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IVF IVF 2.0: 2.0: Ena Enabling ling compr comprehensiv ehensive genetic enetic testing in in Repr productiv ductive Medicine Medicine The Endome ometri trial al Facto ctor: : EM EMMA, MA, ALICE ICE and ER ERA Carlos Simon MD. PhD.
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
Founder & Head of Scientific Advisory Board, Igenomix, S.L. Editorial Editor: External Scientific Advisor:
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)
“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.”
Sir William Osler (1849-1919)
Days 4 - 5 Days 4 - 5 Days 4 - 5 Days 6 - 7 Days 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
Moreno I. et al., AJOG 2016 Moreno I. et al., AJOG 2018
ENDOMETRIAL/VAGINAL ASPIRATION gDNA PURIFICATION
16S rRNA gene
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 on the vagina. It is mainly composed of Lactobacillus species. Significant differences are found between endometrial and vaginal microbiota in 20% of these women. Some bacterial genera are
found in endometrial 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 endometrium is associated with poor reproductive IVF outcomes
Moreno et al., Am J Obstet Gynecol 2016
Prospective Multicenter Observational MICrobiome study
Register: 03330444 First release: Nov 6, 2017 Last Update: Mar 25, 2019 Status: 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.
ASSESSED FOR ELIGIBILITY (n=113)
Not DNA for RT-PCR diagnosis (n=18)
MOLECULAR DIAGNOSIS OF CE (n=95) 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
POS NEG HIS 25 40 HYS 63 2 CUL 34 31 122 73 12 1 Concordant: Total:
Accuracy (13/65): 20.00%
Comparison of 3 classical methods: Histology vs Hysteroscopy vs Microbial culture (n=65)
POS NEG Concordant HIS+HYS+CUL 12 1 Molecular (PCR) 9 4 21 5 9 1 Total: Concordant:
Molecular microbiology vs Concordant classical methods (n=13)
Negative Positive
Accuracy (10/13): 76.92%
ENDOMETRIAL TISSUE
BACTERIAL DNA
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
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
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
Díaz-Gimeno et al., Fertil Steril 2011
Receptive
ERA classifies the molecular receptivity status of the endometrium
Post-Receptive Pre-Receptive
P P+1 P+2 P+3 P+4 P+5 P+6 P+7 P+8 P+9
ERA Publi lica catio tions
2014 Clinical Management of Endometrial Receptivity Semin Reprod Med.32(5):410-4 2014 Timing the window of implantation by nucleolar channel system prevalence matches the accuracy of the endometrial receptivity array Fertility and Sterility. 102(5):1477-81 2015 Human Endometrial Transcriptomics: Implications for Embryonic Implantation Cold Spring Harb Perspect Med. 5(7):a022996 2015 Understanding and improving endometrial receptivity Current Opinion in Obstetrics &
2015 Is endometrial receptivity transcriptomics affected in women with endometriosis? A pilot study ReproductiveBioMedicine Online. 31(5):647-54 2016 Diagnosis of endometrial-factor infertility: current approaches and new avenues for research Geburtshilfe Frauenheilkd.76(6): 699-703 2017 Does an increased body mass index affect endometrial gene expression patterns in infertile patients? A functional genomics analysis Fertility 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 2017 Implantation failure of endometrial origin: it is not pathology, but our failure to synchronize the developing embryo with a receptive endometrium Fertility 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 2017 Window of implantation transcriptomic stratification reveals different endometrial subsignatures associated with live birth and biochemical pregnancy Fertility and Sterility. 108(4):703-710.e3 2018 Implantation failure of endometrial origin: what is new? Current Opinion in Obstetrics and
2018 Inter-cycle consistency versus test compliance in endometrial receptivity analysis test Journal of Assisted Reproduction and
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
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
1978 2010
IVF 1.0
Chromosomal Embryo Factor
15%
IVF 2.0
LIVE BIRTH RATES %
5 15 25 35 45 55 65
10 20 30 40 50 60
Endometrial Factor
15%
NCT035 35302 0254 4 (M (May ay 21 21, , 20 2018 18) NCT355 55839 399 (J (Jun une 15 15, , 20 2018 18)