Implantation window. New insights. Jose Miravet-Valenciano Research - PowerPoint PPT Presentation

Implantation window. New insights. Jose Miravet-Valenciano Research associate ERA team, IGENOMIX

Synchrony

Synchrony

Why a good quality embryo does not implant? SECONDARY VESICULAR FOLLICLE PROLIFERATIVE FOLLICLE SECRETORY FUNCTIONS CELLULAR PROLIFERATION EXTRACELLULAR MATRIX REMODELATION ANGIOGENESIS AND VASCULOGENESIS DNA SYNTHESIS PRYMARY LH peak ADHESION ION CHANNELS FOLLICLE OVULATION DAY 14 METABOLISM GLAND. SECRETION CELL DIFFERENTIATION DAY 0/28 (MENSTRUATION) METABOLISM EXTRA CELLULAR CELL COMMUNICATION TRANSPORT IMMUNE RESPONSE MATRIX CORPUS PROLIFERATION RESPONSE TO STREE DEGRADATION LUTEUM RESPONSE TO WOUNDING INHIBITION INFLAMATORY ADHESION MITOSIS RESPONSE PROTEOLYSIS REGULATION INHIBITION APOPTOSIS MID PROGESTERONE LATE SECRETORY EARLY SECRETORY SECRETORY WOI DAY 21 DAY 19 IMPLANTATION 4

Why a good quality embryo does not implant? P P +1 P +2 P +3 P +4 P +5 P +6 P +7 P +8 P +9 Epithelial PR Progesterone

Reproductive Medicine. The present More than 7 million babies have been born worldwide. ✓ 2 – 6% live births in Europe by ART. ✓ (Mansour et al, Hum Reprod. 2014) 48 million infertile couples in 2010 worldwide. ✓ (Mascarenhas et al, PLoS Med. 2012) 1,5 million IVF cycles/year worldwide. ✓

Reproductive Medicine. Our Mission ONE HEALTHY BABY AT A TIME More and better oocytes?

The cross-talk Mechanisms of implantation: Strategies for successful pregnancy. (Cha et al, Nature Medicine, 2012) 8

The Puzzle of the Endometrial Factor Transcriptomics Riesewijk et al., 2003 Simon et al., 2005 Diaz-Gimeno et al., 2011, 2013 Aghajanova et al., 2012 Secretomics Histology Van der Gaast et al., 2002, 2009 Omics Noyes al., 1950 Vilella et al., 2013 Coutifaris et al., 2004 Murray et al., 2007 Omics Immunohistochemistry Visuals ESC Lessey et al., 1995 Endometrial SC Kliman et al., 2006 Gargett et al., 2009 Microbes Cervello et al., 2010, 2011, 2012 Ultrasound Santamaría et al., 2016 Kasius et al., 2014 Doppler Microbiota Kupesick et al., 2001 Franasiak et al., 2015 Hysteroscopy Moreno et al., 2016 Rambouts et al., 2016

Endometrial Receptivity Array (ERA)

Endometrial Receptivity Analysis (ERA) ERA analyzes the expression of 238 genes in order to determine the personalized window of implantation for each patient. 238 genes Bioinformatic analysis of data Classification and prediction from gene expression Patented in 2009: PCT/ES 2009/000386 LDT with CLIA

Predictor Classifies the Molecular Receptivity Status of the Endometrium Pre-Receptive Receptive Post-Receptive

Endometrial biopsy in a hormonal replacement cycle (HRT) ESTRADIOL (Estradiol valerate 6 mg/day or similar ) PROGESTERONE (vaginal micronized ENDOMETRIAL 400 mg/12 hours or similar) ULTRASOUND BIOPSY >6.5 mm, triple layer < 1 ng/ml endogenous P P+5 (120 ± 3h) P+0 11 12 8 9 10 13 14 1 2 3 4 5 6 7 DAY OF THE CYCLE MENSTRUATION

Endometrial biopsy in a natural cycle hCG ADMINISTRATION (5000 IU) PROGESTERONE (vaginal micronized 200 mg/12 hours or similar) FOLLICLE > 18 mm ENDOMETRIAL BIOPSY hCG ADMINISTRATION (hCG + 0) hCG+7 hCG+2 14 18 20 17 19 21 1 2 15 16 DAY OF THE CYCLE MENSTRUATION

Retraining the ERA Algorithms PROLIFERATIVE POST-RECEPTIVE PRE-RECEPTIVE RECEPTIVE LATE-RECEPTIVE EARLY-RECEPTIVE

Personalized embryo transfer (pET) as a treatment for RIF of endometrial origin pET ET P+3 P+5 P+7 LH+7 LH+5 LH+9

Scientific evidence Fertil Steril. 2013 Fertil Steril. 2011 Fertil Steril. 2013 Hum Reprod. 2014 Fertil Steril. 2014 Hum Reprod. 2014 Hum Reprod. 2014

Clinical data 20,000 PATIENTS 2.4% 12.6% Post- Proliferative receptive 54 Countries >600 Clinics 85.0% Pre- 71.4% 28.6% receptive Receptive Non-receptive

Study Design Prospective, Randomized, Multicenter, International, Open label, Controlled trial (ClinicalTrials.gov Identifier: NCT01954758) Initial estimated number size: 546 Inclusion criteria ✓ Patients undergoing IVF/ICSI with their own oocytes ✓ Age ≤ 37 years ✓ BMI: 18.5-30 ✓ Normal ovarian reserve (AFC > 8; FSH < 8) ✓ Blastocyst transfer (day 5/6) ✓ PGS was not an inclusion criteria ✓ Pathology affecting the endometrial cavity must be previously operated

Study Design Day 5/6 FET DET pET Cryopreserved embryos Transfer ERA HRT Thawed embryos

The ERA-RCT Consorcium UZ Brussels, University Fertility Center, Belgium Genesis IVF, Serbia IVI, Spain (11 sites) Sbalagrm Sofia, Bulgary Oak Clinic, Japan ProcreaTec, Spain Bahceci Health Group, Turkey Stanford University, USA Instituto Vida Matamoros, Mexico IECH Monterrey, Mexico IVI Panama KKH, Singapore ReproTec, Colombia Centro de Infertilidade e Medicina Fetal do Norte Fluminense, Brazil Centro Reproduçao G Mario Covas, Brazil Huntington Medicina Reprodutiva, Brazil Embriofert, Brazil Centro Reproduçao Nilo Frantz, Brazil Recruiting Sites at the Interim (12) Active Sites with EC/IRB approval (28)

Reproductive Outcome FET DET pET Pregnancy rate/ET (%) 61.7 60.8 85.7* (37/60) (45/74) (42/49) 0.003 Implantation rate (%) 35.3 41.4 47.8 (36/102) (53/128) (43/90) 0.21 Biochemical pregnancies (%) 21.6 6.7 11.9 (8/37) (3/45) (5/42) 0.13 Ectopic pregnancies (%) 2.7 0 2.4 (1/37) (0/45) (1/42) 0.55 Clinical miscarriages (%) 5.4 20.0 21.4 (2/37) (9/45) (9/42) 0.10 Ongoing pregnancy/ET (%) 43.3 44.6 55.1 (26/60) (33/74) (27/49) 0.24 Twins (%) 28.6 26.2 19.4 (8/28) (11/42) (7/36) 0.66 Singleton (%) 71.4 73.8 80.6 (20/28) (31/42) (29/36) 0.66 * p value <0.05 by Chi-Square test

The Human Microbiome • Humans have 10x > bacteria than cells • A person of 70 kg weight has 1 Kg of bacteria cohabitants • Our body contains bacteria, particularly abundant in the skin and digestive tract • Between 20 and 60% of these bacteria (depending on location) cannot be cultured

The Questions ✓ Is there a specific endometrial microbiome? And if so ✓ Could the endometrial microbiome play a role in endometrial receptivity and pregnancy outcomes?

Methods ✓ Molecular assessment of endometrial microbiota by NGS gDNA PURIFICATION ENDOMETRIAL/VAGINAL ASPIRATION 16S rRNA gene BARCODED BACTERIAL 16S rRNA PCR DATA ANALYSIS & TAXONOMICAL ASSIGNMENT SEQUENCING

The evidence

Study 1 En Endom omet etrial ial an and vaginal nal mi micr crobiota obiota differ er in so some e as asympto toma matic tic subjec ects ts Subjects: n=13 Paired samples Endometrium-Vagina: n=26 Total samples analyzed: n=52

Endometrial vs vaginal microbiota in fertile subjects Pre-receptive (LH+2) Receptive (LH+7) E: Endometrial fluid V: Vaginal aspirate Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703

Conclusion Study 1 The uterine cavity is not sterile. Endometrial and Vaginal Microbiomes are different in asymptomatic women.

Study 2 Regula ulation tion of en endom domet etrial ial mi micr crobi obiota ota duri ring ng th the e ac acquisi isition tion of en endometri ometrial al rec eceptiv tivity ity Subjects: n= 22 Paired samples LH+2 - LH+7: n=22 Total samples analyzed: n=44

Endometrial microbiota profile of asymptomatic women during the acquisition of endometrial receptivity High percentage of Lactobacillus or Lactobacillus -dominated Microbiota Subjects 1 to 22 Pre-receptive (LH2) Receptive (LH7) Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703

Conclusion Study 2 Endometrial Microbiome is not regulated by hormones during the acquisition of endometrial receptivity.

Study 3 Funct nctional ional impact act of the endomet dometrial ial microbiota obiota compos mposition ition on repr productiv oductive e outco come me in patient ients s under dergoing oing IVF VF Patients analyzed: n=35 Total samples analyzed: n=41

Low abundance of Lactobacillus in endometrium is associated with poor reproductive IVF outcomes BMI: body mass index; LDM: Lactobacillus -dominated microbiota; NLDM: non- Lactobacillus -dominated microbiota ; *Chi Square (χ² test) and Student’s t -test were performed; *p-value<0.05; § : Voluntary termination of pregnancy. Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703

Low abundance of Lactobacillus in endometrium is associated with poor reproductive IVF outcomes 1.00 0.75 0.50 0.25 0.00 Live birth Miscarriage Non-Pregnant Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703

Conclusion Study 3 Endometrial Microbiome affects embryonic implantation and ongoing pregnancy.

Final conclusions Variability is the law of life, so receptivity depends on the patient. ✓ The transcriptomic signature of endometrial receptivity reveals that the ✓ endometrial factor is responsible for 25% of cases of patients with recurrent implantation failure. After correcting the window of implantation, personalized embryo transfer ✓ normalizes clinical results. The microbiological level should be considered to perform an endometrial ✓ receptivity assessment.

Final conclusions “Insanity: doing the same thing over “Insanity: doing the same thing over and over again and expecting LET’S GO PERSONAL and over again and expecting different results” different results”