Disclosure. Dagan Wells University of Oxford Oxford, United Kingdom
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2 Disclosure Dagan Wells University of Oxford Oxford, United Kingdom Disclosure Declared to be member of the advisory board, board of directors or other similar groups of Illumina
3 Objectives Consider Aneuploidy testing Gene mutation testing Endometrial receptivity assessment
4 Recurrent implantation failure How should we investigate? Recurrent miscarriage strategy? Morphology Evaluation of the uterine cavity (HSG, saline scan, HyCoSy, MRI) Coagulation issues Lupus anticoagulant Thrombophilia Clotting screen Immunological Anti-cardiolipin antibodies Anti-thyroid antibodies More than half remain unexplained
5 Recurrent implantation failure Genetic factors Parental karyotypes Incidence of chromosome abnormalities: Neonatal 0.2% Infertile couples 0.6% Severe male factor infertility 3.1% Couples with RPL 9.2% Males and females with RIF 2-3% High order (>6) RIF 10.8% De Sutter et al., 2012; Raziel et al., 2002; Stern et al., 1999
6 Recurrent implantation failure Genetic factors Parental karyotypes There is a clear link between RIF and chromosome abnormality (mostly translocations) Karyotyping is therefore indicated despite relatively high cost Especially clear association with chromosome abnormality for: Women with a history of RIF Males with RIF and severe male-factor infertility Couples with high-order RIF
7 Recurrent implantation failure Genetic factors Parental karyotypes Embryo aneuploidy Gene mutations/polymorphisms Endometrial gene expression profiles
8 Avoiding embryonic aneuploidy: Preimplantation genetic testing for aneuploidy (PGT-A)
9 Aneuploid blastocysts (%) PGT-A The concept Chromosome abnormality is extremely common in oocytes/embryos Problem increases with advancing maternal age At age 35 aneuploid rate is ~40% Thereafter aneuploidy increasing >0.5% per month! Female age Data from >50,000 embryos analyzed by Reprogenetics
10 Aneuploid blastocysts (%) PGT-A The concept Aneuploidy is lethal in the great majority of cases Resulting in failed implantation or miscarriage implantation rate Female age Data from >50,000 embryos analyzed by Reprogenetics
11 PGT-A The concept Standard embryo evaluations do not reveal embryos with the wrong number of chromosomes Ideally, one embryo is transferred to the uterus after chromosome screening or Munne et al., 1993
12 Increasing evidence for PGT-A efficacy 1) 3 published randomized trials and 3 more presented at conferences All show significant improvement in outcome after PGT-A Yang et al. (2012); Scott et al. (2013) Forman et al. (2013) 2) Meta-analyses/systematic reviews conclude that PGT-A is beneficial Dahdouh et al. (2015); Lee et al. (2015) 3) Large published CDC dataset concludes that PGT-A is associated with higher ongoing pregnancy rates and lower miscarriage Chang et al. (2015)
13 Implantation rate PGT-A eliminates the negative effect of maternal age on implantation 80% 70% 60% 50% 40% 30% 20% 10% 0% < >42 Maternal age Ave: 67% PGS * No PGS ** * Harton et al. (2013) Fertil Steril. and unpublished data to 8/2015. N= 2532 followed up cycles of PGT-A by acgh. ** SART 2013
14 Could PGT-A help RIF patients? Increase the chance of a livebirth? Reduce the time taken to livebirth? Most studies retrospective Inconsistent definitions of RIF Poor study design- not intention to treat Existing data mostly from obsolete technologies
15 Could PGT-A help RIF patients? What can we conclude? Specific role of aneuploidy in RIF is unclear RIF is a heterogeneous patient group PGT-A is likely to improve the chances of implantation, but less impact where underlying cause is not aneuploidy Can a subset of RIF be identified where recurrent aneuploidy is the cause? PGT-A more likely to be effective when patients have a favourable ovarian reserve
16 Genetic variation related to RIF: MTHFR gene polymorphism
17 MTHFR
18 Folate in reproduction Folic acid an important B vitamin Appropriate processing of folates is essential for reproduction Amino acid metabolism Purine and pyrimidine synthesis Methylation of proteins, lipids and nucleic acids Folate deficiency (genetic or dietary) Uracil misincorporation into DNA Slower replication, chromosome breakage Impacts implantation, endometrial receptivity, fetal viability
19 MTHFR Enzyme is encoded by the MTHFR gene More than 20 genetic variants identified Most widely studied are c.677c>t and c.1298a>c 677T and 1298C associated with reduced MTHFR activity
20 MTHFR proposed reproductive impacts Increased miscarriage risk (Cao et al., 2013; Nair et al., 2012) Aneuploid conception (Hassold et al., 2001; Kim et al., 2011) IVF treatment - embryo morphology and implantation potential (Laanpere et al., 2011; Soldo et al., 2012) Controversial
21 MTHFR genotype and subfertility IVF patients versus fertile controls 1298A>C Subfertile group have excess of 1298C (P=0.003) More 1298C homozygotes and less wildtype homozygotes Enciso et al., 2016 Human Genetics
22 MTHFR genotype and subfertility 1298C is overrepresented (X4) in couples with RIF (24% vs 6%) 1298C homozygotes frequency is doubled (P<0.001) Impact of 1298C principally when female is a carrier Suggests maternal factor (Oocyte? Endometrial?) MTHFR c.1298a>c genotype has a strong influence on fertility Individuals with compromised MTHFR activity are at risk for RIF
23 MTHFR genotype and subfertility 677C>T displays deviation from Hardy-Weinberg equilibrium Fewer heterozygous RIF patients than expected P<0.01 Enciso et al., 2016 Human Genetics
24 MTHFR genotype and subfertility Deviation from Hardy-Weinberg equilibrium is highly unusual Possible explanations: Alleles do no segregate at random into gametes Gene is under selective pressure (some genotypes less viable)
25 MTHFR genotype and subfertility Couples where male and female are homozygous for different alleles four-fold more common in RIF group than controls Such couples can only produce heterozygous embryos Is there a heterozygote disadvantage? 677T + 677C leads to defective MTHFR dimers?
26 MTHFR genotype in embryos Significant differences in c.677 allele and genotype frequencies in embryos in relation to implantation potential P<0.05
27 MTHFR genotype in embryos Embryonic MTHFR c.677c>t influences implantation capacity May play a role in ~20% of euploid implantation failures Testing MTHFR might assist embryo viability assessment Deviation from H-W equilibrium in patients and embryos Suggests certain genotypes are selected against at some point from gametogenesis and blastocyst formation
28 Genetic determination of the window of implantation
29 Genetic determination of implantation window Endometrial biopsy RNA extraction Endometrial Receptivity Assay (ERA) Analysis of expression levels of 236 genes
30 Genetic determination of implantation window Receptive: The endometrium was found to be receptive at the time of biopsy so embryo transfer should proceed as planned. Pre-receptive: The endometrium was found to be pre-receptive (not ready for implantation yet) at the time of biopsy so embryo transfer should be performed later than planned. Post-receptive: The endometrium was found to be post-receptive (no longer ready for implantation) at the time of biopsy so embryo transfer should be performed earlier than planned. Pre-receptive Receptive Post-receptive Slide courtesy of Gary Harton, igenomix
31 Genetic determination of implantation window Post-receptive Proliferative Early Post-receptive Early Pre-receptive Receptive Pre-receptive F E-PR M-PR R E-T T For an individual patient repeat biopsies show similar results even if taken years apart Slide courtesy of Gary Harton, igenomix
32 Genetic determination of implantation window 1.7% ARN non valid 10,000 PATIENTS 2.7% Invalid sample 65.8% R 2.2% Proliferative 16.5% Post-receptive 29.8% NR* 41 Countries More than 400 Clinics 81.3% Pre-receptive * Not always at P+5/LH+7/hCG+7 Slide courtesy of Gary Harton, igenomix
33 Genetic determination of implantation window Ongoing randomized trial (interim analysis N=365, April 2016 ) Fresh transfer Deferred transfer Personalized transfer Pregnancy rate/et 61.7% (37/60) 60.8% (45/74) 85.7% (42/49)* * p value <0.05 by Chi-Square test Slide courtesy of Gary Harton, igenomix
34 Conclusions RIF is a complex pathology Influenced by multiple independent factors Causes only partially understood Genetics can help reveal the underlying cause in some cases Results can suggest new clinical pathways
Indications for chromosome screening Dagan Wells, PhD, FRCPath dagan.wells@obs-gyn.ox.ac.ukgyn.ox.ac.uk Chromosome imbalance (aneuploidy) Uncontroversial data The incidence of aneuploidy Aneuploidy is
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