Sticky surface a scientific review of endometrial receptivity

Sticky surface: a scientific review of endometrial receptivity Prof. Juan A Garcia-Velasco IVI – Madrid Rey Juan Carlos University Madrid – Spain

Endometrial receptivity o Limiting factor in achieving pregnancy o Multiples embryos transferred: n no implantation? n multiple pregnancy? o Poorly understood

Endometrial receptivity o “Restricted time-related period when the uterus is receptive to blastocyst attachment & invasion” o Concept of WOI – window of implantation n n Temporally frames the period of receptivity 4 -5 day interval with peak Progesterone levels Synchrony between embryo and endometrium Opens day 19/20 by loss of epithelial E 2 & P receptors Revel F&S 2012

Endometrial receptivity E 17 18 19 20 21 22 23 P P+4 P+5 P+6 P+7 P+8 P+9 P+10 Embryos attach ONLY under optimal conditions

When to start progesterone? TIMING is relevant

How to assess ER today? Endometrial dating – fertility status? o o o o IHC Chromatography RT-PCR DNA microarrays micro. RNAs Proteomics Secretomics ……. . Noyes et al. Fertil Steril 1950 - accuracy? - reproducibility? - clinical utility?

Personalized Medicine o o o Doses for COS (AMH, BMI, age…. ) Type of protocol IVF vs ICSI Embryo number and quality ……… However: endometrial status at ET is treated equally neglected customized approach lack of knowledge? Embryo transfer TIMING can be personalized

Technology helps

The age of –OMICS Different subtypes of disease Transcriptome TRANSCRIPTION DNA Transcription regulation m. RNA Alternative splicing Proteome Metabolome TRANSDUCTION Protein Transduction regulation METABOLITES

JCEM MHR HR JCEM Horcajadas JA, Pellicer A, Simón C. Hum Reprod Update 2007.

Endometrial Receptivity Array (ERA)

Endometrial Receptivity Array (ERA) Customized microarray Bioinformatic analysis of data obtained by the customized microarray Classification and prediction from gene expression. Predictors: Characterization of RECEPTIVITY transcriptome

Receptive Profile.

IF Sample NR 1. .

Predictor classifies the molecular receptivity status of the endometrium Non Receptive samples. Proliferative Pre-Receptive Post-Recep

Endometrial Receptivity Array (ERA) - Timing of the biopsy NATURAL CYCLE Endometrial biopsy must be taken on the 7 th day after the LH surge (LH+7) urine or serum preferible HORMONE REPLACEMENT THERAPY CYCLE Endometrial biopsy must be taken on day P+5, after proper E 2 priming E 2 : 6 mg/day P 4 : 800 mg/day

LH surge variants Extremely variable LH surges in configuration, amplitude and duration Short Medium Double Prolonged Single Plateau Double Multiple Direito A, et al. Fertil Steril 2013

LH surge variants When does ovulation happen according to LH surge? Onset End During After Direito A, et al. Fertil Steril 2013

Endometrial Receptivity Array (ERA) – Accuracy In a blinded study ERA classifies better than Noyes criteria Kappa value Pathologist 1 (P 1) Pathologist 2 (P 2) P 1 vs P 2 ERA 0. 618 (0. 446 -0. 791) 0. 685 (0. 545 -0. 824) 0. 622 (0. 435 -0. 839) 0. 922 (0. 815 -1. 000) 0. 61 - 0. 80 - Good Concordance 0. 81 - 1. 00 - Very Good Concordance Díaz-Gimeno P, et al. Fertil Steril 2013

Endometrial Receptivity Array (ERA) – Consistency ERA TEST ANALYZED IN THE SAME PATIENT in two endometrial biopsies up 2 -years apart Code Date First Biopsy Date Second Biopsy Months between First Biopsy Results Second Biopsy Results CON 1 09/2009 02/2012 29 Receptive (0. 908) CON 2 09/2009 03/2012 30 Receptive (0. 908) CON 3 05/2009 04/2012 35 Receptive (0. 908) CON 4 05/2009 05/2012 36 Proliferative Non Receptive (0. 864) CON 5 01/2009 05/2012 40 Proliferative Non Receptive (0. 864) CON 6 07/2009 05/2012 35 Receptive (0. 908) Díaz-Gimeno P, et al. Fertil Steril 2013

Ruiz-Alonso M, et al. Fertil Steril 2013

Ruiz-Alonso M, et al. Fertil Steril 2013

First p. ET outcome after RECEPTIVE ERA in RIF patients (n=310) No relationship with endometrial biopsy 80 IR 70 PR 60 Rate (%) 50 40 30 20 10 0 Months after ERA test Number of patients Implantation Rate (%) Pregnancy Rate (%) 1 91 37. 9 54. 9 2 87 43. 1 59. 8 3 47 44. 6 55. 3 4 30 45. 8 53. 3 5 15 58. 3 73. 3 6 40 48. 6 70. 0

Ruiz-Alonso M, et al. Fertil Steril 2013

Window of Endometrial Receptivity (WOI) P P+1 P+2 Epithelial PR Progesterone P+3 P+4 P+5 P+6 P+7 P+8 P+9

ERA clinical applicability A case report and pilot study comparing routine embryo transfer versus p. ET (Ruiz-Alonso et al. 2014 Hum Reprod 2014).

CASE REPORT Previous ART treatments in our center Routine work-up negative 1. IVF with fresh day-3 ET 2. IVF with fresh day-3 ET 3. IVF with fresh day-5 ET 4. IVF with differed day -5 ET in natural cycle 5. OD with day-3 ET in HRT cycle (P+3) 6. OD with day-3 ET in natural cycle 7. OD with day-5 ET in HRT cycle (P+5) DIAGNOSTIC INTERVENTION ERA pre-receptive at P+5, being receptive at P+7 8. OD with p. ET using day-5 blastocysts in HRT cycle after 7 days of progesterone (P+7) Succesful twin pregnancy Ruiz-Alonso, et al. 2014

Cumulative First attempt CLINICAL OUTCOME Number of patients Source of oocytes Age Number of embryos transferred Implantation rate Pregnancy rate Ongoing pregnancy rate Clinical abortion Biochemical pregnancy Total attempts Number of embryos transferred Implantation rate Pregnancy rate Ongoing pregnancy rate Clinical abortion Biochemical pregnancy ET 17 Ovum donation 40. 7 ± 4. 7 (32 -49) 1. 8 ± 0. 4 12. 9% (4/31) 23. 5% (4/17) 0% (0/4) 100% (4/4) 0. 0% (0/4) 2. 1 ± 1. 3 1. 8 ± 0. 4 10. 8% (7/65) 19. 4% (7/36) 0% (0/7) 71. 4% (5/7) 28. 6% (2/7) Ruiz-Alonso, et al. 2014

Cumulative First attempt CLINICAL OUTCOME Number of patients Source of oocytes Age Number of embryos transferred Implantation rate Pregnancy rate Ongoing pregnancy rate Clinical abortion Biochemical pregnancy Total attempts Number of embryos transferred Implantation rate Pregnancy rate Ongoing pregnancy rate Clinical abortion Biochemical pregnancy ET 17 Ovum donation 40. 7 ± 4. 7 (32 -49) 1. 8 ± 0. 4 12. 9% (4/31) 23. 5% (4/17) 0% (0/4) 100% (4/4) 0. 0% (0/4) 2. 1 ± 1. 3 1. 8 ± 0. 4 10. 8% (7/65) 19. 4% (7/36) 0% (0/7) 71. 4% (5/7) 28. 6% (2/7) ENDOMETRIAL RECEPTIVITY DIAGNOSIS USING ERA ET Receptive 0% (0/17) Pre-receptive 94% (16/17) WOI delayed 1 day 19% (3/16) WOI delayed 2 days 81% (13/16) Post-receptive 6% (1/17) WOI advanced 1 day 100% (1/1)

Cumulative First attempt CLINICAL OUTCOME Number of patients Source of oocytes Age Number of embryos transferred Implantation rate Pregnancy rate Ongoing pregnancy rate Clinical abortion Biochemical pregnancy Total attempts Number of embryos transferred Implantation rate Pregnancy rate Ongoing pregnancy rate Clinical abortion Biochemical pregnancy ET p. ET 17 Ovum donation 40. 7 ± 4. 7 (32 -49) 1. 8 ± 0. 4 1. 7 ± 0. 5 12. 9% (4/31) 34. 5% (10/29) 23. 5% (4/17) 52. 9% (9/17) 0% (0/4) 32. 3% (6/17) 100% (4/4) 0% (0/9) 0. 0% (0/4) 17. 6% (3/17) 2. 1 ± 1. 3 1. 2 ± 0. 4 1. 8 ± 0. 4 10. 8% (7/65) 40. 0% (14/35) 19. 4% (7/36) 60. 0% (12/20) 0% (0/7) 45. 0% (9/20) 71. 4% (5/7) 0% (0/12) 28. 6% (2/7) 15. 0% (3/20) ENDOMETRIAL RECEPTIVITY DIAGNOSIS USING ERA ET Receptive 0% (0/17) Pre-receptive 94% (16/17) WOI delayed 1 day 19% (3/16) WOI delayed 2 days 81% (13/16) Post-receptive 6% (1/17) WOI advanced 1 day 100% (1/1)

Randomized, multicenter, international clinical trial IVF Patients Randomization Group A ET in COS N= 814 Group C Biopsy & ERA N= 814 Group B ET in HRT N= 814 Report Receptive N= 732 Non-receptive N= 82 Randomization Reproductive Follow-up Re. Follow-up productive ET in COS vs. ET in HRT Group C (R) p. ET in HRT N= 732 Group C (NR 1) p. ET in HRT N= 41 Group C (NR 2) ET in HRT N= 41 Reproductive Follow-up p. ET vs. ET

ERA RCT EN EL MUNDO: 21 CENTROS Hannam IVF Toronto Fertility center, Brussels, BELGIUM CANADA Versys clinics, Budapest, IVI SPAIN Genesis IVF, SERBIA HUNGARY Sbalagrm Sofia, BULGARY Stanford University California, USA IVI MEXICO IECH Monterrey Bahceci health group Istambul, TURKEY Seoul Maria Hospital SOUTH KOREA IVI PANAMA KKH, SINGAPORE Fertility Huntington USP Sao Paolo BRASIL

Non-invasive diagnostic methods for the endometrial factor based on secretomics of the EFg Salamonsen 2012 Cytokines Glycoproteins Greenblatt 2011 Signaling Factors Simon 2013 Hamamah 2013 mi. RNAs Proteins Matorras 2011 Simon 2010 Prostaglandins Chemokines

Vilella, et al. 2013

PGs LEVELS IN EF OBTAINED 24 H BEFORE DAY 3 ET Concentration (nmol/g) ** * PGE 2 PGF 2 a *(p<0. 05) **(p<0. 01) Vilella, et al. 2013

Concentration (nmol/g) PGs LEVELS IN EF OBTAINED 24 H BEFORE DAY 5 ET PGE 2 PGF 2 a Vilella, et al. 2013

Conclusions ü The transcriptomic signature of endometrial receptivity (ERA test) revealed that the endometrial factor is responsible for 25% of cases of patients with RIF. ü Personalized Embryo Transfer (p. ET) normalized clinical results. ü We will be able to investigate ER through novel non-invasive biomarkers, such as lipidomics (PG´s) in endometrial secretions

Thank you! juan. garcia. velasco@ivi. es