failure

■ ■ SHGSHG

In general: capability In general: capability of US methods to of US methods to estimate estimate implantation is lowimplantation is low /Cakmak H i Taylor Hs, 2011//Cakmak H i Taylor Hs, 2011/

■ ■ HysHys

PHDPHD- Endometrial dating – no criteria Endometrial dating – no criteria

for window of implantationfor window of implantation- Difficult and unpredictable in Difficult and unpredictable in

stimulated cyclesstimulated cycles- Cannot differentiate fertile from Cannot differentiate fertile from

infertile population infertile population /Coutifaris i /Coutifaris i sur. 2004/sur. 2004/

■ ■ Markers: Markers: - - p53p53 /Goodman et al., 2009//Goodman et al., 2009/ - - gene profiling /microarray/ or proteomic gene profiling /microarray/ or proteomic

analysis- apoA-Ianalysis- apoA-I /Brosens et al., 2011/ /Brosens et al., 2011/ - - alfa, beta integrin, mucin, alfa, beta integrin, mucin,

LIF,LIF, HOXA10, “endometrial HOXA10, “endometrial function test-EFT” function test-EFT” …..still …..still experimental experimental /Dubowy i sur. /Dubowy i sur. 2003/2003/

- - trombophiliatrombophilia

MRI-junctional zone

LIF and IL 11LIF and IL 11 Uterine flushingsUterine flushings – – - LIF levels - estimation of successful implantation in IVF- LIF levels - estimation of successful implantation in IVF /Makkar et /Makkar et

al., 2006/al., 2006/

- low levels in infertile women- low levels in infertile women /Delage et al., 1995//Delage et al., 1995/

- LH +2 can predict IVF success in the next cycle- LH +2 can predict IVF success in the next cycle /Mikolajzyk et al. 2007//Mikolajzyk et al. 2007/

FUTUREFUTURE – estrimation of endometrial function before – estrimation of endometrial function before aspirationaspiration

IL 11mRNA - low levels in trophoblast and plasma in IL 11mRNA - low levels in trophoblast and plasma in anembrionic gestation or miscarriageanembrionic gestation or miscarriage /Koumantaki et al., 2001//Koumantaki et al., 2001/

Therapy??? /Brinsden et al 2009/

Treatment - todayTreatment - today EndometriosisEndometriosis FibroidFibroid PolypPolyp HydrosalpinxHydrosalpinx

PCOSPCOS AdenomyosisAdenomyosis

EndometritisEndometritis TrombophiliaTrombophilia

- Ablation, GNRH - Ablation, GNRH agonistsagonists

- Miomectomy- Miomectomy- Polypectomy- Polypectomy- - Salpingectomy or tubalSalpingectomy or tubal occlusionocclusion- Weight loss, metformin- Weight loss, metformin- - GNRH agonists, surgeryGNRH agonists, surgery -Antibiotics-Antibiotics- LMWH- LMWHHeparin, aspirin – vaskularization

Cortikosteroids – immunological toleranceLIF, progesteron – receptivityIG – immunologic tolerance

Treatment– tomorrowTreatment– tomorrow….…. Reproductive surgeryReproductive surgery Endometrial scratchingEndometrial scratching /Barash A et al., 2003//Barash A et al., 2003/

““PPrimingriming”” - locally instilation - locally instilation or parenteral: or parenteral: granulocyte colony-stimulating factorgranulocyte colony-stimulating factor, HCG , HCG or or piroxicampiroxicam //N. Gleicher N. Gleicher et al., 2011, et al., 2011, R. Mansour R. Mansour et al., 2011, et al., 2011, H.S. Moon H.S. Moon et al., 2004/et al., 2004/

Stem cells Stem cells /Taylor HS, 2004/Taylor HS, 2004, Du H, Taylor HS,2010, Du H, Taylor HS,2010// Heparin, metformin – stimulation of gene expression in Heparin, metformin – stimulation of gene expression in

endometriumendometrium /Germeyer et al., 2011//Germeyer et al., 2011/ Embryo culture with adhaesion promoting factorsEmbryo culture with adhaesion promoting factors ( hyaluronic ( hyaluronic

acid, heparanase, VEGF)acid, heparanase, VEGF) / Revel et al., 2005, Hannan NJ et al., 2011// Revel et al., 2005, Hannan NJ et al., 2011/ PIFPIF /Duzy K., et al., 2011/ /Duzy K., et al., 2011/

…………………………………………………………....

Gene “improvement” Immunomodulation

“Leukonorm”“Lenograstim”

…….