in vitro fertilization and preimplantation genetic diagnosis

Reproductive Genetics Institute

In Vitro Fertilization and Preimplantation Genetic

DiagnosisLama Eldahdah, MS, CGC

Reproductive Genetics InstituteChicago, IL USA


Outline• Normal (in vivo) fertilization & common

inheritance patterns

• IVF (In Vitro Fertilization)

• PGD(Preimplantation Genetic Diagnosis)

• IVF/PGD counseling


Normal FertilizationNormal Fertilization(and common inheritance patterns)(and common inheritance patterns)




Female Karyotype


Male Karyotype


Meiosis


Autosomal Dominant / Recessive


X-Linked Dominant / Recessive


Balanced Reciprocal Translocation


In vitro Fertilization (IVF):In vitro Fertilization (IVF):


Fertilization of egg with sperm outside of the bodyFertilization of egg with sperm outside of the body


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


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


What consists of an IVF cycle?


Outcome of ART

Taken from the CDC 2006 Assisted Reproductive Technology (ART) Report


How successful is ART?



ART trends 1996-2006



VS

ICSI• Intracytoplasmic Sperm Injection


ICSI trends 1996-2006



PreimplantationPreimplantation Genetic Genetic Diagnosis (PGD):Diagnosis (PGD):


Diagnosis of Genetic Disease Before PregnancyDiagnosis of Genetic Disease Before Pregnancy


Autosomal Recessive and Dominant Disorders

X-linked disorders

Advanced Maternal Age (aneuploidy)

Structural chromosomal rearrangements

(translocations)

Traditional Indications for PGD


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


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


First Polar body removal

Microsurgical Techniques

Second Polar body removal

First and Second Polar body removal

Embryo biopsy / Blastomere removal


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


5 Color FISH MultiVysion PB Panel

Normal 1st and 2nd PB for Chromosomes 13,16,18, 21 & 22

Polar Body FISH analysis (normal)


Blastomere FISH analysis (abnormal)


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


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


PGD Categories

• Aneuploidy

• Chromosome Translocations/Inversions

• Single gene conditions


Objectives of PGD for Chromosomal Disorders

• Prevent Chromosomal Disorders

• Reduce Spontaneous Abortions

• Improve Effectiveness of IVF


% 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


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


– 31.9% balanced

– 13.1% normal


FEMALE TRANSLOCATIONS

• Reciprocal


– 12.5% balanced

– 10.7% normal


• Robertsonian


– 23% balanced

– 10.9% normal




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


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


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.


Why are Linked Markers so important?

Detection of Allele Drop Out (ADO)یDetection of RecombinationیDetection of contaminationیDetection of chromosomal aneuploidyی


Clinical outcome of IVF-PGD for single gene disorde rsTur-Kaspa et al, ASRM 2007


IVF/PGD counselingIVF/PGD counseling



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


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


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?


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


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…


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


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


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


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


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?


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?


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?


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


(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.

Reproductive Genetics Institute Questions?


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

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