Reproductive Genetics Institute
In Vitro Fertilization and Preimplantation Genetic
DiagnosisLama Eldahdah, MS, CGC
Reproductive Genetics InstituteChicago, IL USA
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Outline• Normal (in vivo) fertilization & common
inheritance patterns
• IVF (In Vitro Fertilization)
• PGD(Preimplantation Genetic Diagnosis)
• IVF/PGD counseling
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Normal FertilizationNormal Fertilization(and common inheritance patterns)(and common inheritance patterns)
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Female Karyotype
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Male Karyotype
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Meiosis
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Autosomal Dominant / Recessive
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X-Linked Dominant / Recessive
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Balanced Reciprocal Translocation
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In vitro Fertilization (IVF):In vitro Fertilization (IVF):
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Fertilization of egg with sperm outside of the bodyFertilization of egg with sperm outside of the body
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Facts & Figures from the CDCOf approximately 62 million women of reproductive age in 2002:• 2% (1.2 million) had an infertility-related medical appointment within the previous year• 8% had an infertility-related medical visit at some point in the past•7% of married couples (2.1 million couples) in which the woman was of reproductive age reported that they had not used contraception for 12 months and the woman had not become pregnant
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What is ART?
ART includes all fertility treatments in which both eggs and sperm are handled. In general, ART procedures involve surgically removing eggs from a woman’s ovaries, combining them with sperm in the laboratory, and returning them to the woman’s body or donating them to another woman•IVF•GIFT•ZIFT
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What consists of an IVF cycle?
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Outcome of ART
Taken from the CDC 2006 Assisted Reproductive Technology (ART) Report
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How successful is ART?
Taken from the CDC 2006 Assisted Reproductive Technology (ART) Report
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ART trends 1996-2006
Taken from the CDC 2006 Assisted Reproductive Technology (ART) Report
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VS
ICSI• Intracytoplasmic Sperm Injection
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ICSI trends 1996-2006
Taken from the CDC 2006 Assisted Reproductive Technology (ART) Report
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PreimplantationPreimplantation Genetic Genetic Diagnosis (PGD):Diagnosis (PGD):
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Diagnosis of Genetic Disease Before PregnancyDiagnosis of Genetic Disease Before Pregnancy
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Autosomal Recessive and Dominant Disorders
X-linked disorders
Advanced Maternal Age (aneuploidy)
Structural chromosomal rearrangements
(translocations)
Traditional Indications for PGD
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Recurrent Abortion
Repeated IVF failure
Predisposition to Late Onset disorders
Cancer Predisposition genes
Infertility Causing genes
HLA matching (with or without genetic disease)
Maternal-fetal incompatibility
Combined single gene and aneuploidy testing
Expanding Indications for PGD
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Technologies utilized in PGD:
• PCR/fluorescent PCR– Single gene (Mendelian) disorders
• Mutation(s) and linked markers
• FISH (fluorescent in-situ hybridization)– Aneuploidies– Translocations/structural rearrangements– Large deletions/duplications– Specific sequences
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First Polar body removal
Microsurgical Techniques
Second Polar body removal
First and Second Polar body removal
Embryo biopsy / Blastomere removal
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Effect of Micromanipulation on Embryo Development
51.4 44.6 49.9 58.3
010203040506070
% Blastocyst
ICSIICSI / Blastomere Biopsy (1)ICSI / Simultaneous PB1 & PB2, Blastomere Biopsy (2)ICSI / Sequential PB1 & PB2, Blastomere Biopsy (3)
����Mean Age34.3 +/- 4.6 34.6+/-6.1 37.8 +/-3.8 32.8 +/-4.3
51.4 44.6 49.9 58.3
n = 12,022 n = 56 n = 2,341 n = 547
a b c d
a, b, c, d no significant difference
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5 Color FISH MultiVysion PB Panel
Normal 1st and 2nd PB for Chromosomes 13,16,18, 21 & 22
Polar Body FISH analysis (normal)
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Blastomere FISH analysis (abnormal)
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D13S1493
D13S284D13S1303D13S1317D13S800D13S317
D13S631D13S159D13S174
D16S521D16S3024D16S3024D16S3134D16S423
D16S3021D16S520
D18S1104D18S66D18S57D18S1145D18S1127D18S1144D18S386D18S61
D21S1899D21S1914D21S1910D21S1888D21S267D21S268D21S1411D21S1890D21S1903D21S11
D22S1158D22S277D22S283D22S423D22S1157D22S282
AmelX
DXS8083DXS1055
DXS998DXS1215DXS8103DXS8061DXYS154
100 104 110 112
Std
Trisomy 13 detected in blastomereby D13S159 marker
Std Std
160 182186 190 200
Trisomy 21 detected in blastomere by D21S1411 marker
Std StdStd
D21S1411D21S268
122 135 139 150 160 180 182
Disomy 21 in blastomere
139144 150 139 150 160
Monosomy 16 in blastomere
D16S3134D16S423
Std Std
StdStd
Monosomy 13 in blastomere
150 152 160 178 198 200
D13S1317 D13S284D13S
PCR results
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ICSI PBR (1PBR (1stst ) 40 ) 40 –– 42 42 hourshours post HCGpost HCG
PN PBR (2PBR (2ndnd ) / ) / PBR (1st and 2nd) Freezing
Cleavage BBBB Freezing
ET BlastBlast BxBx (option) Freezing
Residual Normal Blastocysts Freezing
Day
2
1
5
4
3
6
0
Undisturbed in culture
Chronology of Procedures
Undisturbed in culture
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PGD Categories
• Aneuploidy
• Chromosome Translocations/Inversions
• Single gene conditions
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Objectives of PGD for Chromosomal Disorders
• Prevent Chromosomal Disorders
• Reduce Spontaneous Abortions
• Improve Effectiveness of IVF
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% Aneuploid Oocytes in relation to Maternal Age% Aneuploid Oocytes in relation to Maternal Age
36.6
43.7
45.0
45.7 45.7
49.2
53.3
52.7
61.3
61.1
65.2
71.9
% Aneuploid Oocytes in Relation to Maternal Age
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Percentages of Unbalanced Embryos by Translocation Type
MALE TRANSLOCATIONS
• Reciprocal
– 72.6% unbalanced
– 16% balanced
– 11.4% normal
Based on 570 embryos from 67 cycles
• Robertsonian
– 54.9% unbalanced
– 31.9% balanced
– 13.1% normal
Based on 213 embryos from 32 cycles
FEMALE TRANSLOCATIONS
• Reciprocal
– 76.8% unbalanced
– 12.5% balanced
– 10.7% normal
Based on 859 embryos from 102 cycles
• Robertsonian
– 66.1% unbalanced
– 23% balanced
– 10.9% normal
Based on 183 embryos from 25 cycles
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Outcome of Pregnancies from 44 Patients Before and After PGD for Chromosomal Translocation
Mean age 32.5 +/- 3.9 years
87.8
%
15.6
79.4
11.5
Non-PGD Pregnancies Pregnancies after PGD
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List of PGD for Single Gene Disorders
• ACHONDROPLASIA• ADENOSINE AMINOHYDROLASEDEFICIENCY
(ADA)• ADRENOLEUKODYSTROPHY; X-LINKED (ALD)• AGAMMAGLOBULINEMIA; X-LINKED, TYPE I• ALOPECIA, CONGENITAL • ALPERS SYNDROME • ALPHA 1 ANTITRYPSIN DEFICIENCY• ALPORT SYNDROME, X-LINKED • ALZHEIMER DISEASE, EARLY-ONSET
FAMILIAL• AMYLOIDOSIS I, HEREDITARY NEUROPATHIC• ANDROGEN INSENSITIVITY SYNDROME• ANGIOEDEMA, HEREDITARY • ARGININOSUCCINIC ACIDURIA (ASL)
• ATAXIA TELANGIECTASIA
• BASAL CELL NEVUS SYNDROME, (BCNS, GORLIN SYNDROME)
• BLEPHAROPHIMOSIS • BRACHYDACTYLY• BRAIN TUMOR, POSTERIOR FOSSA
OF INFANCY, FAMILIAL• BREAST/OVARIAN CANCER,
HEREDITARY (BRCA1)• BREAST/OVARIAN CANCER,
HEREDITARY (BRCA2
…… and thatand that ’’s just the As just the A ’’s and Bs and B ’’ss
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Factors Affecting PGD Accuracy• Cell quality/cell type• Allele-drop-out• Preferential amplification of allele• DNA contamination• Failed amplification -> inconclusive results• Human error
PGD involves a modification of risk –not the elimination of risk. PGD does not replace prenatal diagnosis.
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Why are Linked Markers so important?
Detection of Allele Drop Out (ADO)یDetection of RecombinationیDetection of contaminationیDetection of chromosomal aneuploidyی
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Clinical outcome of IVF-PGD for single gene disorde rsTur-Kaspa et al, ASRM 2007
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IVF/PGD counselingIVF/PGD counseling
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Strategies to Combine IVF & PGD
IVF externally + (biopsy
externally) PGD with RGI
IVF externally + PGD with RGI
(Monitoring externally) IVF
+ PGD with RGI
IVF + PGD with RGI
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The Complexities of the IVF/PGD Patient
• Not all IVF/PGD patients have infertility• Financial burden of IVF and PGD• Lack of knowledge regarding:
– Inheritance of the genetic condition – All reproductive options– Infertility and IVF procedures
• Unrealistic expectations of PGD/IVF• Language and cultural barriers• Lack of resources and support
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The Complexities of the IVF/PGD Patient continued…
• Complex personal, medical and pregnancy histories
• Ethical issues surrounding PGD/IVF• Negative pregnancy test can have different
repercussions• Highly motivated/time consuming patients• Often doing some/all counseling via phone• Privacy/insurance issues—where is genetic
information documented?
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A Framework of PGD Genetic Counselingfor Translocations and Single Gene
Disorders
Pt referred for consultation with PGD genetic counselor
Request and Request and review ofreview ofmedical medical recordsrecords
LaboratoryLaboratorySetupSetup
IVF cycle IVF cycle
review of PGD review of PGD
test resultstest results
FollowFollow --up up consultationconsultation
22--6 weeks6 weeks
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Common PGD questions
1. Will insurance cover IVF/PGD?Sometimes
2. Why do a set-up when the translocation/mutation is known?Many reasons (confirmation, single cell testing)
3. How long does the set-up takeTranslocation 2-4 weeks, Single gene 4-6 weeks
4. Is PGD harmful to embryo development?Risk of arrest after biopsy
5. What about pregnancy rates?See next slide…
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Common questions about Aneuploidy PGD testing
1) Is there a minimum number of embryos that labs will test? No (RGI)
2) Can PGD be done on frozen embryos?Usually
3) Is prenatal testing necessary?Prenatal testing is recommended for confirmation
4) Polar bodies vs. Blastomeres?Center/patient/physician dependent
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PGD Genetic Counseling for TRANSLOCATIONS
• Records review: karyotype, family history• Requires set-up (blood samples-green top)• Discussion FISH technique and expected
percentage of unbalanced embryos• Review of benefits, risks and limitations of
testing (accuracy 90-95%)• Consent forms/informed consents• Cost• IVF cycle dates
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PGD Genetic Counseling for single gene disorders
• Records review: mutation reports, family history• Requires set-up
Need DNA; could be in several forms: blood, cheek swabs, semen sample, stored DNA
• Discussion PCR and DNA linked markers• Percentage of affected embryos• Review of testing strategy (PB vs. BB), benefits, risks
and limitations (accuracy 95-98%)• Consent forms/informed consents• Cost• IVF cycle dates• Review of results and recommendations for transfer
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PGD for Beta Thalassemia, HLA Typing and Aneuploid y testing
Embryo #Embryo #
HBBHBBChromosome 11Chromosome 11
HLAHLAChromosome 6Chromosome 6
HLA(6) Chr.11
1.1 2.1
PGD
1 2 4 6 7 8 9
HLA Markers: D6S1583
RINGD6S2447
LH1TNF A
MIBRF
MOGD6S1624
156156161161151151169169100100121121301301159159178178
1541541551551601601671679494127127252252167167180180
151151159159151151177177100100123123264264167167172172
158158155155147147169169104104100100246246182182178178
139139180180
77IVS2IVS2--11
130130170170165165
13913918018066NN128128168168117117
13913918018066IVS1IVS1--55128128162162159159
144197
6N
128168117
HLA(6) Chr.11
CARRIERMATCH TO 2.2
NORMALNON-MATCH
CARRIERMATCH to 2.1
AFFECTEDNON-MATCH
ETET
HBB Markers:BSTRHBGHPFHMutationBRSAD11S1338D11S1241
0
13,13;13,13;16,1616,1618,1818,1821,21;21,21;
22,22,2222,22,22
011, 011, 013,13;13,13;16,16;16,16;18,18;18,18;21,21;21,21;22,22,22,22,
13,13;13,13;16,16;16,16;18,18;18,18;21,21;21,21;22,22,22,22,
0; 60; 613,13;13,13;16,16;16,16;18,18;18,18;21,21;21,21;22,22,22,22,
13,13;13,13;16,16;16,16;18,18;18,18;21,21;21,21;22,22,22,22,
13,13;13,13;16,16;16,16;18,18;18,18;21,21;21,21;22,22,22,22,
13,13;13,13;16,16;16,16;18,18;18,18;21,21;21,21;22,22,22,22,
ETET
NORMALNON-MATCH
AneuploidyAneuploidyTesting Testing
By FISH or PCRBy FISH or PCR
Trisomy 22
2.1 2.2 2.3
?MATCH TO 2.2
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Audience Response
A 39 year old patient has had trouble conceiving and has been given a diagnosis of infertility. She will be pursuing IVF.
Would you offer PGD to this patient?
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Audience Response
An Ashkenazi Jewish couple is seen at a fertility clinic due to male factor infertility. Carrier screening is offered to them for the Ashkenazi Jewish Screening panel. Both partners are found to be carriers of Tay-Sachs.
Would you offer PGD to this family?
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Audience Response
A couple has had three spontaneous pregnancies all resulting in healthy boys. They would really like to have a girl.
Would you offer IVF/PGD to this family?
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Additional resources
• NSGC (National Society of Genetic Counselors) www.nsgc.org
• Genetests www.genetests.com• CDC http://www.cdc.gov/• On line support groups/chat rooms
• Other PGD patients
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(PREIMPLANTATION) CYTOGENETICSZev Zlatopolsky B.S.; Irina Kirillova, M.D., Ph.D.; Garry Kalmanovich B.S.; Yuri Ilkevitch, Ph.D.
PREIMPLANTATION MOLECULAR GENETICS
Svetlana Rechitsky, Ph.D.; Oleg Verlinsky, B.A.; Irina Barsky, M.S.; Tatiana Sharapova M.S.; Julija Sivakova, B.S.; Tatsiana Pakhalchuk, M.S.; Ekaterina Pomerantseva, Ph.D.
CLINICAL & MOLECULAR GENETICSLama Eldahdah, M.S., C.G.C.Christina Lavin, M.S., C.G.C.Dana Pauling, M.S.. C.G.C.
PRENATAL GENETICS & FETAL MEDICINENorman Ginsberg, M.D.Sandra Concialdi, R.D.M.S.
TISSUE & STEM CELL BANKAnver Kuliev, M.D., Ph.D.Valeri Koukharenko, Ph.D.
EXPERIMENTAL EMBRYOLOGYNikolai Strelchenko, Ph.D.
ASSISTED REPRODUCTION TECHNOLOGY &
MICROMANIPULATIONIlan Tur-Kaspa M.D., Nataliya Tkachenko.M.D.Nathan Katz, Ph.D.; Svetlana Lerner M.S., Natalie Ilkevitch M.S.; Georg Wolf, B.S.; Gloria Enriquez, B.S.
LABORATORY DIRECTOR: Yury Verlinsky, Ph.D.MEDICAL DIRECTOR: Joe Leigh Simpson, M.D.
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LAMA ELDAHDAH, MS, CGCLICENSED GENETIC COUNSELOR
REPRODUCTIVE GENETICS INSTITUTE2825 North Halsted Street
Chicago, IL 60657PHONE: 773-868-2162
FAX: 773-871-5221EMAIL: [email protected]
WEBSITE: www.reproductivegenetics.com