Repeated Implantation Failure Mechanisms Antonis Makrigiannakis MD Ph

Repeated Implantation Failure Mechanisms Antonis Makrigiannakis MD, Ph. D Professor of Obstetrics and Gynecology Medical School, University of Crete

What is RIF • • • How many attempts? How many embryos? Age? Embryo quality? Failure in the presence of potential obstacles?

RECURRENT IMPLANTATION FAILURE • Failure to achieve a pregnancy after 3 completed fresh IVF-ET cycles (Tan et al 2005) • Failure of ≥ 4 -10 embryos to implant • In the era of SET/DET should the definition of RIF be revised

WHY SHOULD IMPLANTATION FAIL TO TAKE PLACE ?

Difficulties in studying the process of implantation Succesfull implantation requires “two-way interaction” blastocyst - endometrium

Implantation - key event in the establishment of pregnancy Apposition Adhesion Invasion Embryo Uterine epithelium Endometrial stroma Invading trophoblast Continuous process from conception to 22 weeks gestation

Factors implicated in RIF Hydrosalpinx/ Uterine alterations Embryos (morphology and genetics) Ovarian stimulation Uterus (morphology) Transfer procedure

TREATMENT STRATEGIES FOR RECURRENT IMPLANTATION FAILURE • Embryo • Endometrium

EMBRYO • • • Preimplantation genetic diagnosis Blastocyst transfer Assisted hatching Co-culture of embryos with endometrium Other methods of embryo selection Donor oocyte/embryo

TREATMENTS OF PROVEN BENEFIT embryo : assisted hatching

ENDOMETRIUM • Hysteroscopy • Hydrosalpinges • Reproductive immunology • Novel approaches

Histopathology-Immunology of implantation site T=interstitial EVT, E=intravascular EVT, K=u. NK cells, M=macrophage, L=T cells

Th 1/Th 2 balance in Normal Invasion NK cell “Paternal” Antigen APC Embryo IL-12 IL-18 Th 0 Cell IL-4 IL-10 Th 1 Helper cell -- X IFN Cytotoxic T cell X Cell mediated immunity M Th 2 Helper cell IL- 4 IL-10 B cell Antibody mediated immunity

Th 1/Th 2 balance in failed invasion NK cell “Paternal” Antigen IL-12 IL-18 APC Embryo Th 0 Cell IL-4 IL-10 Th 1 Helper cell IFN Cytotoxic T cell - Cell mediated immunity M Th 2 Helper cell IL- 4 IL-10 B cell Antibody mediated immunity

ENDOMETRIUM • Hysteroscopy • Hydrosalpinges • Reproductive immunology • Novel approaches

Does endometrial scratching promote implantation and live birth rates in patients with RIF?

The first evidence was reported at the beginning of the 20 th century! Mechanical manipulation has been first shown to be associated with decidual formation in guinea pig. scratching the uterus during the progestational phase of the estrous cycle provoked a rapid growth of decidual cells Loeb L. Zentralblatt fur allgemeine Pathologie und pathologische Anatomie 18 563– 565. 1907

Systematic local endometrial injury & IVF outcome Local endometrial injury improves clinical pregnancy rate Reproductive Bio. Medicine Online (2012) 25, 345– 354

Summary of the live birth/ ongoing pregnancy rate for the 5 studies included in the systematic review Improvement of live birth/ongoing pregnancy rate Reproductive Bio. Medicine Online (2012) 25, 345– 354

Endometrial scratching is beneficial to live birth and ongoing pregnancy rates

Endometrial scratching is beneficial especially to RIF: Clinical pregnancy rate

Endometrial scratching is beneficial especially to RIF: Live birth rate

Issues to be addressed • Which is the best cycle? – Evidence support the performance of endometrial biopsy one cycle prior to IVF – It is advisable not to perform endometrial injury on the day of oocyte retrieval because it appears to significantly reduce clinical and ongoing pregnancy rates. Cochrane Database Syst Rev. 2012 Jul 11; 7: CD 009517.

Possible mechanisms Genes up-regulated by endometrial biopsy Up-regulation of receptivity molecules Kalma et al. Fertil Steril 2009; 91: 1042

Endometrial biopsy of macrophages/dendritic cells • tumor necrosis factor-a (TNF-a), • growth-regulated oncogene-a (GROa), • interleukin-15 (IL-15), • macrophage inflammatory protein 1 B (MIP-1 B), • Osteopontin A positive correlation was found between the levels of macrophages/dendritic cells, MIP-1 B expression, and TNF-a expression & the pregnancy outcome. Gnainsky et al. Fertil & Steril 2010; 94: 2030

Suggested mechanism for the role of endometrial injury-dependent inflammation in implantation Reproduction (2012) 144 661– 668

Does intrauterine administration of PBMCs promote implantation & pregnancy rates in patients with RIF?

Intrauterine administration of autologous PBMCs promote clinical pregnancy, implantation and live birth rates in patients with repeated failure of IVF-embryo transfer 41. 2% vs 11. 1% and 23. 4% vs 4. 1% and 35. 3% vs 5. 5% Yoshioka et al. Hum Reprod 2006; 21: 3290

• 253 cycles were studied • All women received frozen/thawed embryos. • PBMCs were not treated with HCG Okitsu et al. Journal of Reproductive Immunology 92 (2011) 82– 87

Proposed mechanisms for PBMC actions within the uterine cavity • Activated PBMC that are administered into the uterine cavity can induce adequate endometrial differentiation for embryo implantation. • PBMC can evoke favorable inflammatory reactions in the uterine cavity, for example, secreting proteases that may effectively change the function or structure of surface molecules expressed on the endometrial luminal epithelial cells. • PBMC may move from the uterine cavity toward the endometrial stromal tissue, creating a leading pathway for subsequent embryo attachment and invasion J. Mamm. Ova Res. 26, 122– 128, 2009 J. Reprod. Immunol. 81, 1– 8. , 2009

Issues to be addressed • What is the biological impact? • When is the appropriate time to administer the PBMCs? • Should PBMCs be pre-treated with HCG? • Apart from RIF? • Differential (Better) Activation of PBMCs?

DOES INTRAUTERINE ADMINISTRATION OF PBMCs PRETREATED WITH CRH PROMOTE IMPLANTATION RATES IN PATIENTS WITH RIF?

Background • Implantation sites in rat uterus contains increased CRH concentrations. Makrigiannakis et al, JCEM 1995

* *P<0. 001

Maternal Age Previous Cycles Endometrial Thickness on HCG day (mm) Av. Number of Oocytes Av. Number of MII oocytes Fertilisation (%) Grade 1 Embryos on D 3 (%) Av. Number of ET embryos Clinical Pregnancy Rate (%) Abortion Rate (%) Live Birth Rate (%) 50 Control P value Group (n=99) 34. 19 34. 09 ns 3. 13 3. 45 ns 9. 71 9. 5 ns 8. 73 7. 93 ns 6. 94 5. 97 ns 84. 02 85. 22 51. 56 ns 1. 8 1. 9 24. 27 0. 01 17. 94 41. 67 0. 01 34. 40 14. 14 0. 01 * 30 Control PBMC 20 ns 42. 00 40 Results ns 52. 6 Pregnancy Rate (%) PBMC Group (n=93) 10 0 Day 3 ET P< 0. 05

CRH induces IL 6

The expression of adhesion molecules by endometrial epithelial cells is regulated by blood monocyte-derived DCs. Freshly isolated day-12 epithelial cells were treated with either control culture medium (C) or with conditioned medium collected from cultures of blood monocyte-derived DCs (DC).

CRH decreases IFN

Intrauterine PBMCs administration & IVF outcome in RIF patients CRH & cytokine production CRH added to primary cultures of PBMCs significantly increased IL-6 (Th 2 -type immunity) release and decreased IFN-γ (Th 1 -type immunity) levels in a dose dependent manner Makrigiannakis et al, EJCI, 2015

DOES INTRAUTERINE ADMINISTRATION OF PBMCs PRETREATED WITH CRH PROMOTE IMPLANTATION RATES IN PATIENTS WITH RIF? YES!

Intrauterine PBMCs administration & IVF outcome in RIF patients CRH & endometrium • CRH induces stromal decidualization and potentiates the decidualizing effect of progesterone Makrigiannakis et al, MHR 1999 • CRH regulates local modulators of the decidualization process; it inhibits the enhancer PGE 2, induces the inhibitor interleukin (IL) 1 and stimulates the inducer IL-6. Zoumakis et al, 2000; Makrigiannakis et al 1999

DOES INTRAUTERINE ADMINISTRATION OF HCG PROMOTE IMPLANTATION RATES IN PATIENTS WITH RIF?

Ye et al 2015: HCG significantly increases biochemical and pregnancy rates Biochemical pregnancy rate Clinical pregnancy rate

However concurrent evidence on blastocyst transfer did not support this finding

No impact of HCG administratio n on Clinical & Live Birth rates

Currently meta-analysis: impossible • For the analyses of live birth and clinical pregnancy, there was considerable heterogeneity (I 2 greater than 75%). • Exploration for the sources of heterogeneity identified two key pre-specified variables as important determinants: – stage of ET (cleavage versus blastocyst stage) – Dose of IC-h. CG (less than 500 international units (IU) versus 500 IU or greater).

Endometrium-Embryo Cross-talking: Basic Science evidence

Developmentally incompetent embryos induce proteotoxic stress to the decidua Brosens et al, Scientific Rep 2014

The extended role of the Decidualised Endometium Endometrial receptivity Embryo quality control A Immunomodulation Trophoblast invasion B Tissue haemostasis Oxidative stress resistance Menstruation

From Implantation window to Selection window

The Embryo Selection Hypothesis The endometrium has an important role in embryo selection • Decidual cells selectively recognize impaired embryos and inhibit implantation • Undecidualised cells do not mount such a response. • Ability of stromal cells to express the decidual phenotype is impaired in women with recurrent miscarriage

Receptivity vs. Selectivity Hyper-Fertility Recurrent PL Macklon & Brosens, Biol Reprod 2014

Endometrial receptivity: Prediction via gene expression profiles

Signature discovery (303 genes) Validation (303 genes) Gene profiling can predict RIF with significant accuracy

Future Management of RIF? • GM-CSF

No effect in older women undergoing IVF

Future Management of RIF? • Intralipids

Need for RCT

Conclusions • Local endometrial injury, HCG, PBMC and PBMC & CRH use may improve pregnancy outcomes in women with unexplained RIF • ? ? How it works: not entirely known (Inflammation) • Use under approved clinical trials with appropriate patient consent • Need for appropriate randomized trials comparing standardized research interventions with no intervention in a well-defined RIF patient population

Conclusions CSF-1 is to be used only under strict research protocols Intralipids needs both basic science and observational evidence to support a clinical trial

THANK YOU!