Implantation window. New insights. Jose Miravet-Valenciano Research associate ERA team, IGENOMIX
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Implantation window. New insights.
Jose Miravet-Valenciano
Research associate ERA team, IGENOMIX
Synchrony
Synchrony
4
WOI
PROLIFERATIVE
EARLY SECRETORYLATE SECRETORYMID
SECRETORY
OVULATION
DAY 0/28 (MENSTRUATION)
DAY 14
IMPLANTATION
SECRETORY FUNCTIONSCELLULAR PROLIFERATIONEXTRACELLULAR MATRIX REMODELATIONANGIOGENESIS AND VASCULOGENESISDNA SYNTHESISADHESION ION CHANNELS
METABOLISMTRANSPORTPROLIFERATIONINHIBITIONMITOSISINHIBITION
METABOLISMGLAND. SECRETIONCELL DIFFERENTIATIONCELL COMMUNICATIONIMMUNE RESPONSERESPONSE TO STREERESPONSE TO WOUNDINGADHESIONPROTEOLYSIS REGULATION
EXTRA CELLULAR MATRIX DEGRADATIONINFLAMATORY RESPONSEAPOPTOSIS
LH peak
PROGESTERONE
PRYMARYFOLLICLE
SECONDARY FOLLICLE VESICULAR
FOLLICLE
CORPUS
LUTEUM
DAY 19DAY 21
Why a good quality embryo does not implant?
Why a good quality embryo does not implant?
Progesterone
Epithelial
PR
P P+1 P+2 P+3 P+4 P+5 P+6 P+7 P+8 P+9
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
More and better oocytes?
ONE HEALTHY BABY AT A TIME
The cross-talk
8
Mechanisms of implantation:
Strategies for successful pregnancy.
(Cha et al, Nature Medicine, 2012)
The Puzzle of the Endometrial Factor
Omics
Visuals
UltrasoundKasius et al., 2014
HistologyNoyes al., 1950
Coutifaris et al., 2004
Murray et al., 2007
Endometrial SCGargett et al., 2009
Cervello et al., 2010, 2011, 2012
Santamaría et al., 2016
SecretomicsVan der Gaast et al., 2002, 2009
Vilella et al., 2013
TranscriptomicsRiesewijk et al., 2003
Simon et al., 2005
Diaz-Gimeno et al., 2011, 2013
Aghajanova et al., 2012
Microbes
MicrobiotaFranasiak et al., 2015
Moreno et al., 2016
DopplerKupesick et al., 2001
Omics
ESC
HysteroscopyRambouts et al., 2016
ImmunohistochemistryLessey et al., 1995
Kliman et al., 2006
Endometrial Receptivity Array (ERA)
Endometrial Receptivity Analysis (ERA)
ERA analyzes the expression of 238 genes in order to determine thepersonalized window of implantation for each patient.
Patented in 2009: PCT/ES 2009/000386
LDT with CLIA
238 genes
Bioinformatic analysis of data
Classification and prediction from gene expression
Receptive
Predictor Classifies the Molecular ReceptivityStatus of the Endometrium
Post-ReceptivePre-Receptive
DAY OF THE CYCLE
ENDOMETRIAL BIOPSY
PROGESTERONE (vaginal micronized400 mg/12 hours or similar) ULTRASOUND
>6.5 mm, triple layer< 1 ng/ml endogenous P
P+0 P+5 (120 ± 3h)
ESTRADIOL (Estradiol valerate6 mg/day or similar )
1 2 3 4 5 6 7 8 10 11 12 13 149
MENSTRUATION
Endometrial biopsy in a hormonal replacement cycle (HRT)
DAY OF THE CYCLE
ENDOMETRIAL BIOPSY
hCG+2
1 2
hCG ADMINISTRATION (hCG + 0)
FOLLICLE > 18 mm
19 20 211817161514
PROGESTERONE (vaginal micronized200 mg/12 hours or similar)
MENSTRUATION
hCG+7
hCG ADMINISTRATION (5000 IU)
Endometrial biopsy in a natural cycle
Retraining the ERA Algorithms
RECEPTIVE
LATE-RECEPTIVE
EARLY-RECEPTIVE
POST-RECEPTIVE
PRE-RECEPTIVE
PROLIFERATIVE
Personalized embryo transfer (pET) as a treatment for RIF of endometrial origin
P+5P+3 P+7
LH+7LH+5 LH+9
ETpET
Scientific evidence
Fertil Steril. 2011 Fertil Steril. 2013
Fertil Steril. 2014
Fertil Steril. 2013 Hum Reprod. 2014
Hum Reprod. 2014 Hum Reprod. 2014
20,000 PATIENTS
71.4%
Receptive
28.6%
Non-receptive
12.6% Post-
receptive
2.4%
Proliferative
85.0% Pre-
receptive
54 Countries
>600 Clinics
Clinical data
Study Design
Prospective, Randomized, Multicenter, International,
Open label, Controlled trial
(ClinicalTrials.gov Identifier: NCT01954758)
Initial estimated number size: 546
✓ 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
Inclusion criteria
Day 5/6
DET pETFET
ERATransfer
Cryopreservedembryos
Thawedembryos
HRT
Study Design
The ERA-RCT Consorcium
Stanford University, USA
IECH Monterrey, Mexico IVI Panama
Centro Reproduçao Nilo
Frantz, Brazil
IVI, Spain (11 sites)
UZ Brussels, University Fertility Center, Belgium
Bahceci Health Group, Turkey
KKH, Singapore
Genesis IVF, Serbia
Oak Clinic, Japan
Instituto Vida Matamoros, Mexico
ReproTec, Colombia
ProcreaTec, Spain
Centro de Infertilidade e Medicina Fetal
do Norte Fluminense, Brazil
Embriofert, BrazilHuntington Medicina Reprodutiva, Brazil
Centro Reproduçao G Mario Covas, Brazil
Sbalagrm Sofia, Bulgary
Active Sites with EC/IRB approval (28)
Recruiting Sites at the Interim (12)
Reproductive Outcome
* p value <0.05 by Chi-Square test
FET DET pET
Pregnancy rate/ET (%) 61.7 (37/60) 60.8 (45/74) 85.7* (42/49) 0.003
Implantation rate (%) 35.3 (36/102) 41.4 (53/128) 47.8 (43/90) 0.21
Biochemical pregnancies (%) 21.6 (8/37) 6.7 (3/45) 11.9 (5/42) 0.13
Ectopic pregnancies (%) 2.7 (1/37) 0 (0/45) 2.4 (1/42) 0.55
Clinical miscarriages (%) 5.4 (2/37) 20.0 (9/45) 21.4 (9/42) 0.10
Ongoing pregnancy/ET (%) 43.3 (26/60) 44.6 (33/74) 55.1 (27/49) 0.24
Twins (%) 28.6 (8/28) 26.2 (11/42) 19.4 (7/36) 0.66
Singleton (%) 71.4 (20/28) 73.8 (31/42) 80.6 (29/36) 0.66
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?
✓ Molecular assessment of endometrial microbiota by NGS
ENDOMETRIAL/VAGINAL
ASPIRATION
gDNA
PURIFICATION
16S rRNAgene
BARCODED BACTERIAL 16S rRNA PCR
SEQUENCING DATA ANALYSIS & TAXONOMICAL ASSIGNMENT
Methods
The evidence
Study 1
Endometrial and vaginal microbiota differ in some asymptomatic subjects
Subjects: n=13Paired samples Endometrium-Vagina: n=26Total samples analyzed: n=52
Pre-receptive (LH+2)Receptive (LH+7)E: Endometrial fluidV: Vaginal aspirate
Endometrial vs vaginal microbiota in fertile subjects
Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703
The uterine cavity is not sterile.
Endometrial and Vaginal Microbiomes
are different in asymptomatic women.
Conclusion Study 1
Study 2
Regulation of endometrial microbiota during the acquisition of endometrial receptivity
Subjects: n= 22Paired samples LH+2 - LH+7: n=22Total samples analyzed: n=44
Subjects 1 to 22Pre-receptive (LH2)Receptive (LH7)
High percentage of LactobacillusorLactobacillus-dominated Microbiota
Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703
Endometrial microbiota profile of asymptomatic womenduring the acquisition of endometrial receptivity
Endometrial Microbiome is not
regulated by hormones during the
acquisition of endometrial receptivity.
Conclusion Study 2
Study 3
Functional impact of the endometrial microbiota composition on reproductive outcome in patients undergoing IVF
Patients analyzed: n=35Total samples analyzed: n=41
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.
Low abundance of Lactobacillus in endometrium isassociated with poor reproductive IVF outcomes
Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703
1.00
0.00
0.75
0.50
0.25
Live birth Non-PregnantMiscarriage
Low abundance of Lactobacillus in endometrium isassociated with poor reproductive IVF outcomes
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.
LET’S GO PERSONAL“Insanity: doing the same thing over
and over again and expecting
different results”
“Insanity: doing the same thing over
and over again and expecting
different results”
Final conclusions
Our Team
SPAIN
Scientists
AL-ASMAR, NASSER
ALONSO VALERO, ROBERTO
BLESA JARQUE, DAVID
BOVER CATALA, ANA
CAMPOS GALINDO, INMACULADA
CERVERO SANZ, ANA CRISTINA
CLEMENTE CISCAR, MONICA
DIEZ JUAN, ANTONIO
GARCIA HERRERO, SANDRA
GIL SANCHIS, CLAUDIA
GOMEZ DE LA CRUZ, CARLOS ALFONSO
GOMEZ SANCHEZ, EVA MARIA
HERVAS LORENTE, ARANTXA
JIMENEZ ALMAZAN, JORGE
LOPEZ IGLESIAS, PILAR
MARTIN RODRIGUEZ, JULIO CESAR
MARTINEZ CONEJERO, JOSE ANTONIO
MARIN LOPEZ DE CARVAJAL, LUCIA
MARIN VALLEJO, CARLOS
MATEU BRULL, EMILIA
MILAN SANCHEZ, MIGUEL
MIR PARDO, PERE
MIRAVET VALENCIANO, JOSE ALBERTO
MORENO GIMENO, INMACULADA
NAVARRO GAYA, ROSER
PEINADO CERVERA, MARIA VANESSA
POO LLANILLO, MARIA EUGENIA
RINCON BERTOLIN, ALEJANDRO
RODRIGO VIVO, LORENA
RODRIGUEZ IGLESIAS, BEATRIZ
RUBIO LLUESA, CARMEN
RUIZ ALONSO, MARIA
SANCHEZ PIRIS, MARIA ISABEL
SANTAMARIA COSTA, JAVIER
SANZ SALVADOR, LUCIA
SIMON VALLES, CARLOS
VALBUENA PERILLA, DIANA
VERA RODRIGUEZ, MARIA
Collaborators:
SPAIN
Technicians
AGUILA CLARES, BEGOÑA
AYALA ALVAREZ, GUSTAVO LEONARDO
BERMELL JUNCOS, SOLEDAD
BOSCH IBAÑEZ, ALVARO
BURGOS LUJAN, INES
CENTELLES PASTOR, VICENTE
COLOMA MARCO, MARIA DOLORES
ESCOBEDO LUCEA, MILAGROS
ESCORCIA MORA, PATRICIA
FERRO BARBERO, AZARINA
GALVEZ VIEDMA, MARTA
GARCIA BAYARRI, VANESSA
GARCIA MORENO, MIRIAM
GOMEZ LOPEZ, MARIA
HERRERO BAENA, MARIA
IÑIGUEZ QUILES, LAURA
MARTINEZ BENITO, TANTRA
MARTINEZ ESCRIBANO, SEBASTIAN
MARTINEZ FERNANDEZ, MARIA
ASUNCION
MARTINEZ MERINO, LUCIA
MATEOS GREGORIO, PABLO
MOLES SELMA, SARA
MORATA GARCIA, MARIA JESUS
NIETO ALFANI, JESSICA
PERIS PARDO, LAURA
POZO CRUZ, ANA MARIA
SANCHEZ GONZALEZ, ESTELA
USA & CANADA
Scientists
AKINWOLE, ADEDOYIN
CINNIOGLU, CENGIZ
DARVIN, TRISTAN
HARTON, GARY
JAKUBOWSKA MILENA
KAYALI, REFIK
MAE HOOVER, LARISSA
PHILLIPS, KIMBER
SNEIDER, ALYSSA
STANKEWICZ, TIFFANY
YEH, CHRISTINE
Technicians
ALVAREZ, INALVIS
BAUTISTA, ABELARD
BOJI NESCU, ANCA
DUENAS, FRANCISCO
CUI, KATHY
GRIFFIN, MARISA
HAGHI, GHAZAL
LAYNE, NICOLE
LOANIDIS, ALEXANDROS
NGUYEN, VI
PENA, DAYTERNA
PHAM, QUON
SANTI, ANNAI
SEN, GURKAN
SHAIBI, DEREK
TUYEN, KENNY
BRASIL
Scientists
COPRERSKI, BRUNO
ESTEVES MORAES, CAMILA
RIBOLDI, MARCIA
UEHARA DE SOUZA, MARIANE
Technicians
DE GODOI IGLESIAS LIMA, GABRIELLE
SANTANA, LEILA
DUBAI
Scientists
CHOPRA, RUPALI
Technicians
ALNIMS, HAYA
ROY CHOWDGHURY, SHEWETA
SHARMA, SHEWETA
WARRIER EDAKUNNY, SHRUTI
MEXICO
Scientists
BALLESCA ESTRADA, ADRIANA
GARCIA PASCUAL, CARMEN MARÍA
Technicians
CRISTINA COYOTECATL
INDIA
Scientists
KHAJURIA, RAJNI
SINGH BUTTAR, BRINDERJIT
Technicians
UPADHYAY, DIVYESH
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