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

Genetic TestingGenetic Testing

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

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

Causes of InfertilityCauses of Infertility

Female Reproductive OrgansFemale Reproductive Organs

FSH (+)

FSH=Follicle Stimulating Hormone

PhysiologyPhysiology

E2 (-)

E2=Estradiol

Developing follicle

Causes of Female InfertilityCauses of Female Infertility

OvaryOvary TubesTubes UterusUterus CervixCervix HormonesHormones ChromosomesChromosomes

Causes of Female Infertility - OvaryCauses of Female Infertility - Ovary

AGEAGE

Problems with ovulation Problems with ovulation

Premature ovarian failurePremature ovarian failure

Ovary - Female AgeOvary - Female Age

Women are born with their lifetime egg supplyWomen are born with their lifetime egg 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 Women Percentage of Married Women Who are InfertileWho are Infertile

From 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 TestingOvarian Reserve Testing

Day 3 FSH/EDay 3 FSH/E22 FSH (mIU/mL)FSH (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 cycleClinical pregnancy per cycleas function of both FSH and ageas function of both FSH and age

Pearlstone et al, Fertil Steril 1992:58:674

Prevalence of genetically abnormal Prevalence of genetically abnormal oocytes in infertile womenoocytes in infertile women

Abnorm

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 ng/mLMid-luteal serum progesterone > 10 ng/mL Endometrial biopsyEndometrial biopsy

Ovary - Causes of AnovulationOvary - Causes of Anovulation

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

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

Ovary – Premature Ovarian FailureOvary – Premature Ovarian Failure

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 TubesFallopian Tubes

Infection (chlamydia)Infection (chlamydia)

EndometriosisEndometriosis

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

Open TubesOpen Tubes

Blocked TubesBlocked Tubes

Female Infertility - UterusFemale Infertility - Uterus

UterusUterus FibroidsFibroids PolypsPolyps

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

Asherman’s syndromeAsherman’s syndrome

Female Reproductive OrgansFemale Reproductive Organs

Female InfertilityFemale Infertility

Uterine muscle tumorUterine muscle tumor

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

25-30% of women25-30% of women

Normal Shape of UterusNormal Shape of Uterus

Fibroid UterusFibroid Uterus

Female Infertility - UterusFemale Infertility - Uterus

UterusUterus TumorsTumors

FibroidsFibroids PolypsPolyps

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

Asherman’s syndromeAsherman’s syndrome

Mullerian DefectMullerian Defect

Treatment with HysteroscopyTreatment with Hysteroscopy

Treatment with LaparoscopyTreatment with Laparoscopy

Female Infertility - UterusFemale Infertility - Uterus

UterusUterus TumorsTumors

FibroidsFibroids PolypsPolyps

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

Asherman’s syndromeAsherman’s syndrome

Female Infertility - CervixFemale Infertility - Cervix

CervixCervix Post-surgicalPost-surgical

StenosisStenosis Mucus changesMucus changes

Female Infertility - HormonesFemale Infertility - Hormones

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 InfertilityOther Causes of Female Infertility

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 OrgansMale Reproductive Organs

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 MorphologyNormal Sperm Morphology

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 oocytePenetration of oocyte Decondensation and Decondensation and

aster formationaster formation 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 ProductionAbnormalities of Sperm Production

GeneticGenetic Y chromosome microdeletionsY chromosome microdeletions

Damage to testes – anatomicalDamage to testes – anatomical CryptorchidismCryptorchidism VaricoceleVaricocele

InfectionInfection Mumps orchitisMumps orchitis

GonadotoxinsGonadotoxins

Abnormalities of Sperm FunctionAbnormalities of Sperm Function

Antisperm antibodiesAntisperm antibodies Genital tract inflammationGenital tract inflammation

prostatitisprostatitis

VaricoceleVaricocele Failure of acrosome reactionFailure of acrosome reaction Problems with sperm binding/penetrationProblems with sperm binding/penetration

Obstructions in Ductal SystemObstructions in Ductal System

VasectomyVasectomy

Congenital bilateral absence of the vas deferensCongenital bilateral absence of the vas deferens

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

Male Infertility - LifestyleMale Infertility - Lifestyle

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

Infertility: Initial EvaluationInfertility: Initial Evaluation

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 insemination Clomiphene citrate + intrauterine insemination

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

Intrauterine Insemination (IUI)Intrauterine Insemination (IUI)

Clomiphene Citrate (Clomid)Clomiphene Citrate (Clomid)

Anti-estrogenAnti-estrogen

↑ ↑ FSHFSH

↑ ↑ egg productionegg production

$$

Estrogen AntagonistEstrogen Antagonist

FSH (+)

FSH=Follicle Stimulating Hormone

PhysiologyPhysiology

E2 (-)

E2=Estradiol

Developing follicle

Clomid

ART: Treatment OptionsART: Treatment Options

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

ObservationObservation CC + IUICC + IUI

CyclesCycles 150150 148148

PregnanciesPregnancies 55 1414

FecundityFecundity 0.0330.033 0.0950.095

Deaton et al, Fertil Steril 1990;54:1083

FSH +IUIFSH +IUI

Daily injections Daily injections

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

$$$$

FSH (+)

FSH=Follicle Stimulating Hormone

PhysiologyPhysiology

E2 (-)

E2=Estradiol

Developing follicle

Infertility Treatment OptionsInfertility Treatment Options

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

Serhall et al, Fertil Steril 1988;49:602

TreatmentTreatment CyclesCycles PregnancyPregnancy Pregnancy per Pregnancy per cyclecycle

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 - HistoryIn Vitro Fertilization - History

1978 – First “test tube” baby was born in England 1978 – First “test tube” baby was born in 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 to uterus2004 - >98% IVF with transfer of embryo to uterus

In Vitro Fertilization (IVF)In Vitro Fertilization (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 HyperstimulationOvarian Hyperstimulation

Egg RetrievalEgg Retrieval

Good EggGood Egg

Bad EggBad Egg

FertilizationFertilization

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

1 day after egg retrieval1 day after egg retrieval

Day 3 EmbryoDay 3 Embryo

Day 3 EmbryoDay 3 Embryo

Day 3 EmbryoDay 3 Embryo

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 (ICSI)Intracytoplasmic sperm injection (ICSI) Preimplantation genetic diagnosis (PGD)Preimplantation genetic diagnosis (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, or a poor For patients with 2 or more failed IVF cycles, or a poor prognosis, can add more based on clinical judgementprognosis, can add more based on clinical 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 PregnancyIVF and Multiple Pregnancy

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

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

Current status of ART in the USACurrent status of ART in the 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 = $10,000-15,000IVF cycle + medications = $10,000-15,000 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 donationEgg donation

IVF for twoIVF for two

DonorDonor Standard controlled Standard controlled

ovarian hyperstimulationovarian hyperstimulation Egg retrievalEgg retrieval

RecipientRecipient Embryo transferEmbryo transfer

Egg DonationEgg Donation

Known/anonymous donorKnown/anonymous donor

<35 years old<35 years old

Current status of ART in the Current status of ART in the USAUSA

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

Egg DonationEgg Donation

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

by Jessica Cohen by Jessica Cohen

Egg DonationEgg Donation

October 23, 1999October 23, 1999 Selling Fashion Models' Eggs Online Raises Ethics IssuesSelling Fashion Models' Eggs Online Raises Ethics Issues 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 photographer mainstream infertility groups, a longtime fashion photographer has begun offering up models as egg donors to the highest has begun offering up models as egg donors to the highest bidders, auctioning their ova via the Internet to would-be parents bidders, auctioning their ova via the Internet to would-be parents willing to pay up to $150,000 in hopes of having a beautiful willing to pay up to $150,000 in hopes of having a beautiful child. child.

Egg Donation - Ethical IssuesEgg Donation - Ethical Issues

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 Decade of Pregnancy in the Sixth Decade of LifeLife

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 Decade of Pregnancy in the Sixth Decade of LifeLife

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

Of the 77 women, 42 (54.5%) had live birthsOf the 77 women, 42 (54.5%) had live births

45 deliveries in 42 women45 deliveries in 42 women

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

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

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

Offered to all womenOffered to all women Prenatal vitamins – 400 micrograms folic acid/dayPrenatal vitamins – 400 micrograms folic acid/day Rubella immunityRubella immunity Varicella immunityVaricella immunity Rh statusRh status HIVHIV Hepatitis B screenHepatitis B screen Cystic Fibrosis screeningCystic Fibrosis screening

Preconception CounselingPreconception Counseling

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 disordersAshkenazi Jews – 7 autosomal recessive disorders

Gaucher disease (1/13), Tay-Sachs (1/30), Familial Gaucher disease (1/13), Tay-Sachs (1/30), Familial dysautonomia (1/30), Canavan disease (1/40), Fanconi dysautonomia (1/30), Canavan disease (1/40), Fanconi anemia (1/89), Niemann-Pick disease (1/90), Bloom anemia (1/89), Niemann-Pick disease (1/90), Bloom syndrome (1/100)syndrome (1/100)

Prenatal TestingPrenatal Testing

UltrasoundsUltrasounds

Serum screensSerum screens

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

AmniocentesisAmniocentesis

Prenatal Tests - UltrasoundPrenatal Tests - Ultrasound

Nuchal translucency screeningNuchal translucency screening

Performed between 10-13 weeks gestationPerformed between 10-13 weeks gestation

Screen for Down SyndromeScreen for Down Syndrome

Nuchal TranslucencyNuchal Translucency

Prenatal Tests - UltrasoundPrenatal Tests - Ultrasound

Second trimester ultrasoundSecond trimester ultrasound Detailed examDetailed exam Down Syndrome, other trisomiesDown Syndrome, other trisomies Cardiac, renal, spinal, limb, brain deformitiesCardiac, renal, spinal, limb, brain deformities Cleft lip/palateCleft lip/palate

Ultrasound AbnormalitiesUltrasound Abnormalities

Ultrasound AbnormalitiesUltrasound Abnormalities

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 and Neural tube defects, Down syndrome and

trisomy 18trisomy 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 defectPrevious child or pregnancy with birth defect 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 cellsCatheter/needle biopsy of placental cells Performed through cervix or abdomenPerformed through cervix or abdomen Can tests for aneuploidy and enzyme defectsCan tests for aneuploidy and enzyme defects 1-2% miscarriage rate1-2% miscarriage rate Digit/limb deficiencies (10 weeks)Digit/limb deficiencies (10 weeks)

Prenatal Tests - AmniocentesisPrenatal Tests - Amniocentesis

Performed at 15-18 weeks gestationPerformed at 15-18 weeks gestation 10 cc amniotic fluid10 cc amniotic fluid Living cells from fetus in amniotic fluidLiving cells from fetus in amniotic fluid 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 Hemoglobinopathies Sickle Cell Disease (SCD) and Hemoglobinopathies

Postnatal Screening – Tandem Mass Postnatal Screening – Tandem Mass SpectrometySpectromety

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 Diagnosis Preimplantation Genetic Diagnosis (PGD)(PGD)

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

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

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

PGD – Clinical IndicationsPGD – Clinical Indications

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 (FISH)Fluorescence in situ hybridization (FISH) Aneuploidy/translocations and sexing (5-9 Aneuploidy/translocations and sexing (5-9

chromosomes)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

•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 Disorders - PGD for Single Gene Disorders - AdvantagesAdvantages

Safer than elective terminationSafer than elective termination More psychologically acceptable for couplesMore psychologically acceptable for couples Provides couples with another optionProvides couples with another option

AdoptionAdoption SterilizationSterilization Donor gametesDonor gametes

PGDPGD

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

PGDPGD

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

PGD – Timing of BiopsyPGD – Timing of Biopsy

<67 hours post-retrieval<67 hours post-retrieval

Implantation rates Implantation rates significantly lower if >70 significantly lower if >70 hourshours

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

PGDPGD

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

PGDPGD

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

PGDPGD

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

Balanced TranslocationsBalanced Translocations

PGD – Female EmbryoPGD – Female Embryo

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

Sex-linked diseasesSex-linked diseases

““Family balancing”Family balancing”

PGD – Male EmbryoPGD – Male Embryo

Uses FISH to identify Uses FISH to identify XY embryoXY embryo

Prenatal vs. Preimplantation Prenatal vs. Preimplantation DiagnosisDiagnosis

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 From PGD?Who Would Benefit From PGD?

Couples with a history of --Couples with a history of -- Abnormal numbers of chromosomesAbnormal numbers of chromosomes 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 cellsUse of nuclear material from somatic cells 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 embryoWrongful death” of discarded embryo

Stem Cell Stem Cell Research:Research:

PromiseandControversy

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