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

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Implantation window. New insights.

Jose Miravet-Valenciano Research associate ERA team, IGENOMIX

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Synchrony

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Synchrony

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WOI

PROLIFERATIVE EARLY SECRETORY LATE SECRETORY MID SECRETORY OVULATION DAY 0/28 (MENSTRUATION) DAY 14 IMPLANTATION

SECRETORY FUNCTIONS CELLULAR PROLIFERATION EXTRACELLULAR MATRIX REMODELATION ANGIOGENESIS AND VASCULOGENESIS DNA SYNTHESIS ADHESION ION CHANNELS METABOLISM TRANSPORT PROLIFERATION INHIBITION MITOSIS INHIBITION

METABOLISM

GLAND. SECRETION

CELL DIFFERENTIATION CELL COMMUNICATION IMMUNE RESPONSE RESPONSE TO STREE RESPONSE TO WOUNDING ADHESION PROTEOLYSIS REGULATION

EXTRA CELLULAR MATRIX DEGRADATION INFLAMATORY RESPONSE APOPTOSIS

LH peak PROGESTERONE PRYMARY FOLLICLE SECONDARY FOLLICLE VESICULAR FOLLICLE CORPUS LUTEUM DAY 19 DAY 21

Why a good quality embryo does not implant?

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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

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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.

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Reproductive Medicine. Our Mission

More and better oocytes?

ONE HEALTHY BABY AT A TIME

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The cross-talk

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Mechanisms of implantation: Strategies for successful pregnancy. (Cha et al, Nature Medicine, 2012)

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The Puzzle of the Endometrial Factor

Omics Visuals

Ultrasound

Kasius et al., 2014

Histology

Noyes al., 1950 Coutifaris et al., 2004 Murray et al., 2007

Endometrial SC

Gargett et al., 2009 Cervello et al., 2010, 2011, 2012 Santamaría et al., 2016

Secretomics

Van der Gaast et al., 2002, 2009 Vilella et al., 2013

Transcriptomics

Riesewijk et al., 2003 Simon et al., 2005 Diaz-Gimeno et al., 2011, 2013 Aghajanova et al., 2012

Microbes

Microbiota

Franasiak et al., 2015 Moreno et al., 2016

Doppler

Kupesick et al., 2001

Omics

ESC

Hysteroscopy

Rambouts et al., 2016

Immunohistochemistry

Lessey et al., 1995 Kliman et al., 2006

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Endometrial Receptivity Array (ERA)

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Endometrial Receptivity Analysis (ERA)

ERA analyzes the expression of 238 genes in order to determine the personalized 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

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Receptive

Predictor Classifies the Molecular Receptivity Status of the Endometrium

Post-Receptive Pre-Receptive

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DAY OF THE CYCLE

ENDOMETRIAL BIOPSY PROGESTERONE (vaginal micronized 400 mg/12 hours or similar)

ULTRASOUND >6.5 mm, triple layer < 1 ng/ml endogenous P P+0 P+5 (120 ± 3h)

ESTRADIOL (Estradiol valerate 6 mg/day or similar )

1 2 3 4 5 6 7 8 10 11 12 13 14 9

MENSTRUATION

Endometrial biopsy in a hormonal replacement cycle (HRT)

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DAY OF THE CYCLE

ENDOMETRIAL BIOPSY

hCG+2

1 2

hCG ADMINISTRATION (hCG + 0) FOLLICLE > 18 mm

19 20 21 18 17 16 15 14

PROGESTERONE (vaginal micronized 200 mg/12 hours or similar)

MENSTRUATION

hCG+7

hCG ADMINISTRATION (5000 IU)

Endometrial biopsy in a natural cycle

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Retraining the ERA Algorithms

RECEPTIVE LATE-RECEPTIVE EARLY-RECEPTIVE POST-RECEPTIVE PRE-RECEPTIVE PROLIFERATIVE

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Personalized embryo transfer (pET) as a treatment for RIF of endometrial origin

P+5 P+3 P+7 LH+7 LH+5 LH+9

ET pET

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Scientific evidence

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

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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

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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

perated

Inclusion criteria

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Day 5/6

DET pET FET

ERA Transfer

Cryopreserved embryos Thawed embryos

HRT

Study Design

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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, Brazil Huntington 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)

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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) (0/45)

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

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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

n

location) cannot be cultured

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The Questions ✓ Is there a specific endometrial microbiome?

And if so

✓ Could the endometrial microbiome play a role in endometrial receptivity and pregnancy outcomes?

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✓ Molecular assessment of endometrial microbiota by NGS

ENDOMETRIAL/VAGINAL ASPIRATION gDNA PURIFICATION

16S rRNA gene

BARCODED BACTERIAL 16S rRNA PCR SEQUENCING DATA ANALYSIS & TAXONOMICAL ASSIGNMENT

Methods

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The evidence

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Study 1 En Endom

met

etrial ial an and vaginal nal mi micr crobiota

biota 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

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Pre-receptive (LH+2) Receptive (LH+7) E: Endometrial fluid V: Vaginal aspirate

Endometrial vs vaginal microbiota in fertile subjects

Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703

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

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Study 2 Regula ulation tion of en endom domet etrial ial mi micr crobi

biota
ta duri

ring ng th the e ac acquisi isition tion of en endometri

metrial

al rec eceptiv tivity ity

Subjects: n= 22 Paired samples LH+2 - LH+7: n=22 Total samples analyzed: n=44

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Subjects 1 to 22 Pre-receptive (LH2) Receptive (LH7)

High percentage of Lactobacillus

r

Lactobacillus-dominated Microbiota

Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703

Endometrial microbiota profile of asymptomatic women during the acquisition of endometrial receptivity

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

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Study 3

Funct nctional ional impact act of the endomet dometrial ial microbiota

biota compos

mposition ition on repr productiv

ductive

e outco come me in patient ients s under dergoing

ing IVF

VF

Patients analyzed: n=35 Total samples analyzed: n=41

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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 is associated with poor reproductive IVF outcomes

Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703

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1.00 0.00 0.75 0.50 0.25

Live birth Non-Pregnant Miscarriage

Low abundance of Lactobacillus in endometrium is associated with poor reproductive IVF outcomes

Moreno et al., 2016. Am J Obstet Gynecol. 215:684-703

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

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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.

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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

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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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