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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 attentionAndres.Salumets@ccrmb.ee