MELBOURNE IVF

PRE-IMPLANTATION GENETIC DIAGNOSIS AND ASSISTED

REPRODUCTIVE TECHNOLOGY IN HAEMOPHILIA

DR PENELOPE FOSTER

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WHAT IS PGD ?

early embryo diagnosisallows selection of unaffected embryos for transfer to patientalternative to antenatal testing and termination of affected pregnancy

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TECHNIQUE OF PGD

standard IVF cyclebiopsy of 1 or 2 cells from day 3 embryodiagnostic testing on biopsied cellsselection of embryos for transfer

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PGD IN HAEMOPHILIA

OPTIONS Sex selection:

if affected husband, all male offspring unaffected, all females carriers = select male embryos for transferif carrier wife, 1/2 males affected, 1/2 females carrier = select female embryos for transfer

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PGD IN HAEMOPHILIA

OPTIONSSpecific gene detection

avoids discarding unaffected male embryosavoids transfer of carrier female embryos

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SEX SELECTION - FISH

FLUORESCENT IN-SITU HYBRIDISATIONdetects chromosome number cell from embryo fixed to slideapply FISH probesDNA sequences complementary to small segment of particular chromosomeprobes labelled with coloured fluorochromescoloured spots indicate presence of sequence8-probe FISH – chromosomes 4,13,16,18,21,22,X,Yselect euploid XX or XY embryos for transfer

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13, 16, 18, 21, 22 X, Y, 4

FISH ON BLASTOMERES

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XX XY

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PGD FOR SPECIFIC GENE DETECTION

DNA amplification by PCR2 cells from embryofragment analysis on DNA sequencerinclusion of informative markersindividualised tests for each couplesignificant time and effort required for each test

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Markers AllelesDXS1073 124, 126, 128DXS8061 139, 145, 147Factor VIII wt, mut

AMEL 111 = X c.some

117 = Y c.some

128147wt

126139mut

124145wt

124145wt

126139mut

128147wt

Add title

Unaffected Male

Unaffected Female

Carrier Female

Affected Male

Husband Mother

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MOTHER

AFFECTED MALE

RESULTS OF PCR ANALYSIS FOR HAEMOPHILIA A

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MONOGENIC PGD AT MELBOURNE IVF

Tests developed to date:Cystic fibrosis β-thalassaemiaα-thalassaemiaDuchennes muscular dystrophyα-1-antitrypsin deficiencyKennedys disease Fragile–X Motor neurone disease (exclusion)Huntington’s disease (direct)Huntington’s disease (exclusion)Neurofibromatosis 1Hirschprung’s diseaseX-linked hydrocephalusMyotonic dystrophyChronic granulomatous diseaseNiemann-Pick type COpitz syndromeRapp-Hodgkin ectodermal dysplasia

Multiple cases for many of these

Tests being developed:Congenital adrenal hyperplasiaTuberous sclerosisMultiple exostosis

Waiting:BRCA2Waardenburg syndromeNeurofibromatosis 2AR polycystic kidney diseaseTreacher-Collins syndromeMenkes diseaseFamilial adenomatous polyposisRetinoblastomaWHIM syndromeHaemophilia A

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Cystic fibrosisTay Sachs diseaseβ-thalassaemiaSickle cell anaemiaRh blood typingSpinal muscular atrophyAdrenogenital syndromeCongenital adrenal hyperplasiaPlakophilin-1 (PKP1)MCADCDG1C Epidermolysis bullosaGaucher’s disease Hyperinsulinemic hypoglycemia PHH1Fanconis anemiaHLA matchingFragile XMyotonic dystrophyHuntingtonsWiscott-Aldrich syndromeIncontinentia pigmentiOrnithine transcarbamylase def.Myotubular myopathyHunter syndromeFabry diseaseChoroideraemiaKallman syndromeCoffin-Lowy syndromeBarth syndromeHypospadiasGolabi-Rosen syndrome

Marfans syndromeCharcot-Marie-Tooth disease (type 1A)Amyloid polyneuropathyCrouzons syndromeNF2Osteogenesis imperfecta I and IVStickler syndromeTuberous sclerosis Central core disease Familial adenomatous polyposis coliLi Fraumeni syndromeLesch Nyhan syndromeDuchenne muscular dystrophyBecker muscular dystrophyHaemophilia ACharcot-Marie-Tooth disease Retinitis pigmentosaOrnithine Transcarbamylase DeficiencyAgammaglobulinemiaAlport syndrome Hunter’s syndrome MPSII Oro-facial-digital syndrome type 1AdrenoleukodystrophyChronic granulomatous diseaseMenkes diseaseLowe syndromeEctodermal dysplasiaEpilepsyBRCA1AtaxiaRenal agenesisNorrie disease

Conditions that have been diagnosed by PGD – worldwide

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IVF Cycle

Pituitary down – regulation with OCP & GNRH agonist (gonadotrophin –releasing hormone)Ovarian stimulation with r FSH (follicle stimulating hormone)hCG triggerVaginal ultrasound – assisted OPU (ovum pick up)Embryo transfer (ET) 2 or 3 days after OPU

Monitor follicular maturation with vaginal ultrasoundAim for cohort of “leading follicles” of 18-20mm diameterAverage egg No./OPU = 11Fertilisation ~60%

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. . . . . . . . . . . . . . . . . . . . .

Period Synarel

O C P F S H O P U E T

IVF Cycle

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Pipette loaded with acidified culture media – pH 2.4

Hole digested in zona by acid

Acid drilling

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PGD OUTCOMES – “FERTILE”PATIENTS (all data up to end 2006)

13.6

14.3

52

21.4

35.2

29

ROB TRANS

1621.818.328.134.8IMP. RATE

2426.825.029.238.9% CLIN PREG

1944.29.75158% NO ET

36.025.35211.514.3%“NORMAL”

EMBRYOS

~3434.435.433.534.5AGE

730183624943CYCLES

ESHRE DATA V

MIVF TOTALMONOGENICREC

TRANSSEXING

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Request for PGD submitted

PGD Committee•Senior PGD scientists•IVF doctor•Clinical geneticist•Genetics counsellor•PGD nurse

PGD refused

PGD counselling with genetics counsellor

PGD counselling with clinical geneticist

IVF counselling (mandatory in Victoria)

PGD cycle starts

Access to PGD at Melbourne IVF

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Although the degree of accuracy of these tests is high, all tests have a failure rate, and the test results could be wrong.

A full genetic analysis is not being carried out and there are many other genetic conditions that are not being analysed or tested for.

Finding a normal cell using FISH testing does not mean that a baby resulting from the embryo will have the normal number of chromosomes or be of the expected sex.

In single gene defect testing, we cannot guarantee that the embryo will not have the disorder being tested for.

Consent to PGD

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Consent to PGDIt is strongly recommended that all women with PGD pregnancy consider DNA testing in early pregnancy ( CVS or amniocentesis ) to confirm the early embryo diagnosis.

Spontaneous conception may occur during a PDG cycle, and all couples having PGD should avoid any form of unprotected sex during the treatment cycle.

Rarely, some embryos may be destroyed during the biopsy procedure.

Rarely, it may not be possible to obtain a result on an embryo.

Embryos that are very poor quality will not be subjected to embryo biopsy and will be discarded.

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PGD - BENEFITSRELIABLE97% embryos diagnosed

ACCURATEmisdiagnosis rate ~ 2%

RAPIDembryo biopsy and diagnostic testing completed 8 –

30 hours

TREATMENT OPTIONalternative to antenatal testing and TOP

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PGD – PITFALLS

InvasiveHighly medicalised, requires IVFExpensiveSpecific feasibility testing can take monthsNo guarantee of pregnancy

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HIV AND ASSISTED REPRODUCTIVE TECHNOLOGY

Chronic Viral Illness Clinic at Royal Women’s Hospital Melbourne established 2002principle of harm minimisation (reduced risk of HIV transmission to partner and baby)Use of assisted reproductive technology (intra-uterine insemination or IVF/ICSI)

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HIV +ve MALE

Good healthUndetectable viral load for 2 monthsSemen screening for HIV

2 successive samples <50 copies=semen storage for IUI /IVF (all

semen samples tested for HIV RNA and DNA)risk of transmission to partner <1/2000

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CVI PROGRAMME RWH

33 referrals27 male HIV+ve4 female HIV+ve1 couple both HIV+ve

20 patients treated16 male HIV+ve - 12 pregnancies(7 delivered, inc 2 sets twins, 2 ongoing)3 female HIV+ve - 1 ongoing pregnancy

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ACKNOWLEDGEMENTSMIVF PGD TEAM

Leeanda WiltonSharyn Stock-MyerPam MatthewsMirjana MarticGreta GilliesKay OkeKate Pope

John McBainPenelope FosterMac GardnerDavid AmorStacey RoePeter ColemanRiddhi Marfatia

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ACKNOWLEDGEMENTSRWH CVI COMMITTEE

Janell ArcherGordon BakerHarold BourneKate CherryGary ClarkeSuzanne CrowePenelope FosterSuzanne Garland

Rachael Knight Hayley MaticAnne MijchSam PernaSepehr TabriziLoreto ValentVicky GreengrassMichelle Giles