The Implantation Gap

Home , Luteal support

ESHRE Campus

Early Pregnancy Winter Course

Recurrent Implantation Failure

Nick Macklon

The Implantation Gap

100

80 60 ? 40

0 Implantation Oocyte retrieval Fertilised oocytes Embryo transfer

Lecture Overview

•Current approaches to diagnosis

•Current approaches to therapy

•Novel approaches The Iceberg of Pregnancy Loss

(Macklon et al, Hum Reprod Update, 2002)

Clinical Live birth Miscarriage

Post-implantation Pre-clinical

Pre-implantation

Conceptions

Causes of implantation failure

Hydrosalpinx/ COH Endom. cavity

Trombophilias

Embryo Endometrium (morphologic, genetic) (morphologic, genetic)

Embryo transfer

Current approaches to diagnosis

•Inherited Thrombophilia Increased incidence in RIF •Aquired thrombophilia Qublan et al, Hum Reprod 2006

•Thyroid abnormalities

•Hydrosalpinges

•Uterine anomaly

•Infections Dam et al Gyn Obstet Invest 2006

Pre-implantation genetic screening Voullaire et al, Fertil Steril. 2007. Current approaches to diagnosis

Current approaches to management

•Treatment of thrombophilias

•Empirical therapies

•Surgical interventions

•Assisted hatching/prolonged in-vitro culture

•Pre-implantation genetic screening

Boomsma and Macklon 2006

Aspirin: the evidence from RCTS

Study n Dose Pregnancy Rate p

Rubenstein 1999 298 100mg from CD 21 45% vs 28% NS

Waldenström 2004 1380 75mg from ET 35% vs 30% NS

Pakkila 2005 374 100mg from stim 25% vs 28% NS

Duvan 2006 100 100mg from ET 24% vs 23% NS Aspirin: evidence in implantation failure

•RCT double blind, placebo controlled trial, 143 women

Aspirin (100 mg/day) + heparin (5000 IU b.d.) vs placebo

Treatment Control OR 95% CI

Implantation rate 6.8% 8.5% 0.65 (0.33-1.28)

Miscarriage rate 21% 18% 1.2 (0.8-2.0)

Stern et al, 2003

Nitric oxide donors: evidence

• Sildanefil from Day 3 improves uterine blood flow and endometrial development (Sher & Fisch 2000) • RCT of NO donors (5mg NTG patch; 1 day prior to ET) Vs Placebo - Ohl et al 2002

NO donor Placebo group group (n = 68) (n = 70) No of oocytes retrieved 9.5 ± 4.1 10.2 ± 5.0 No of ‘good’ quality embryos 2.8 ± 0.7 2.8 ± 0.6 / trf Pregnancy rate / transfer 28.6% 27.9% Clinical pregnancy rate 22.9% 26.5%

High NO levels increase fragmentation

(n=7) 50 (n=17) 80

)

(

O 25 40 (n=10)

(n=8) O

Poor Good Pregnant Non

Embryo quality Lee et al, 2004 Progesterone treatment:rationale

Blois et al, J. Immunol 2004

Background levels of P4 in IVF

41 IVF pregnancies vs 43 spontaneous pregnancies

IVF pregnancy

Spontaneous pregnancy •

*P<0.05

(Costea et al, 2000)

Progesterone supplements during early IVF gestation: evidence

Impact on delivery rate after 3 weeks progesterone* after a positive hCG test

Study Control RCT 100 Two centre RCT % 80 200 per matched group 60 40 Both groups luteal support 20 0 s s s s s e k k k ie *600mg/day PV ci e e e r n e e e e a w w w iv n l 7 7 7 e eg g > D r n n n P io i o l rt o ti a o g r ic b n o m A O b e A ch io B (Nyboe Anderson et al, 2002) The rationale for glucocorticoids

•Defect in cytokine network and excess of NK cell activity implicated in implantation failure (Ledee 2004,2005)

•Reduce the NK cell count (Pountain, 1993)

•Normalise the cytokine expression profile

•Suppression of endometrial inflammation (Hill 1990)

The data? •Variations in results •Inadequately powered studies

Results of meta-analysis

12 studies , 1669 patients

Pregnancy rate per couple

Review: Peri-implantation glucocorticoid administration for assisted reproductive technology cycles Comparison: 01 Glucocorticoids versus no glucocorticoids/ placebo Outcome: 02 Pregnancy rate per couple

Study Glucocorticoids Control OR (fixed) Weight OR (fixed) or sub-category n/N n/N 95% CI % 95% CI

Kemeter-a 16/73 6/73 3.23 3.13 [1.15, 8.54] Tan 7/17 5/14 2.22 1.26 [0.29, 5.42] Catt 8/56 6/55 3.58 1.36 [0.44, 4.22] Moffit 49/131 47/136 19.89 1.13 [0.69, 1.87] Ando 12/23 16/35 4.18 1.30 [0.45, 3.72] Bider-a 9/54 4/24 3.18 1.00 [0.28, 3.63] Mottla 17/39 12/36 4.85 1.55 [0.60, 3.95] Bider-b 7/52 6/47 3.76 1.06 [0.33, 3.42] Kim CH-a 33/43 29/44 4.59 1.71 [0.66, 4.38] Botti 13/39 17/52 6.69 1.03 [0.43, 2.49] Ubaldi-b 21/50 24/50 9.59 0.78 [0.36, 1.73] Ezzeldin 66/267 65/259 34.23 0.98 [0.66, 1.46]

Total (95% CI) 844 825 100.00 1.16 [0.93, 1.45] Total events: 258 (Glucocorticoids), 237 (Control) Test for heterogeneity: Chi² = 6.71, df = 11 (P = 0.82), I² = 0% Test for overall effect: Z = 1.33 (P = 0.18)

0.1 0.2 0.5 1 2 5 10 Favours control Favours Steroids

Live birth per couple

Study Glucocorticoids Control OR (fixed) Weight OR (fixed) or sub-category n/N n/N 95% CI % 95% CI

Ando 7/23 14/35 100.00 0.66 [0.21, 2.00]

Total (95% CI) 23 35 100.00 0.66 [0.21, 2.00] Total events: 7 (Glucocorticoids), 14 (Control) Test for heterogeneity: not applicable Test for overall effect: Z = 0.74 (P = 0.46)

0.1 0.2 0.5 1 2 5 10 Favours controls Favours Steroids Miscarriage rate per couple

Study Glucocorticoids Control OR (fixed) Weight OR (fixed) or sub-category n/N n/N 95% CI % 95% CI

Kemeter-a 4/73 3/73 12.84 1.35 [0.29, 6.27] Moffit 14/131 12/136 47.62 1.24 [0.55, 2.78] Ando 5/23 2/35 5.62 4.58 [0.81, 26.05] Mottla 5/39 2/36 8.21 2.50 [0.45, 13.79] Bider-b 1/52 1/47 4.66 0.90 [0.05, 14.84] Kim CH-a 4/43 3/44 12.18 1.40 [0.29, 6.67] Ubaldi-b 1/50 2/50 8.87 0.49 [0.04, 5.58]

Total (95% CI) 411 421 100.00 1.48 [0.87, 2.53] Total events: 34 (Glucocorticoids), 25 (Control) Test for heterogeneity: Chi² = 3.11, df = 6 (P = 0.80), I² = 0% Test for overall effect: Z = 1.44 (P = 0.15)

0.1 0.2 0.5 1 2 5 10 Favours Steroids Favours control Can PGS reduce EPL?

Prospective matched controlled study in 74 women

PGS for 10 chromosomes No PGS in two blastomeres.

Day 3 or 4: embryo transfer

Day 6: start daily urine collection

Day 15: end urine collection and pregnancy test

Urine analysis with high sensitivity hCG assay, corrected for creatinine levels

Cut off level of hCG determined by oocyte donor curves

Urinary hCG concentrations and outcome hCG Profile

10000 Ongoing Pregnancy

1000 Miscarriage

EPL

100

No mU hCG/mmol mU Kreat 10 implantation

1 6 7 8 9 10 11 12 13 14 15 days after OPU mean hCG singleton pregn mean hCG donor mean hCG EPL mean hCG no EPL mean hCG sp abortions

PGS reduces peri-implantation loss Experimental approaches

• hrLIF • NK cell tests • IVIG infusion • Allogenic lymphocyte therapy

New approaches in Implantation Failure

•New approaches to understanding human implantation

•New approaches to studying the endometrial factor

•New therapeutic approaches

Embryo-endometrial cross talk: in vitro model

Thawing cryo-preserved Day 4 embryos

in vitro implantation Day 5 after extended culture Day 8 Human Embryo

Assessment of Cytokine profile Day 8 in vitro implantation

G. Tecklenberg et al Significanty lower cytokine/trophic factor expression in arrested embryo implantation in vitro

IL-1β HB-EGF

18,00 12,00

16,00 10,00

14,00 8,00

12,00

6,00

10,00

4,00 8,00

2,00 6,00

6 4,00 0,00

arrested control developed arrested control developed Tecklenberg et al 2007

The embryo dysregulates the endometrial stromal cell response

receptive endometrium

Endometrial Embryo Stromal Cells

Receptive Maintainance of cytokines/trophic factors cytokines trophic factors receptors Non receptive Downregulation of cytokines/trophic factors

Tecklenberg et al 2007

Ovarian Stimulation and the endometrium

• Ovarian stimulation affects endometrial receptivity

• Supraphysiological sex steroid levels as cause

• Evidence for action at level of gene expression

•hMG and GnRH agonist versus natural cycle Horjacas et al 2004

•recFSH and GnRH antagonist/agonist and P4 versus natural cycle Mirkin et al 2004

•recFSH and GnRH antagonist only versus natural cycle Macklon et al 2008 Δgene expression = Δ Endometrial receptivity?

Endometrial secretion aspiration

aspiration control (N=66) (N=66) Implantation rate 23 % 18 % Positive pregnancy test 36 % 33 %

Pregnancy confirmed by 33 % 30 % NS ultrasound

Van der Gaast et al. Reprod Biomed Online, 2002.

The intrauterine environment

Pro-inflammatory cytokines IFN-γ IL-1, IL-12, IL-15, IL-17, TNFα

Anti-inflammatory cytokines IL-5, IL-6, IL-10

Chemokines CXCL 10, MCP-1, MIF, Eotaxin

Growth Factors VEGF, HB-EGF

Signaling Factors DKK-1 Impact of ovarian stimulation?

•240 women undergoing first cycle IVF

•Exclusion criteria: >41 years of age

Endometrial Oocyte pick-up Endometrial aspiration (N=40) aspiration

0.25mg GnRH ant. ET 150 IU rec FSH hCG

LH peak+6 CD 2 CD 6 Culture day 4 Boomsma et al

The impact of ovarian stimulation

32:1

16:1

8:1

4:1

2:1

1:1

1:2

1:4 Fold change in expression in IVF versus natural cycle

1:8 IL1 IL5 IL12 IL17 TNF VEGF MIF EOtax DKK1 Boomsma et al 2007

Combined diagnostic techniques

• Ledee et al J Reprod Immunol. 2007 Mild stimulation as a therapeutic option

Exogenous FSH Exogenous HMG/FSH threshold

FSH thres level window hold window window

20 menses Follicle 15 size (mm) 10

5 (atresia) 1 7 14 1 7 14 Cycle day Stimulation day menses

Macklon et al, Endocrine Reviews, April 2006

Mild Stimulation and Embryo Quality

% embryos score 1 Hohmann et al JCEM 2003 80 P = 0.008

60 61

29 20

0 Conventional Mild

protocol

Effect of ovarian stimulation on embryo aneuploidy

Conventional (n=44) GnRH Agonist

rFSH 225 IE/day 2 wks Ultrasound 0 1 2 3 3 21 3 5 8

Ultrasound rFSH 150 IE/day hCG hCG hCG

GnRH embryos normal of Transfer Mild (n=67) Antagonist

111 patients fertilization of Assessment Oocyte retrieval and insemination retrieval Oocyte 528 fertilized oocytes and FISH analysis biopsy Embryo 302 embryos FISHed Baart, Human Reproduction 2007 Chromosomally competent embryos generated after mild versus conventional stimulation

20 ** 18 Conventional 16 Mild 14 * 12 10 8 6

Average number 4 2 0 s s l o a s te y y r m o c b r ry o o b m N O E m e Baart, Human Reproduction 2007

Conclusions

•Implantation failure remains the major challenge in IVF

•Multifactorial

•No effective evidence-based treatment

•Requires new approaches:

Careful phenotypic and genetic studies

Better understanding of human implantation

Rational therapies

Realistic expectations The UMCU Periconception Group

Research Fellows National collaborators Carolien Boomsma Annemieke Kavelaars Gijs Teklenberg Cobi Heijnen Monique Sterrenberg Gerard Visser Benoit Jacod Frank Holstege Margarida Avo Regine Steegers-Thuinissen Lan Chao Guo Meng

Research co-supervisors International collaborators Renee Eijkemans Helen Mardon Oxford Dagmaar Gutknecht Linda Giudice San Francisco Aaron Hsueh Stanford Jan Brosens London Zi-Jiang Chen Jinan