Infertility, In Vitro Fertilization (IVF) and Genetic Testing

Infertility, In Vitro Infertility, In Vitro Fertilization (IVF) Fertilization (IVF)

and Genetic and Genetic TestingTesting

Michele Evans, M.D.Michele Evans, M.D.

OutlineOutline

InfertilityInfertility Treatment optionsTreatment options

In Vitro FertilizationIn Vitro Fertilization Genetic TestingGenetic Testing

PreconceptionPreconception PreimplantationPreimplantation Prenatal Prenatal

ControversiesControversies

Normal FertilityNormal Fertility

0

0.2

0.4

0.6

0.8

1

1 2 3 4 5 6 7 8 9 10 11 12

Months of Follow Up

Cumulative Pregnancy

Rates

Hull, et al: Br Med J 1985:291;1693

Overview of InfertilityOverview of Infertility

Definition: 1 year of well-timed, Definition: 1 year of well-timed, unprotected intercourse without a unprotected intercourse without a pregnancypregnancy

10-15% of population is infertile 10-15% of population is infertile (subfertile)(subfertile)

Causes of InfertilityCauses of Infertility

Female Reproductive Female Reproductive OrgansOrgans

FSH (+)

FSH=Follicle Stimulating Hormone

PhysiologyPhysiology

E2 (-)

E2=Estradiol

Developing follicle

Causes of Female Causes of Female InfertilityInfertility

OvaryOvary TubesTubes UterusUterus CervixCervix HormonesHormones ChromosomesChromosomes

Causes of Female Infertility Causes of Female Infertility - Ovary- Ovary

AGEAGE

Problems with ovulation Problems with ovulation

Premature ovarian failurePremature ovarian failure

Ovary - Female AgeOvary - Female Age

Women are born with their lifetime Women are born with their lifetime egg supplyegg supply 4 million at 20 weeks gestation4 million at 20 weeks gestation 400,000 at birth400,000 at birth

100,000 eggs left at time of puberty100,000 eggs left at time of puberty Fertility initially declines at age Fertility initially declines at age 2727 Significant decline at age 37-38Significant decline at age 37-38 Rare pregnancies after age 44Rare pregnancies after age 44

Percentage of Married Percentage of Married Women Who are InfertileWomen Who are InfertileFrom 3 national U.S. surveysFrom 3 national U.S. surveys

Age (years)Age (years) InfertileInfertile

20-2420-24 7.07.0

25-2925-29 8.98.9

30-3430-34 14.614.6

35-3935-39 21.921.9

40-4440-44 28.728.7

Menken et al, Science 1989;23:1389

Ovarian Reserve Ovarian Reserve TestingTesting

Day 3 Day 3 FSH/EFSH/E22

FSH FSH (mIU/mL)(mIU/mL)

EE22 (pg/mL) (pg/mL)

NormalNormal <10<10 <100<100

BorderlineBorderline 10-2010-20 NANA

AbnormalAbnormal >20>20 >100>100

Clinical pregnancy per Clinical pregnancy per cyclecycle

as function of both FSH as function of both FSH and ageand age

Pearlstone et al, Fertil Steril 1992:58:674

Prevalence of genetically Prevalence of genetically abnormal oocytes in abnormal oocytes in

infertile womeninfertile womenA

bn

orm

al

(%)

Ovary - OvulationOvary - Ovulation

Ovary - OvulationOvary - Ovulation

HistoryHistory Regular menses (90%)Regular menses (90%) Premenstrual symptomsPremenstrual symptoms

Patient self-testingPatient self-testing BBT, urinary ovulation kits (LH)BBT, urinary ovulation kits (LH)

Laboratory testsLaboratory tests Mid-luteal serum progesterone > 10 Mid-luteal serum progesterone > 10

ng/mLng/mL Endometrial biopsyEndometrial biopsy

Ovary - Causes of Ovary - Causes of AnovulationAnovulation

Hormone imbalanceHormone imbalance ObesityObesity AnorexiaAnorexia Significant stressSignificant stress Patients display:Patients display:

Irregular menstrual cyclesIrregular menstrual cycles Skipped cyclesSkipped cycles Minimal or absent premenstrual Minimal or absent premenstrual

symptomssymptoms

Ovary – Premature Ovarian Ovary – Premature Ovarian FailureFailure

Menopause prior to age 40Menopause prior to age 40 ↓↓EstrogenEstrogen ↑↑FSHFSH

CausesCauses AutoimmuneAutoimmune GeneticGenetic Idiopathic Idiopathic

1-2% pregnancy rate1-2% pregnancy rate

Causes of Female Infertility Causes of Female Infertility – Fallopian Tubes– Fallopian Tubes

Infection (chlamydia)Infection (chlamydia)

EndometriosisEndometriosis

Tubal ligation (female sterilization)Tubal ligation (female sterilization)

Open TubesOpen Tubes

Blocked TubesBlocked Tubes

Female Infertility - Female Infertility - UterusUterus

UterusUterus FibroidsFibroids PolypsPolyps

Mullerian (congenital) defectsMullerian (congenital) defects AbsentAbsent Bicornuate/SeptumBicornuate/Septum

Asherman’s syndromeAsherman’s syndrome

Female Reproductive Female Reproductive OrgansOrgans

Female InfertilityFemale Infertility

Uterine muscle Uterine muscle tumortumor

Benign (>95%)Benign (>95%)

25-30% of women25-30% of women

Normal Shape of UterusNormal Shape of Uterus

Fibroid UterusFibroid Uterus


UterusUterus TumorsTumors

FibroidsFibroids PolypsPolyps

Mullerian defects (congenital)Mullerian defects (congenital) Absent uterusAbsent uterus Bicornuate/septateBicornuate/septate


Mullerian DefectMullerian Defect

Treatment with Treatment with HysteroscopyHysteroscopy

Treatment with Treatment with LaparoscopyLaparoscopy


UterusUterus TumorsTumors

FibroidsFibroids PolypsPolyps

Mullerian defects (congenital)Mullerian defects (congenital) Absent uterusAbsent uterus Bicornuate/septateBicornuate/septate


Female Infertility - Female Infertility - CervixCervix

CervixCervix Post-surgicalPost-surgical

StenosisStenosis Mucus changesMucus changes

Female Infertility - Female Infertility - HormonesHormones

Endocrine abnormality (hormones)Endocrine abnormality (hormones) ThyroidThyroid ProlactinProlactin Polycystic ovary syndrome (PCOS)Polycystic ovary syndrome (PCOS)

Estrogen, insulinEstrogen, insulin Hypothalamic hypogonadismHypothalamic hypogonadism

StressStress Exercise (ballet dancer)Exercise (ballet dancer)

Other Causes of Female Other Causes of Female InfertilityInfertility

OthersOthers Chromosome abnormalitiesChromosome abnormalities

Turner’s syndrome (XO)Turner’s syndrome (XO) Androgen Insensitivity (XY)Androgen Insensitivity (XY)

Male pseudohermaphroditeMale pseudohermaphrodite Female phenotypeFemale phenotype Blind vaginal canalBlind vaginal canal Inguinal hernia (50%)Inguinal hernia (50%)

Causes of Male InfertilityCauses of Male Infertility

Abnormality in sperm productionAbnormality in sperm production

Abnormality in sperm functionAbnormality in sperm function

Obstruction in the ductal systemObstruction in the ductal system

Male Reproductive Male Reproductive OrgansOrgans

Sperm: Semen AnalysisSperm: Semen Analysis

Volume: Volume: >> 2 mL 2 mL Concentration: Concentration: >> 20,000,000 per mL 20,000,000 per mL Motility: Motility: >> 50% 50% Normal morphology: Normal morphology: >> 40% normal 40% normal

Krueger strict criteria: Krueger strict criteria: >> 14% normal 14% normal Best predictor of fertilizing ability Best predictor of fertilizing ability

Normal Sperm Normal Sperm MorphologyMorphology

Abnormal MorphologyAbnormal Morphology

Abnormal MorphologyAbnormal Morphology

SpermSperm

How many are needed for fertilization?How many are needed for fertilization? Natural conceptionNatural conception

20,000,00020,000,000 Intra-uterine inseminationIntra-uterine insemination

1,000,0001,000,000 In-vitro fertilization (IVF)In-vitro fertilization (IVF)

10,00010,000 Intra-cytoplasmic sperm injection (ICSI)Intra-cytoplasmic sperm injection (ICSI)

11

FertilizationFertilization

Binding to zona Binding to zona pellucidapellucida

Penetration of Penetration of oocyteoocyte

Decondensation Decondensation and aster and aster formationformation

Pronucleus Pronucleus formationformation

Causes of Male InfertilityCauses of Male Infertility

Abnormality in sperm productionAbnormality in sperm production

Abnormality in sperm functionAbnormality in sperm function

Obstruction in the ductal systemObstruction in the ductal system

Abnormalities of Sperm Abnormalities of Sperm ProductionProduction

GeneticGenetic Y chromosome microdeletionsY chromosome microdeletions

Damage to testes – anatomicalDamage to testes – anatomical CryptorchidismCryptorchidism VaricoceleVaricocele

InfectionInfection Mumps orchitisMumps orchitis

GonadotoxinsGonadotoxins

Abnormalities of Sperm Abnormalities of Sperm FunctionFunction

Antisperm antibodiesAntisperm antibodies Genital tract inflammationGenital tract inflammation

prostatitisprostatitis VaricoceleVaricocele Failure of acrosome reactionFailure of acrosome reaction Problems with sperm Problems with sperm

binding/penetrationbinding/penetration

Obstructions in Ductal Obstructions in Ductal SystemSystem

VasectomyVasectomy

Congenital bilateral absence of the Congenital bilateral absence of the vas deferensvas deferens

Epididymis/ejaculatory ductsEpididymis/ejaculatory ducts Congenital or acquiredCongenital or acquired

Male Infertility - Male Infertility - LifestyleLifestyle

TobaccoTobacco MarijuanaMarijuana AlcoholAlcohol CocaineCocaine Steroids (can be permanent)Steroids (can be permanent) HeatHeat ExerciseExercise

Infertility: Initial Infertility: Initial EvaluationEvaluation

EggsEggs OvulationOvulation Egg qualityEgg quality

SpermSperm PresencePresence QualityQuality

Gamete transport/ImplantationGamete transport/Implantation HysterosalpingogramHysterosalpingogram

Unexplained InfertilityUnexplained Infertility

Work-up is negativeWork-up is negative

15-20 % of couples15-20 % of couples

TreatmentsTreatments

Observation (improve timing)Observation (improve timing) Clomiphene citrate + intrauterine Clomiphene citrate + intrauterine

insemination (IUI)insemination (IUI) FSH + IUIFSH + IUI IVFIVF Egg donationEgg donation

Intrauterine Intrauterine Insemination (IUI)Insemination (IUI)

Clomiphene Citrate Clomiphene Citrate (Clomid)(Clomid)

Anti-estrogenAnti-estrogen

↑ ↑ FSHFSH

↑ ↑ egg productionegg production

$$

Estrogen AntagonistEstrogen Antagonist

FSH (+)



E2 (-)

E2=Estradiol

Developing follicle

Clomid

ART: Treatment ART: Treatment OptionsOptions

Clomiphene citrate + IUIClomiphene citrate + IUI Prospective randomized trialProspective randomized trial Patients with unexplained infertilityPatients with unexplained infertility

ObservatioObservationn

CC + IUICC + IUI

CyclesCycles 150150 148148

PregnanciePregnanciess

55 1414

FecundityFecundity 0.0330.033 0.0950.095Deaton et al, Fertil Steril 1990;54:1083

FSH +IUIFSH +IUI

Daily injections Daily injections

Goal is to make multiple follicles Goal is to make multiple follicles (eggs)(eggs)

$$$$

FSH (+)



E2 (-)

E2=Estradiol

Developing follicle

Infertility Treatment Infertility Treatment OptionsOptions

IUI, FSH or FSH + IUIIUI, FSH or FSH + IUI Patients with unexplained infertilityPatients with unexplained infertility

Serhall et al, Fertil Steril 1988;49:602

TreatmeTreatmentnt

CyclesCycles PregnancPregnancyy

Pregnancy Pregnancy per cycleper cycle

IUIIUI 3030 11 2.7%2.7%

FSHFSH 4949 33 6.1%6.1%

FSH+IUIFSH+IUI 3434 99 26.4%26.4%

In Vitro Fertilization - In Vitro Fertilization - HistoryHistory

1978 – First “test tube” baby was born in 1978 – First “test tube” baby was born in England England

1981 – IVF in U.S.1981 – IVF in U.S.

Started with GIFT and ZIFTStarted with GIFT and ZIFT

2004 - >98% IVF with transfer of embryo 2004 - >98% IVF with transfer of embryo to uterusto uterus

In Vitro Fertilization In Vitro Fertilization (IVF)(IVF)

Who Needs IVF?Who Needs IVF?

Failed other treatmentsFailed other treatments Tubal damageTubal damage Significant male factorSignificant male factor Absent uterusAbsent uterus Carriers of genetic diseasesCarriers of genetic diseases Cancer patients?Cancer patients?

Ovarian Ovarian HyperstimulationHyperstimulation

Egg RetrievalEgg Retrieval

Good EggGood Egg

Bad EggBad Egg

FertilizationFertilization

2 Pronuclei (2PN)2 Pronuclei (2PN)

1 day after egg 1 day after egg retrievalretrieval

Day 3 EmbryoDay 3 Embryo




Blastocyst – Day 5Blastocyst – Day 5

Hatching BlastocystHatching Blastocyst

Embryo TransferEmbryo Transfer

Embryo TransferEmbryo Transfer

Special IVF ProceduresSpecial IVF Procedures

Assisted hatchingAssisted hatching Intracytoplasmic sperm injection Intracytoplasmic sperm injection

(ICSI)(ICSI) Preimplantation genetic diagnosis Preimplantation genetic diagnosis

(PGD)(PGD) FreezingFreezing Egg donationEgg donation SurrogacySurrogacy

Assisted HatchingAssisted Hatching

Empty ZonaEmpty Zona

ICSIICSI

ICSIICSI

ICSIICSI

How Many Embryos are How Many Embryos are Transferred?Transferred?

Related to age and embryo qualityRelated to age and embryo quality <35 = 2<35 = 2 35-37 = 2-335-37 = 2-3 38-40 = 3-438-40 = 3-4 >40 = up to 5>40 = up to 5

For patients with 2 or more failed IVF cycles, For patients with 2 or more failed IVF cycles, or a poor prognosis, can add more based on or a poor prognosis, can add more based on clinical judgementclinical judgement

IVF Success Rates - 2000IVF Success Rates - 2000

U.S. Fertility Centers (SART/CDC)U.S. Fertility Centers (SART/CDC) Female ageFemale age

<35 – 33%<35 – 33% 35-37 – 27%35-37 – 27% 38-40 – 18.6%38-40 – 18.6% >40 – 7.8%>40 – 7.8%

IVF Statistics - 2000IVF Statistics - 2000

65.1% singletons (16,533)65.1% singletons (16,533)

30.8% twins (7,817)30.8% twins (7,817)

3.9% triplets (1,000)3.9% triplets (1,000)

0.2% higher order multiples (44)0.2% higher order multiples (44)

Singleton PregnancySingleton Pregnancy

Twin PregnancyTwin Pregnancy

Triplet PregnancyTriplet Pregnancy

IVF and Multiple IVF and Multiple PregnancyPregnancy

Maternal complicationsMaternal complications Fetal complicationsFetal complications CostCost ““Selective reduction”Selective reduction” Single embryo transfer vs. success Single embryo transfer vs. success

ratesrates

IVF Statistics - 2000IVF Statistics - 2000

383 U.S. programs offer IVF383 U.S. programs offer IVF

99,989 cycles of ART treatment99,989 cycles of ART treatment

7,581 donor oocyte cycles7,581 donor oocyte cycles

25,394 deliveries (birth of 35,345 25,394 deliveries (birth of 35,345 neonates)neonates)

Current status of ART in Current status of ART in the USAthe USA

SART registry, 1998

Pregnancy and live birth ratesfor ART cyclesusing fresh,non-donor eggs,by age of woman

Cost of IVFCost of IVF

IVF cycle + medications = $10,000-IVF cycle + medications = $10,000-15,00015,000

Assisted hatching = $500Assisted hatching = $500 ICSI = $1,500ICSI = $1,500 Freezing = $650Freezing = $650 Storage = $360 Storage = $360 Egg Donor = $5,000Egg Donor = $5,000 Surrogate = $10,000-15,000Surrogate = $10,000-15,000

Egg DonationEgg Donation

Egg Egg donationdonation

IVF for twoIVF for two

DonorDonor Standard controlled Standard controlled

ovarian ovarian hyperstimulationhyperstimulation

Egg retrievalEgg retrieval

RecipientRecipient Embryo transferEmbryo transfer


Known/anonymous donorKnown/anonymous donor

<35 years old<35 years old

Current status of ART Current status of ART in the USAin the USA

SART registry, 1998

Live birth rates per transferfor fresh embryos from own anddonor eggs, by age of recipient

Cumulative pregnancy ratesafter oocyte donation

Human Reprod 1997;12:835


Grade A: The Market for a Yale Grade A: The Market for a Yale Woman’s EggsWoman’s Eggs When a Yale undergraduate explored When a Yale undergraduate explored becoming an egg donor for a wealthy becoming an egg donor for a wealthy couple willing to pay top dollar to the couple willing to pay top dollar to the right candidate, she didn't realize how right candidate, she didn't realize how unsettling the process of candidacy unsettling the process of candidacy would prove to bewould prove to be

by Jessica Cohen by Jessica Cohen

Egg DonationEgg Donation October 23, 1999October 23, 1999 Selling Fashion Models' Eggs Online Raises Ethics Selling Fashion Models' Eggs Online Raises Ethics

IssuesIssues By CAREY GOLDBERG By CAREY GOLDBERG

CAMBRIDGE, Mass. -- To the horror and disgust of CAMBRIDGE, Mass. -- To the horror and disgust of mainstream infertility groups, a longtime fashion mainstream infertility groups, a longtime fashion photographer has begun offering up models as egg photographer has begun offering up models as egg donors to the highest bidders, auctioning their ova donors to the highest bidders, auctioning their ova via the Internet to would-be parents willing to pay via the Internet to would-be parents willing to pay up to $150,000 in hopes of having a beautiful up to $150,000 in hopes of having a beautiful child. child.

Egg Donation - Ethical Egg Donation - Ethical IssuesIssues

Egg Donor Egg Donor Known or anonymousKnown or anonymous How many times to donate?How many times to donate?

RecipientRecipient How old is too old?How old is too old?

Pregnancy in the Sixth Pregnancy in the Sixth Decade of LifeDecade of Life

USC experience: 1991-2000USC experience: 1991-2000

77 recipients of egg donation77 recipients of egg donation

Mean age 52.8 + 2.9 yearsMean age 52.8 + 2.9 years

Paulson, Tourgeman, Boostanfar et al, JAMA 2002:228;2320.

Pregnancy in the Sixth Pregnancy in the Sixth Decade of LifeDecade of Life

Medical screeningMedical screening EKG and treadmillEKG and treadmill Mammogram, PapMammogram, Pap Chest x-rayChest x-ray Glucose tolerance testGlucose tolerance test Complete blood countComplete blood count Blood chemistry with cholesterolBlood chemistry with cholesterol

Pregnancy in the Sixth Pregnancy in the Sixth Decade of Life: Pregnancy Decade of Life: Pregnancy

OutcomeOutcome Of the 77 women, 42 (54.5%) had Of the 77 women, 42 (54.5%) had

live birthslive births

45 deliveries in 42 women45 deliveries in 42 women

Pregnancy in the Sixth Pregnancy in the Sixth Decade of Life: Pregnancy Decade of Life: Pregnancy

OutcomeOutcome 45 Live births45 Live births

78% Cesarean delivery78% Cesarean delivery 31 Singletons31 Singletons

68% Cesarean delivery68% Cesarean delivery 6% Vacuum assisted6% Vacuum assisted 26% NSVD26% NSVD

14 Multiple gestations14 Multiple gestations 100% Cesarean delivery100% Cesarean delivery

Pregnancy in the Sixth Pregnancy in the Sixth Decade of Life: Obstetric Decade of Life: Obstetric

ComplicationsComplications

35% Pre-eclampsia35% Pre-eclampsia 25% mild25% mild 10% severe10% severe

Background incidenceBackground incidence 3-5% in young women3-5% in young women 10% in women over age 4010% in women over age 40

Pregnancy in the Sixth Pregnancy in the Sixth Decade of Life: Decade of Life:

Obstetric ComplicationsObstetric Complications 20% gestational diabetes20% gestational diabetes

17.5% diet controlled17.5% diet controlled 2.5% insulin2.5% insulin

Background incidenceBackground incidence 5% overall, increasing with age5% overall, increasing with age

<20 years of age: 3.7%<20 years of age: 3.7% 20-30 years of age: 7.5%20-30 years of age: 7.5% >30 years of age: 13.8%>30 years of age: 13.8%

Is 55 a “physiological limit”?Is 55 a “physiological limit”?

Marked increase in pre-eclampsiaMarked increase in pre-eclampsia

Increase in diabetesIncrease in diabetes

How old is too old?How old is too old?

Genetic TestingGenetic Testing

PreconceptionPreconception

PreimplantationPreimplantation

PrenatalPrenatal

PostnatalPostnatal

Preconception Preconception CounselingCounseling

Offered to all womenOffered to all women Prenatal vitamins – 400 micrograms Prenatal vitamins – 400 micrograms

folic acid/dayfolic acid/day Rubella immunityRubella immunity Varicella immunityVaricella immunity Rh statusRh status HIVHIV Hepatitis B screenHepatitis B screen Cystic Fibrosis screeningCystic Fibrosis screening

Preconception Preconception CounselingCounseling

Offered to certain ethnic groupsOffered to certain ethnic groups Mediterranean – thalassemiaMediterranean – thalassemia African-American – sickle-cell anemiaAfrican-American – sickle-cell anemia Caucasian/Hispanic – cystic fibrosisCaucasian/Hispanic – cystic fibrosis Ashkenazi Jews – 7 autosomal recessive Ashkenazi Jews – 7 autosomal recessive

disordersdisorders Gaucher disease (1/13), Tay-Sachs (1/30), Gaucher disease (1/13), Tay-Sachs (1/30),

Familial dysautonomia (1/30), Canavan disease Familial dysautonomia (1/30), Canavan disease (1/40), Fanconi anemia (1/89), Niemann-Pick (1/40), Fanconi anemia (1/89), Niemann-Pick disease (1/90), Bloom syndrome (1/100)disease (1/90), Bloom syndrome (1/100)

Prenatal TestingPrenatal Testing

UltrasoundsUltrasounds

Serum screensSerum screens

Chorionic villus sampling (CVS)Chorionic villus sampling (CVS)

AmniocentesisAmniocentesis

Prenatal Tests - Prenatal Tests - UltrasoundUltrasound

Nuchal translucency screeningNuchal translucency screening

Performed between 10-13 weeks Performed between 10-13 weeks gestationgestation

Screen for Down SyndromeScreen for Down Syndrome

Nuchal TranslucencyNuchal Translucency

Prenatal Tests - Prenatal Tests - UltrasoundUltrasound

Second trimester ultrasoundSecond trimester ultrasound Detailed examDetailed exam Down Syndrome, other trisomiesDown Syndrome, other trisomies Cardiac, renal, spinal, limb, brain Cardiac, renal, spinal, limb, brain

deformitiesdeformities Cleft lip/palateCleft lip/palate

Ultrasound Ultrasound AbnormalitiesAbnormalities

Ultrasound Ultrasound AbnormalitiesAbnormalities

Prenatal Tests - SerumPrenatal Tests - Serum

15-20 weeks gestation15-20 weeks gestation

Quad ScreenQuad Screen Tests for AFP, hCG, uE3 and inhibin ATests for AFP, hCG, uE3 and inhibin A Neural tube defects, Down syndrome Neural tube defects, Down syndrome

and trisomy 18and trisomy 18 Readjusts age-related risksReadjusts age-related risks

Prenatal TestsPrenatal Tests

Who is offered further testing?Who is offered further testing? Advanced maternal ageAdvanced maternal age Previous child or pregnancy with birth Previous child or pregnancy with birth

defectdefect Suggestive screening test resultsSuggestive screening test results Family historyFamily history

Prenatal Tests – CVSPrenatal Tests – CVS

Chorionic Villus SamplingChorionic Villus Sampling 11-13 weeks gestation11-13 weeks gestation Catheter/needle biopsy of placental Catheter/needle biopsy of placental

cellscells Performed through cervix or abdomenPerformed through cervix or abdomen Can tests for aneuploidy and enzyme Can tests for aneuploidy and enzyme

defectsdefects 1-2% miscarriage rate1-2% miscarriage rate Digit/limb deficiencies (10 weeks)Digit/limb deficiencies (10 weeks)

Prenatal Tests - Prenatal Tests - AmniocentesisAmniocentesis

Performed at 15-18 weeks gestationPerformed at 15-18 weeks gestation 10 cc amniotic fluid10 cc amniotic fluid Living cells from fetus in amniotic Living cells from fetus in amniotic

fluidfluid Cells grown in lab for 1-2 weeksCells grown in lab for 1-2 weeks Results in 3 weeksResults in 3 weeks

Postnatal TestingPostnatal Testing

Most done during first day of lifeMost done during first day of life Heel stickHeel stick CaliforniaCalifornia

GalactosemiaGalactosemia Hypothyroidism (congenital) Hypothyroidism (congenital) Phenylketonuria (PKU) Phenylketonuria (PKU) Sickle Cell Disease (SCD) and Sickle Cell Disease (SCD) and

Hemoglobinopathies Hemoglobinopathies

Postnatal Screening – Postnatal Screening – Tandem Mass SpectrometyTandem Mass Spectromety

Fatty Acid Oxidation Disorders Carnitine/Acylcarnitine Translocase Deficiency (Translocase)

Carnitine Palmitoyl Transferase Deficiency Type I (CPT-I)2

3-Hydroxy Long Chain Acyl-CoA Dehydrogenase Deficiency (LCHAD)2,4-Dienoyl-CoA Reductase Deficiency2

Medium Chain Acyl-CoA Dehydrogenase Deficiency (MCAD)Multiple Acyl-CoA Dehydrogenase Deficiency (MADD or Glutaric Acidemia-Type II)Neonatal Carnitine Palmitoyl Transferase Deficiency-Type II(CPT-II)Short Chain Acyl-CoA Dehydrogenase Deficiency (SCAD)Short Chain Hydroxy Acyl-CoA Dehydrogenase Deficiency (SCHAD)Trifunctional Protein Deficiency (TFP Deficiency)Very Long Chain Acyl-CoA Dehydrogenase Deficiency (VLCAD)

Organic Acid Disorders 3-Hydroxy-3-Methylglutaryl-CoA Lyase Deficiency (HMG)

Glutaric Acidemia-Type I (GA I)Isobutyryl-CoA Dehydrogenase DeficiencyIsovaleric Acidemia (IVA) Acute onset Chronic2-Methylbutryl-CoA Dehydrogenase Deficiency3-Methylcrotonyl-CoA Carboxylase Deficiency (3MCC Deficiency)3-Methylglutaconyl-CoA Hydratase DeficiencyMethylmalonic Acidemias Methylmalonyl-CoA Mutase Deficiency 0 Methylmalonyl-CoA Mutase Deficiency + Some Adenosylcobalamin Synthesis Defects Maternal Vitamin B12 DeficiencyMitochondrial Acetoacetyl-CoA Thiolase Deficiency (3-Ketothiolase Def.)Propionic Acidemia (PA) Acute onset Late onsetMultiple-CoA Carboxylase DeficiencyMalonic Aciduria

Amino Acid Disorders Argininemia

Argininosuccinic Aciduria (ASA Lyase Deficiency) Acute onset Late onsetCarbamoylphosphate Synthetase Deficiency (CPS Def.)2

Citrullinemia (ASA Synthetase Deficiency) Acute onset Late onsetHomocystinuriaHypermethioninemiaHyperammonemia, Hyperornithinemia, Homocitrullinemia Syndrome (HHH)2

Hyperornithinemia with Gyral Atrophy2

Maple Syrup Urine Disease (MSUD) Classical MSUD Intermediate MSUD5-Oxoprolinuria (pyroglutamic Aciduria)2

Phenylketonuria (PKU) Classical PKU Hyperphenylalaninemia Biopterin Cofactor Deficiencies (4)TyrosinemiaTransient Neonatal Tyrosinemia Tyrosinemia Type I (Tyr I)2

Tyrosinemia Type II (Tyr II) Tyrosimenia Type III (Tyr III)

Other Abnormal Profiles Hyperalimentation

Liver DiseaseMedium Chain Triglyceride (MCT) Oil AdministrationPresence of EDTA Antigoagulants in blood specimenTreatment with Benzoate, Pyvalic Acid, or Valproic AcidCarnitine Uptake Deficiency2

Preimplantation Genetic Preimplantation Genetic Diagnosis (PGD)Diagnosis (PGD)

Can test embryos for genetic Can test embryos for genetic abnormalities prior to implantationabnormalities prior to implantation

Uses single cell (blastomere) at 8-Uses single cell (blastomere) at 8-cell stagecell stage

Which Embryo is Which Embryo is Disease-Free?Disease-Free?

PGD – Clinical PGD – Clinical IndicationsIndications

Single gene defectsSingle gene defects Balanced translocationsBalanced translocations Advanced maternal age (aneuploidy)Advanced maternal age (aneuploidy) Repetitive IVF failureRepetitive IVF failure Recurrent pregnancy lossRecurrent pregnancy loss Embryo selectionEmbryo selection

Fluorescence in situ hybridization Fluorescence in situ hybridization (FISH)(FISH) Aneuploidy/translocations and Aneuploidy/translocations and

sexing (5-9 chromosomes)sexing (5-9 chromosomes) PCRPCR

specific single gene disordersspecific single gene disorders

PGD

•Achondroplasia •ADPKD1 •ADPKD2 •Adrenoleukodystroph •Age-related aneuploidies •Alpha-thalassemia •Alpha-1-antitrypsin •Alport disease •Amyloid precursor protein (APP) mutation •ARPKD •Becker muscular dystrophy •Beta-thalassemia •Charcot Marie Tooth disease •Chromosomal translocations •Congenital adrenal hyperplasia •Cystic fibrosis •Down syndrome •Duchenne muscular dystophy •Dystonia •Epidermolysis bullosa •Familial dysautonomia •Fanconi anemia •FAP •Fragile X syndrome •Gaucher disease •Hemophilia A and B •HLA genotyping •HSNF5 mutation

•Achondroplasia •ADPKD1 •ADPKD2 •Adrenoleukodystroph •Age-related aneuploidies •Alpha-thalassemia •Alpha-1-antitrypsin •Alport disease •Amyloid precursor protein (APP) mutation •ARPKD •Becker muscular dystrophy •Beta-thalassemia •Charcot Marie Tooth disease •Chromosomal translocations •Congenital adrenal hyperplasia •Cystic fibrosis •Down syndrome •Duchenne muscular dystophy •Dystonia •Epidermolysis bullosa •Familial dysautonomia •Fanconi anemia •FAP •Fragile X syndrome •Gaucher disease •Hemophilia A and B •HLA genotyping •HSNF5 mutation

•Huntington disease •Hypophosphatasia •Incontinentia pigmenti •Kell disease •Klinefelter syndrome •LCHAD •Lesch Nyhan syndrome •Marfan syndrome •Multiple epiphysial dysplasia •Myotonic dystophy •Myotubular myopathy •NF1 and NF2 •Norrie disease •Osteogenesis imperfecta •OTC deficiency •P53 mutations •PKU •Retinitis pigmentosa •SCA6 •Sickle cell anemia •Sonic hedgehog mutations •Spinal muscular atrophy (SMA) •Tay-Sachs disease •Tuberous sclerosis •Turner syndrome •Von Hippel Lindau •X-linked hydrocephaly •X-linked hyper IgM syndrome

•Huntington disease •Hypophosphatasia •Incontinentia pigmenti •Kell disease •Klinefelter syndrome •LCHAD •Lesch Nyhan syndrome •Marfan syndrome •Multiple epiphysial dysplasia •Myotonic dystophy •Myotubular myopathy •NF1 and NF2 •Norrie disease •Osteogenesis imperfecta •OTC deficiency •P53 mutations •PKU •Retinitis pigmentosa •SCA6 •Sickle cell anemia •Sonic hedgehog mutations •Spinal muscular atrophy (SMA) •Tay-Sachs disease •Tuberous sclerosis •Turner syndrome •Von Hippel Lindau •X-linked hydrocephaly •X-linked hyper IgM syndrome

PGD for Single Gene PGD for Single Gene Disorders - AdvantagesDisorders - Advantages

Safer than elective terminationSafer than elective termination More psychologically acceptable for More psychologically acceptable for

couplescouples Provides couples with another optionProvides couples with another option

AdoptionAdoption SterilizationSterilization Donor gametesDonor gametes

PGDPGD Pre-implantation Pre-implantation

genetic diagnosis genetic diagnosis (PGD)(PGD) has been has been successfully used in successfully used in diagnosing and diagnosing and preventing inherited preventing inherited genetic diseases like genetic diseases like Cystic Fibrosis, Tay Cystic Fibrosis, Tay Sach’s, Thalassemia, Sach’s, Thalassemia, Sickle Cell Anemia Sickle Cell Anemia and may be potentially and may be potentially used to screen for used to screen for cancer mutations.cancer mutations.

PGDPGD

After a cycle of After a cycle of in- vitro in- vitro fertilization, fertilization, biopsy of a single biopsy of a single cell can be cell can be performed from performed from an 8 cell embryo an 8 cell embryo obtained after 3 obtained after 3 days of culture in days of culture in the laboratory.the laboratory.

PGD – Timing of BiopsyPGD – Timing of Biopsy

<67 hours post-<67 hours post-retrievalretrieval

Implantation rates Implantation rates significantly lower if significantly lower if >70 hours>70 hours

Probably represents Probably represents technical issues with technical issues with compacting embryocompacting embryo

PGDPGD

The genetic The genetic material of this material of this single cell can be single cell can be amplified by PCR amplified by PCR and the and the chromosomal chromosomal mutation or an mutation or an aneuploidy can be aneuploidy can be identified in the identified in the embryo that embryo that underwent a biopsy.underwent a biopsy.

PGDPGD The embryos would The embryos would

continue to grow for continue to grow for 2 more days in the 2 more days in the laboratory, awaiting laboratory, awaiting genetic analysis, genetic analysis, and confirmation of and confirmation of which embryos were which embryos were unaffected with the unaffected with the mutation or mutation or aneuploidy.aneuploidy.

PGDPGD

The unaffected The unaffected embryos are then embryos are then transferred to the transferred to the uterus at the uterus at the blastocyst stage on blastocyst stage on day 5 of embryo day 5 of embryo culture and culture and subsequently a child subsequently a child would be born would be born unaffected from the unaffected from the screened genetic screened genetic disease.disease.

Balanced TranslocationsBalanced Translocations

PGD – Female EmbryoPGD – Female Embryo

Uses fluorescence Uses fluorescence in-situ in-situ hybridization hybridization (FISH) technique (FISH) technique to identify XXto identify XX

Sex-linked diseasesSex-linked diseases

““Family balancing”Family balancing”

PGD – Male EmbryoPGD – Male Embryo

Uses FISH to Uses FISH to identify XY embryoidentify XY embryo

Prenatal vs. Prenatal vs. Preimplantation DiagnosisPreimplantation Diagnosis

PNDPND PGD PGD

CellsCells >100,000 >100,000 11

Time Time 2 weeks2 weeks 6-10 hrs6-10 hrs

AccuracyAccuracy 99%99% 90%90%

Who Would Benefit Who Would Benefit From PGD?From PGD?

Couples with a history of --Couples with a history of -- Abnormal numbers of Abnormal numbers of

chromosomeschromosomes Single gene disordersSingle gene disorders Balanced translocationsBalanced translocations

Couples who --Couples who -- Desire an offspring of a certain sex Desire an offspring of a certain sex

Future considerationsFuture considerations

Cytoplasmic transferCytoplasmic transfer Donation of enucleated oocytesDonation of enucleated oocytes

Oocyte cryopreservationOocyte cryopreservation ““Pausing the biological clock”Pausing the biological clock”

Reproduction without gametesReproduction without gametes Use of nuclear material from somatic Use of nuclear material from somatic

cellscells Donated or synthetic cytoplasmDonated or synthetic cytoplasm Reconstituted oocytesReconstituted oocytes

CasesCases

Sperm donorSperm donor Female coupleFemale couple Huntington’s diseaseHuntington’s disease Surrogacy – Baby “M”Surrogacy – Baby “M” Single womanSingle woman Sex-linked diseaseSex-linked disease Family balancingFamily balancing ““Wrongful death” of discarded Wrongful death” of discarded

embryoembryo

Stem Cell Stem Cell Research:Research:

PromiseandControversy

Thank Thank youyou

Documents

Infertility, In Vitro Fertilization (IVF) and Genetic Testing