ΠΡΟΕΜΦΥΤΕΥΤΙΚΗ ΓΕΝΕΤΙΚΗ ΙΑΓΝΩΣΗ ΠΟΤΕ, ΓΙΑ ΠΟΙΟΝ ΚΑΙ ΓΙΑΤΙ ΓΕΩΡΓΙΟΣ ΜΑΡΟΥΛΗΣ ΚΑΘΗΓΗΤΗΣ ΜΑΙΕΥΤΙΚΗΣ & ΓΥΝΑΙΚΟΛΟΓΙΑΣ ΗΜΟΚΡΤΙΤΕΙΟΥ ΠΑΝΕΠΙΣΤΗΜΙΟΥ ΘΡΑΚΗΣ

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1 ΠΡΟΕΜΦΥΤΕΥΤΙΚΗ ΓΕΝΕΤΙΚΗ ΙΑΓΝΩΣΗ ΠΟΤΕ, ΓΙΑ ΠΟΙΟΝ ΚΑΙ ΓΙΑΤΙ ΓΕΩΡΓΙΟΣ ΜΑΡΟΥΛΗΣ ΚΑΘΗΓΗΤΗΣ ΜΑΙΕΥΤΙΚΗΣ & ΓΥΝΑΙΚΟΛΟΓΙΑΣ ΗΜΟΚΡΤΙΤΕΙΟΥ ΠΑΝΕΠΙΣΤΗΜΙΟΥ ΘΡΑΚΗΣ

2 ΠΡΟΓΕΝΕΤΙΚΗ ΙΑΓΝΩΣΗ ΑΜΝΙΟΠΑΡΑΚΕΝΤΙΣΗ ΒΙΟΨΙΑ ΧΟΡΙΑΚΩΝ ΛΑΧΝΩΝ ΠΡΟΕΜΦΥΤΕΥΤΙΚΗ ΓΕΝΕΤΙΚΗ ΙΑΓΝΩΣΗ

3 Π-Γ- ΕΝ ΕΙΞΕΙΣ ΕΝΤΟΠΙΣΜΟΣ ΓΕΝΕΤΙΚΩΝ ΝΟΣΗΜΑΤΩΝ ΠΟΥ ΕΜΦΑΝΙΖΟΝΤΑΙ ΣΤΗ ΝΕΟΓΝΙΚΗ ΗΛΙΚΙΑ 1. ΜΟΝΟΓΟΝΙ ΙΑΚΑ ΝΟΣΗΜΑΤΑ α. ΑΥΤΟΣΩΜΑΤΙΚΑ ΥΠΟΛΛΕΙΠΟΜΕΝΑ (ΚΥΣΤΙΚΗ ΙΝΩΣΗ) β. ΑΥΤΟΣΩΜΑΤΙΚΑ ΕΠΙΚΡΑΤΟΥΝΤΑ (HUNTIGTON DΙSEASE) γ. ΦΥΛΛΟΣΥΝ ΕΤΑ (ΜΥΑΣΘΕΝΕΙΑ DUCHENE) 2. ΧΡΩΜΟΣΩΜΙΚΕΣ ΑΝΩΜΑΛΙΕΣ α. ΣΥΝ ΡΟΜΟ DOWN, XXY, XYY ΕΝΤΟΠΙΣΜΟΣ ΑΙΤΙΩΝ ΚΑΘ ΕΞΙΝ ΑΠΟΒΟΛΩΝ ΕΝΤΟΠΙΣΜΟΣ ΑΙΤΙΩΝ ΑΠΟΤΥΧΙΩΝ ΕΞΩΣΩΜΑΤΙΚΗΣ ΓΟΝΙΜΟΠΟΙΗΣΗΣ ΠΡΟΧΩΡΗΜΕΝΗ ΗΛΙΚΙΑ ΤΗΣ ΓΥΝΑΙΚΑΣ

4 PREIMPLANTATION GENETIC DIAGNOSIS WHEN, FOR WHOM AND WHY GEORGE B MAROULIS PROFESSOR OF OBSTETRICS AND GYNECOLOGY DEMOCRITUS UNIVERSITY OF THRACE

5 PRENATAL DIAGNOSIS AMNIOCENTESIS CHORIONIC VILLI BIOPSY PRE-IMPLANTATION GENETIC DIAGNOSIS

6 PRE-IMPLATATION GENETIC DIAGNOSIS (PGD) Provides the ability to detect chromosomal abnormalities in the embryo before implantation Can detect genetically inherited diseases in the embryo before implantation PGD only on embryos obtained from IVF

7 PGD INDICATIONS Detection of genetic diseases that appear in early neonatal life (thalassemias, cystic fibrosis) Diagnosis of single gene defects: A. autosomal recessive (cystic fibrosis) B. autosomal dominant (Huntington s disease) C. sex-linked (Duchenne myotonic dystrophy) Diagnosis of chromosomal abnormalities that: Result in major health problems (Down, XXY, XYY) Cause recurrent abortions Are associated with repeated IVF failure

8 PGD POLAR BODY BIOPSY BLASTOMERE BIOPSY PCR, FISH

9 PCR DETECTION OF GENETIC ABNORMALITIES CAN DETECT GENE MUTATIONS ON GENETIC CAN DETECT GENE MUTATIONS ON GENETIC MATERIAL OBTAINED FROM ONLY ONE CELL, IN A FEW HOURS TIME

10 PGD AS Single cell biopsied FISH successful 99% Biopsy d3 Transfer d4 or d5 Determine aneuploidy in 85% of embryos

11 PGD TECHNIQUES PGD/AS Investigates certain Chromosomes : X, Y, 13,14,15,16,18,21,22,,,,, C ti G i H b idi ti I ti t ll Comparative Genomic Hybridization Investigates all Chromosomes

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17 CGH ADVANTAGE EVALUATES ALL CHROMOSOMES DETECTS MORE CHROMOSOME ABNORMALITIES (1,3,4,6,8,9,10,11,12) (IN 30% OF CASES) PARTIAL ANEUPLOIDY DUE TO CHROMOSOME BREAKAGE (15% OF CASES ONLY FREEZE EMBRYOS DISADVANTAGE VOULLAIRE L, Mol Hum Reprod 8:1035,2002

18 CGH IN REPEATED IVF FAILURE CHROMOSOME ABNORMALITY 60% ANEUPLOIDY 71% OBSERVED: 1,3,4,6,7,8,9,10,11,12,13,14,15,16,21,22,XY COMPLEX ABNORMALITY 29% FISH WOULD HAVE DIAGNOSED 85% <37 years >37 years 40% NORMAL 28% NORMAL VOULLAIRE L, Mol Hum Reprod 8:1035,2002

19 Rates of Chromosome Abnormalities Assessed by Multiprobe Multicolor FISH in Multiple Blastomeres of Developing and Arrested Cleaved Monospermic Embryos EMBRYOS CHROMOSOME ABNORMALITY (%) ANALYZED ABNORMALITY TYPE OF EMBRYO (n) POLYPLOIDY HAPLOIDY ANEUPLOIDY a MOSAICISM RATE 6 (%) Arrested Growing slowly Developing normally a Aneuploidy for chromosomes X, Y, 13, 18, and 21; average maternal age affects this rate. b Total rates are based on embryos with multiple abnormalities and exclude those with mosaicism in a small proportion of cells.

20 Staessen C et al, Hum Reprod 19;2849, 2004

21 PGD IN REPEATED IVF FAILURE FAILURE IN IVF MOST COMMONLY DUE TO: 1. ADVANCED AGE (CHROMOSOMAL ABNORMALITIES) 2. POOR OVARIAN RESPONCE 3. POOR ENDOMETRIUM 4. UNKNOWN

22 CHROMOSOMALLY ABNORMAL EMBRYOS FOR EIGHT CHROMOSOME PAIRS Age Embryos analyzed % Normal % Aneuploid %Other abnormal Note: The data are from Saint Barnabas Medical Center. Note: The data are from Saint Barnabas Medical Center. Munne. PGD of aneuploidy. Fertil Steril 2002.

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26 In vitro oocyte maturation and subsequent delayed fertilization is associated with increased embryo aneuploidy Emery RB et al, Fertil Steril 84:1027;2005

27 ABORTION Due to abnormal embryos Even normal embryos may be lost due to the abortion of an abnormal embryo

28 Chromosomal Complements in First Trimester Spontaneous Abortions COMPLEMENT CHROMOSOME FREQUENCY (%) Normal 52 Triploidy 7.7 Tetraploidy 2.15 Monosomy X 8.6 Structural abnormalities 13 Sex chromosome polysomy 47 XXX XXY 0.15 Autosomal trisomies Mosaicism 1.3 Simpson 1987

29 PGD significantly significantly reduces pregnancy loss in infertile couples: a multicenter analysis Munne S et al, Fertil Steril 85:326;2006

30 RECURRENT MISCARRIAGE CAUSES ABNORMAL KARYOTYPE 60-70% HORMONAL CAUSES 10-12% STRUCTURAL ABNORMALITIES 20-25% CERVIX INCOMPETENCE IMMUNOLOGIC FACTORS 10-12%

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32 Rubio et al, Human Reproduction, Vol. 18, No. 1, , 188 January 2003

33 Human Reproduction, Vol. 18, No. 1, , 188 January 2003

34 Munne S.:Fertil Steril 84:2005

35 PATIENTS AND METHOD 140 WOMEN Group A (n =24): at least 3 recurrent abortions Group B (n=29): recurrent IVF failures Group C (n=87): women who had children with sex-linked diseases or those who were not included in Groups A, B All women underwent IVF or ICSI Fetal biopsy on 3 rd day FISH was performed on chromosomes X, Y, 13,18,21 and 16 obtained from one blastomere of each embryo Embryo-transfer the next day Kapetanakis E, Maroulis G, ASRM 2005

36 Incidence of abnormal embryos according to age Abnormal embryos (%) Age Group A Group B Group C (RA) (RIF) (C) < >

37 Group A Group B Group C Cases Embryos examine with FISH % Normal embryos No of Embryos/ET ,4 37,6 68 2,8 2,7 2,9 IR 12,5 2,8 9,9 PR 37,5 7,7 32,3 Abortion Rate ,0 Kapetanakis E, Maroulis G, ASRM 2005

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39 ABNORMAL EMBRYOS AND ALTERED GENE EXPRESSION Genes such as BRCA 1,TP53, ATM are expressed during fragmentation of embryos. They may or may not be responsible for it but since their expression is correlated with this important indicator of viability suggests that these may be used as markers to assess embryo health Wells D.:Fertil Steril 84:

40 Summary of tentative relationships between specific morphologies and altered gene expression. Genes are noted as being upregulated (+) or repressed ( ) Morphology BUB1 MAD2 APC TP53 RBI ATM BRCA1 BRCA2 β-actina Polar body abnormalities Pronuclear abnormalities Misshapen oocytes Condensed organelles + Granular cytoplasm Fragmentation Disorganized + + Multinucleation Vacuoles + Wells. Gene expression and embryo morphology. Fertil Steril 2005.

41 Low expression levels of all genes in 2 and 3 cell embryos; [2] upon activation of the genome, embryos containing cells with DNA damage express genes responsible for DNA repair, including ATM, BRCA1, BRCA2, and TP53. The ATM protein is an upstream regulator of DNA repair pathways and consequently ATM is one of the first genes expressed. BRCA1 and BRCA2 proteins interact to repair the damaged DNA. Additionally, BRCA1 stabilizes the p53 protein and directs its transcription factor activity toward the stimulation of DNA repair and away from apoptosis; [3] p53 protein begins to accumulate and acts to downregulate BRCA1 transcription; (4) as BRCA1 levels fall, p53 is able to activate apoptotic pathways. Upregulation of TP53 maintains i high h levels l of p53 protein, despite the loss of BRCA1-mediated stabilization. Blastomeres that have not successfully repaired their DNA by this stage may undergo apoptosis, perhaps leading to fragmentation

42 CONCLUSIONS The results suggest that in both the recurrent abortion (RA) and in the repeated IVF failure (RIF) groups there is a common underlying factor which is the significant increase in the rate of chromosomally abnormal embryos. The results also suggest that while in women with recurrent abortions the transfer of normal embryos improves the pregnancy rate and live-birth rate, the improvement in women with IVF failure is less, which suggests that in these women there are additional problems with the embryos other than abnormalities in chromosomes X, Y, 13, 14,15,16, 18, 21, 22 which may account for the decrease in the IR. Therefore, PGD helps identifying problems in women with RA and RIF, improves outcome in RA and may not be as helpful in RIF but can point to use of alternatives such as the use of donor oocytes. In the future identification of gene expression related to abnormal embryo development may also be used as markers for preimplantation detection of defective embryos.

43 ΠΡΟΕΜΦΥΤΕΥΤΙΚΟΣ ΓΕΝΝΕΤΙΚΟΣ ΕΛΕΓΧΟΣ ΣΥΜΠΕΡΑΣΜΑΤΑ ΤΑ ΑΠΟΤΕΛΕΣΜΑΤΑ ΜΑΣ, ΕΙΧΝΟΥΝ ΠΩΣ ΤΟΣΟ ΣΤΙΣ ΚΑΘ ΕΞΙΝ ΑΠΟΒΟΛΕΣ ΟΣΟΣ ΚΑΙ ΣΤΙΣ ΑΠΟΤΥΧΗΜΕΝΕΣ ΠΡΟΣΠΑΘΕΙΣ IVF, ΚΟΙΝΟΣ ΠΑΡΑΓΟΝΤΑΣ ΕΙΝΑΙ Η ΠΑΡΟΥΣΙΑ ΧΡΩΜΟΣΩΜΙΚΩΝ ΑΝΩΜΑΛΙΩΝ ΤΟΥ ΕΜΒΡΥΟΥ ΤΑ ΑΠΟΤΕΛΕΣΜΑΤΑ ΜΑΣ, ΕΝΩ ΕΙΧΝΟΥΝ ΒΕΛΤΙΩΣΗ ΤΩΝ ΠΟΣΟΣΤΩΝ ΤΕΛΕΙΟΜΗΝΗΣ ΕΓΚΥΜΟΣΥΝΗΣ ΜΕΤΑ ΤΗΝ ΜΕΤΑΦΟΡΑ ΥΓΕΙΩΝ ΕΜΒΡΥΩΝ ΣΕ ΓΥΝΑΙΚΕΣ ΜΕ ΙΣΤΟΡΙΚΟ ΚΑΘ ΕΞΙΝ ΑΠΟΒΟΛΩΜ, ΕΝ ΤΟΥΤΟΙΣ ΕΝ ΠΑΡΟΥΣΙΑΖΟΥΝ ΑΝΑΛΟΓΗ ΕΠΙΤΥΧΙΑ ΣΕ ΓΥΝΑΙΚΕΣ ΜΕ ΕΠΑΝΕΙΛΛΗΜΕΝΕΣ ΑΠΟΤΥΧΙΕΣ IVF ΠΡΟΣΠΑΘΕΙΩΝ, ΓΕΓΟΝΟΣ ΠΟΥ ΥΠΟ ΗΛΩΝΕΙ ΠΩΣ ΑΥΤΈΣ ΜΠΟΡΕΙ ΝΑ ΩΦΕΙΛΩΝΤΑΙ ΣΕ ΑΝΩΜΑΛΙΕΣ ΑΛΛΕΣ ΑΠΟ ΑΥΤΕΣ ΤΩΝ ΜΕΧΡΙ ΤΩΡΑ ΕΛΕΓΧΟΜΕΝΩΝ ΧΡΩΜΟΣΩΜΑΤΩΝ ΕΤΣΙ ΛΟΙΠΟΝ, Η ΠΡΟΕΜΦΥΤΕΥΤΙΚΗ ΙΑΓΝΩΣΗ ΒΟΗΘΑ ΣΤΟΝ ΕΝΤΟΠΙΣΜΟ ΤΩΝ ΑΝΩΜΑΛΩΝ ΕΜΒΡΥΩΝ ΩΣΤΕ ΝΑ ΑΠΟΦΕΥΧΘΟΥΝ ΚΛΗΡΟΝΟΜΙΚΕΣ ΑΣΘΕΝΕΙΕΣ, ΝΑ ΒΕΛΤΙΩΘΕΙ Η ΠΙΘΑΝΟΤΗΣ ΕΓΚΥΜΟΣΥΝΗΣ ΣΕ ΠΡΟΒΛΗΜΑΤΙΚΕΣ ΚΑΤΑΣΤΑΣΕΙΣ ΑΠΟΒΟΛΩΝ Ή ΥΠΟΓΟΝΙΜΟΤΗΤΑΣ Ή ΤΕΛΟΣ ΝΑ ΜΑΣ ΚΑΤΕΥΘΥΝΕΙ ΠΡΟΣ ΠΙΘΑΝΕΣ ΑΝΑΛΛΑΚΤΙΚΕΣ ΛΥΣΕΙΣ ΌΠΩΣ Η ΧΡΗΣΗ ΩΑΡΙΩΝ Ή ΣΠΑΡΜΑΤΟΖΩΑΡΙΩΝ ΑΠΟ ΟΤΗ ΣΤΟ ΜΕΛΛΟΝ ΕΊΝΑΙ ΠΙΘΑΝΟ ΑΝΤΙ ΤΩΝ ΧΡΩΜΟΣΩΜΑΤΩΝ ΝΑ ΧΡΗΣΙΜΟΠΟΙΟΥΜΕ ΤΗΝ ΥΠΑΡΞΗ ΟΡΙΣΜΕΝΩΝ ΓΟΝΙ ΙΩΝ ΣΑΝ ΕΙΚΤΕΣ ΓΙΑ ΤΗΝ ΠΑΡΟΥΣΙΑ ΑΝΩΜΑΛΙΩΝ ΣΤΑ ΕΜΒΡΥΑ.