IVF 2.0: Enabling comprehensive genetic...Professor Ob/Gyn, University of Valencia. Spain Adjunct...
Transcript of IVF 2.0: Enabling comprehensive genetic...Professor Ob/Gyn, University of Valencia. Spain Adjunct...
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)