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We are Leading IVF Clinic India offer IVF treatments with state of art facility and High Success rate. We are pioneering the field of Fertility Managment
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Genomics of oocyte and embryo-endometrium interaction at implantation
ANDRES SALUMETS
Competence Centre on Reproductive Medicine and Biology;Women’s Clinic and Institute of Bio- and Translational Medicine, University of Tartu, ESTONIA
November / 2013
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• 1993 - Graduated from University of Tartu as biochemist• 2003 - Doctoral degree from University of Helsinki
Väestöliitto Helsingin Lapsettomuusklinikka, supervised by prof. T. Tuuri and prof. AM Suikkari
• 2010 - ... Professor of reproductive medicine at Women’s Clinic and Institute of Bio- and Translational Medicine, University of Tartu
• 2010 - ... Head of Competence Centre on Reproductive Medicine and Biology (CCRMB)
• 2010 - ... President of Baltic Fertility Society
• 2013 - ... Member of Executive Committee of ESHRE
• Consultant to IVF clinics in Tartu, Tallinn, Tbilisi and Delhi
Andres Salumets – Short CV
3UNIVERSITAS TARTUENSIS and HOSPITAL
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Karl Ernst von Baer (1792-1876)
1826
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Humans are relatively infertile species
o The chance for pregnancy per menstrual cycle is 1:3
o The cumulative pregnancy rate:
3 months - 60%6 months - 75%12 months - 85%24 months - 95%
o Average time to pregnancy 3-4 months
o Infertility exists in 10% of couples
o In vitro fertilization (IVF) is actively used in infertility treatment
Infertile couples
The Nobel Prize in Physiology or Medicine 2010
Robert G. Edwards
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Major shortcomings of IVF• IVF implantation and pregnancy results have remained almost
unchanged for 30 years. Clinical pregnancy rate of ONLY 25-30%.• The selection of the best embryos is limited – no clear criteria to
select the best embryos for transfer. Limited use of single embryo transfers.
• Too many multiple pregnancies, 25-30%.• A few possibilities to help couples with recurrent IVF failure.• Limited possibilities to analyse and modulate the endometrial
quality (receptivity).• Poor responders and limited amount of oocytes. How to coupe
with the higher age of IVF patients? • IVF complications, like ovarian hyperstimulation syndrome.• Limited sources of donor oocytes and embryos.• Too expensive.
100,0
80,0
60,0
15,03,0 2,5
0,0
20,0
40,0
60,0
80,0
100,0
120,0
Oocytes Zygotes Embryos with good
quality
Embryos replaced
Embryos implanted
Children
1200
1000
700
200
40
30
%
No.
IVF is inefficient
CA 100 IVF CYCLES8
Recent achievements• Human Genome Project – HGP,
2000 y– 46 chromosomes, XX and XY– ca 30 000 genes– 10 million single nucleotide
polymorphisms (SNPs) – CNVs – DNA copy number
variations (12% of our genome)– Gene and chromosomal mutations
Female and male fertility and infertility phenotypes 9
High-throughput OMICs analysing platforms for genetics, epigenetics, transcriptomics, proteomics and metabolomics
Recent achievements – useful for IVF
TRANSCIPTOMICS Gene expression RNA splicing
GENETICS Genes, gene variations DNA modifications
METABOLOMICS
PROTEOMICS
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OMICS in reproductive medicine
Gene variations related to the ovarian reserve
and stimulation outcome
Polar body biopsy and genetic analysis – oocyte chromosomal
aberrations. Preimplantation genetic diagnosis
Gene expression of cumulus cells and proteome analysis of follicular
fluid samples
Gene expression analysis of sperm cells
Biopsy and analysis of embryo cells for chromosomal aberrations and gene expression pattern
Proteome and metabolome analysis of embryo’s culture media
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• Genome-wide association study (GWAS) of women of European ancestry, with 38,968 women in the discovery stage, and 14,435 women in the replication stage
• Gene donors from - EGCUT (Estonian Genome Center of University of Tartu)
• In this large 2-stage GWAS analysis we confirmed 4 previously identified menopause loci and identified and replicated 13 novel loci associated with age age at natural menopause p<5e-08
Stolk et al, Nature Genetics, 2012
OVARIAN AGING = SOMATIC AGING
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Menopausal age ... ... links to DNA repair and aging pathways
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Genes and ovarian stimulation in IVF
• Predicting fertility potential, infertility treatment outcome and the duration of the reproductive period• Fertility prediction testing (ultrasound, hormones and genetic predisposition) – FERTILITY PRESERVATION AND OOCYTE FREEZING
CUMULATED GENETIC VARIATIONS >> IMPAIRED IVF STIMULATION OUTCOME >> EARLY MENOPAUSE
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Oocytes in ovaries ... IVF and fertility & menopause
Genetic variations DISCOVERED leading to earlier menopause and accelerated follicular death (earlier age-related infertility)
Genetic variations leading to more oocytes in IVF.Genetic testing of IVF patients and personalised stimulation protocols
Oocyte stem cells in ovaries – renewal of oocytes? Better drugs for IVF
hormonal stimulation (more oocytes from poor responders)
Drugs postponing the menopause and prolonging the reproductive lifespan
Oocyte freezing and biobankingGENE TESTING
and IMPROVED IVF OUTCOME
Sperm cells - 1-2%
Oocytes - ca 20% (POSITIVE RELATIONSHIP WITH WOMAN’S AGE!)
Preimplantation stage embryos - 20-50%
Spontaneous abortions (6-20) - 35%
Stillbirths (20-40) - 4%
Live births - 0,3%
Stage of conception and pregnancy (weeks)
6-8 20 400
Chromosomal aberrations
Woman’s age ↑More chromosomal aberrations in
oocytes ↑Fewer embryo implantations and
pregnancies ↓More spontaneous abortions ↑
DOWN SYNDROME TRISOMY 21
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Genomic analysis in IVF
• Polar body and embryo diagnosticsFISH – limited number of chromosomes• Array and sequencing based technologies
- Analysis of all chromosomes and gene transcripts from single cell!
• 90% of the embryos are chromosomally abnormal (polar body vs embryo diagnostics)
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Microscopics
Oocytes
Embryos
Genomics
Chromosomes
SNPs and mutations
Transcriptomics
Single mRNAs
mRNA arrays
Proteomics
Immuno-techniques
Mass spectrometry, MS
MetabolomicsHPLC
MS and spectroscopy
EMBRYO SELECTION and -OMICS
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Ultra-sensitive sensors for embryo selection
HLA-G and HCG
HLA-G “sandwich”
TIRF measurement Image
Sensor overview
SENSOR OVERVIEW
The window of implantation (WOI)
ES MS LS
TR1ES, no pinopodes MS, fully developed pinopodes
LS, no pinopodes Inf, few pinopodes
WOI 20.-24. cycle day 6-8 days after
ovulation 48 hours
PINOPODES
Altmäe et al, Reprod Sci, 2011 19
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Fertile women vs women with unexplained infertilityAbnormalities in gene expression pathways
Altmäe et al., MHR, 2010
GENE EXPRESSION
HISTOLOGY 20
We identified a number of genes in the embryo-endometrial interactions that are known to be involved in the implantation process, like LEP, LEPR, VCAN, TGFB1, laminins, collagens and fibulins In addition, several new molecules involved in the interactions were identified, like APP, BMP2, DSC2, PDGFRA, ADAMST1 and others
ENDOMETRIAL RECEPTIVITY MARKERS
Altmäe et al., 2012, Mol Endocrinology21
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Individualised day for embryo transfer
Women 1
Women 2
Women 3
D1 D2 D3 D4 D5
Endometrial biopsy and gene expression analysis
Array analysis
Selection of the BEST day for (frozen) embryo transfer!
Women 1
Women 2
Women 3
DAYS
FUTURE VIEW WITHOUT LIMITATIONS
Granulosa cells
Oocytes
Spermatogonia and sperm cells
Sertoli cells
Genetic analysis of oocytes, polar bodies and granulosa
cells
Oocyte freezing and banking
In vitro produced oocyte!
DIFFERENTIATION OF:• Embryonal stem cells• Induced pluripotent stem cells, iPS cells
In vitro produced sperms!
IVF
Endometrial cell transplantation and improved implantation
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Thank you for the [email protected]