IVF 2.0: Enabling comprehensive genetic

testing in Reproductive Medicine

The Endometrial Factor:

EMMA, ALICE and ERA

Carlos Simon MD. PhD.

Professor Ob/Gyn, University of Valencia. Spain

Adjunct Clinical Professor Ob/Gyn. Baylor College,USA

Head of the Scientific Advisory Board, Igenomix

Disclosure Carlos Simon

Founder & Head of Scientific Advisory Board, Igenomix, S.L.

Editorial Editor:

External Scientific Advisor:

ART remains inefficient

Globally, live birth rates in ART range between 25% and 30% per

initiated cycle

The most fundamental reason why IVF treatments are not successful is

because transfer of good quality embryos or even euploid embryos

into the endometrial cavity does not lead to pregnancy.

Malizia B et al., N Engl J Med. 2009

Adamson et al., Fertil Steril. 2011

The contribution of the embryonic and endometrial factor

Embryo Aneuploidies

Drosophila 0.01 %

Mouse 0.01 %

Human 20 – 100 %

Implantation Rate (IR)

Natural cycle 35 %

Euploid embryos 60 %

Rodents 95 %

Rabbits 96%

(Rubio et al. RCT Fertil Steril 2017)

The main difference between humans and rodents lies in

The endometrial/decidual control in human implantation

versus

The embryo control in rodent implantation (embryonic diapause)

Personalized Medicine

“Variability is the law of life, as no two faces are the

same, so no two bodies are alike, and no two

individuals react alike, and behave alike under the

abnormal conditions which we know as disease.”

H. Sapiens 150,000 y

Sir William Osler

(1849-1919)

Days 4 - 5Days 4 - 5Days 4 - 5Days 6 - 7Days 8 - 9

Endometrial

Microbiota

Moreno et, al. AJOG 2016

Moreno et, al. AJOG 2018

Endometrial Receptivity

Diaz-Gimeno et, al. F&S 2011

Ruiz-Alonso et, al. F&S 2013

Garrido-Gomez et. al, HR 2014

Von Grothusen et. al, HR 2018

Wang W. et al. Cell. 2018 in press

Decidualization Decidualization

Garrido-Gomez et. al, JCEM 2011

Garrido-Gomez et. al, Development 2017

Garrido-Gomez et. Al, PNAS 2017

Vilella et, al. Development 2015

Balaguer et, al. MHR 2018

The Maternal Contribution

Learning objetives

Materno-Embryonic Crosstalk

Endometrial

microbiota

Endometrial

receptivity

Decidualization

Materno-embryonic crosstalk

Endometrial microbiome

Moreno I. et al., AJOG 2016

Moreno I. et al., AJOG 2018

Residents, Tourists, or Invaders?

Molecular assessment of endometrial microbiota by NGS

ENDOMETRIAL/VAGINALASPIRATION

gDNAPURIFICATION

16S rRNAgene BARCODED BACTERIAL 16S rRNA PCR

SEQUENCING DATA ANALYSIS & TAXONOMICAL ASSIGNMENT

Average bacterial communities in endometrial and vaginal microbiota of fertile subjects

In fertile and asymptomatic women:

The endometrial microbiota is similar to that onthe vagina.

It is mainly composed of Lactobacillus species.

Significant differences are found betweenendometrial and vaginal microbiota in 20% ofthese women.

Some bacterial genera are only found inendometrial samples.

Endometrium Vagina

Moreno et al., Am J Obstet Gynecol 2016

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 endometriumis associated with poor reproductive IVF outcomes

Moreno et al., Am J Obstet Gynecol 2016

Prospective Multicenter Observational MICrobiome study

Register: 03330444First release: Nov 6, 2017Last Update: Mar 25, 2019Status: Active. Not recruiting

First IRB/EC approval May 29, 2017

Last IRB/EC approval March 27, 2018

FPFI August 4, 2017

LPLI February 26, 2019

Recruitment length 1.5 years (finished)

Study length 2.5 years

MIC study:452 patients were enrolled at 13 participant sites from 8 countries worldwide

16S bacterial library

RT-PCR 16S METAGENOMICS

Enterobacteria

Escherichia coli

Enterococcus faecalis

Staphylococcus spp.

Streptococcus spp.

Gardnerella vaginalis

Neisseria gonorrhoeae

Chlamydia trachomatis

Ureaplasma urealyticum

Mycoplasma hominis

Specific amplification of CE pathogens

MICROBIOME PROFILE

OBJECTIVE: We sought to develop a molecular diagnostic method for CE equivalent to the 3 classic methods together, overcoming the bias of using any of them alone.

Figure 1.

ASSESSED FOR ELIGIBILITY (n=113)

Not DNA for RT-PCR diagnosis (n=18)

MOLECULAR DIAGNOSIS OF CE

(n=95)

SET

CE DIAGNOSED BY 3 CLASSICAL METHODS

(n=65)

Concordant CE diagnosis by

the 3 classical methods

(n=13)

Excluded for uncomplete clinical diagnosis:

Non informative (n=9)

Only 1 classical diagnosis method (n=3)

Only 2 classical diagnosis methods (n=18)

Discordant CE diagnosis by

the 3 classical methods

(n=52)

Discordant

histology+hysteroscopy

with microbial culture

(n=14)

Discordant

hysteroscopy+microbial

culture with histology

(n=21)

Discordant

histology+microbial

culture with hysteroscopy

(n=17) Moreno et al., AJOG 2018

Molecular microbiological diagnosis of chronic endometritis

POS NEG

HIS 25 40

HYS 63 2

CUL 34 31

122 73

12 1Concordant:

Total:

Accuracy (13/65): 20.00%

Comparison of 3 classical methods: Histology vs Hysteroscopy vs Microbial culture (n=65)

POS NEG

Concordant

HIS+HYS+CUL12 1

Molecular

(PCR)9 4

21 5

9 1

Total:

Concordant:

Molecular microbiology vs Concordant classical methods (n=13)

NegativePositive

Chronic endometritis

Accuracy (10/13): 76.92%

New diagnostic posibilities

ENDOMETRIAL

TISSUE

BACTERIAL DNA

16S bacterial library

RT-PCR16S

METAGENOMICS

Enterobacteria

Escherichia coli

Enterococcus faecalis

Staphylococcus spp.

Streptococcus spp.

Gardnerella vaginalis

Neisseria gonorrhoeae

Chlamydia trachomatis

Ureaplasma urealyticum

Mycoplasma hominis

Specific amplification of

CE pathogens

MICROBIOME PROFILE

HUMAN RNA ERA

Take home message:

ENDOMETRIAL MICROBIOTA

The uterine cavity is not a sterile site.

Endometrial microbiota is not a carry-over from the vagina.

The percentage of Lactobacillus together with specific pathogens

are significant variables to predict reproductive success.

The molecular diagnosis of pathogens causing CE is equivalent to

using histology, hysteroscopy and microbial culture together

Endometrial

microbiota

Endometrial

receptivity

Decidualization

Materno-embryonic crosstalk

The Maternal Contribution

Endometrial receptivity

Plasma membrane transformation

Riesewijk et al., 2003 (HG-U133 2.0) WOI

Ponnapalam et al., 2004 (Home-made array) menstrual cycle

Talbi et al., 2005 (HG-U133 2.0) menstrual cycle

WOI

Molecular medicine: human endometrial transcriptome

Díaz-Gimeno et al., Fertil Steril 2011

Receptive

ERA classifies the molecular receptivity status of the endometrium

Post-ReceptivePre-Receptive

The symphony of syncronization

Progesterone

Epithelial

PR

P P+1 P+2 P+3 P+4 P+5 P+6 P+7 P+8 P+9

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

P+5P+3 P+7

LH+7LH+5 LH+9

ETpET

ERA Publications

2014 Clinical Management of Endometrial Receptivity Semin Reprod Med. 32(5):410-4

2014Timing the window of implantation by nucleolar channel system prevalence matches the accuracy of the

endometrial receptivity arrayFertility and Sterility. 102(5):1477-81

2015 Human Endometrial Transcriptomics: Implications for Embryonic ImplantationCold Spring Harb Perspect Med.

5(7):a022996

2015 Understanding and improving endometrial receptivityCurrent Opinion in Obstetrics &

Gynecology. 27(3):187-92

2015 Is endometrial receptivity transcriptomics affected in women with endometriosis? A pilot studyReproductive BioMedicine Online.

31(5):647-54

2016 Diagnosis of endometrial-factor infertility: current approaches and new avenues for research Geburtshilfe Frauenheilkd.76(6): 699-703

2017Does an increased body mass index affect endometrial gene expression patterns in infertile patients? A functional

genomics analysisFertility and Sterility.107(3):740-748.e2

2017 Endometrial function: facts, urban legends, and an eye to the future Fertility and Sterility. 108(1):4-8

2017Implantation failure of endometrial origin: it is not pathology, but our failure to synchronize the developing embryo

with a receptive endometriumFertility and Sterility. 108(1):15-18

2017 Meta-signature of human endometrial receptivity: a meta-analysis and validation study of transcriptomic biomarkers Scientific Reports. 7(1):10077

2017Window of implantation transcriptomic stratification reveals different endometrial subsignatures associated with live

birth and biochemical pregnancyFertility and Sterility. 108(4):703-710.e3

2018 Implantation failure of endometrial origin: what is new?Current Opinion in Obstetrics and

Gynecology. PMID: 29889670

2018 Inter-cycle consistency versus test compliance in endometrial receptivity analysis testJournal of Assisted Reproduction and

Genetics. 2018 May 26. PMID: 29804174.

YEAR TITLE JOURNAL

MORE CLINICAL WORK PRO ERA RCT – Study protocol overview

01954758(1st Release: Sep 26th, 2013)(Last update: Nov 4th, 2018)

Main objective Secondary objectives

LB rate and cumulative LB rate at

1-year follow-up (pET versus FET

and pET versus ET)

Implantation rate, clinical

pregnancy, biochemical

pregnancy, spontaneous

abortion, ectopic pregnancy

obstetrical and neonatal

outcome

First IRB/EC approval June 25th 2013

EC FWA# 00027749

Last IRB/EC approval April 28th 2016

FPFI October 2013

LPLV (Last LB) September 2018

Study length 5 years

Recruitment length 4 years

My view

1978 2010

IVF 1.0

ChromosomalEmbryo Factor1

5%

IVF 2.0

LIV

E B

IRTH

RA

TES %

5

15

25

35

45

55

65

10

20

30

40

50

60

Endometrial Factor1

5%

NCT03530254 (May 21, 2018)NCT3558399 (June 15, 2018)