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Textbook of Assisted Reproductive Technologies Laboratory and Clinical Perspectives Third EdiTion Edited by
david K GardnEr
ariEL WEissman
CoLin m hoWLEs
ZEEv shoham
Textbookof AssistedReproductive Technologies Theeditors(fromlefttoright:DavidKGardner,ColinMHowles,ZeevShoham andArielWeissman)attheannualmeetingofESHRE,Barcelona,2008
Theeditorswouldliketomakeaspecialacknowledgmenttotheirrespectivechildren,whoare aconstantreminderofthejoyandhappinessthatworkinginthisfieldofmedicinecanbring tofamilies,friends,andcommunities
Textbookof AssistedReproductive Technologies LaboratoryandClinicalPerspectives ThirdEdition Editedby
DavidKGardner DPhil ChairofZoology,UniversityofMelbourne,Victoria,AustraliaandScientificDirector, ColoradoCenterforReproductiveMedicine,USA
ArielWeissman MD SeniorPhysician,IVFUnit,DepartmentofObstetricsandGynecology,EdithWolfson MedicalCenter,HolonandSacklerFacultyofMedicine,TelAvivUniversity TelAviv,Israel
ColinMHowles PhD, FRSM VicePresident,ScientificAffairsFertility,GlobalMedicalAffairs,MerckSerono InternationalSA,Geneva,Switzerland
ZeevShoham MD Director,ReproductiveMedicineandInfertilityUnit,DepartmentofObstetricsand Gynecology,KaplanMedicalCenter,Rehovot,Israel
FirstpublishedintheUnitedKingdomin2004
ThirdeditionpublishedintheUnitedKingdomin2009byInformaHealthcare,TelephoneHouse,69–77 PaulStreet,London,EC2A4LQ.InformaHealthcareisatradingdivisionofInformaUKLtd.Registered Office:37/41MortimerStreet,LondonW1T3JH.RegisteredinEnglandandWalesnumber1072954
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2.Qualitycontrol:maintainingstabilityinthelaboratory
DavidHMcCulloh
3.TheARTlaboratoryintheeraofISO1000andGLP
CeceliaSjöblomandChristophKeck
HaroldBourne,JanellArcher,DavidHEdgarandHWGordonBaker 6.Spermchromatinassessment
AshokAgarwal,JurisErenpreissandRakeshSharma
7.Oocyteretrievalandselection
LauraFRienziandFilippoMUbaldi
8.PreparationandevaluationofoocytesforICSI
IritGranotandNavaDekel
9.Oocyteinvitromaturation
DanielaNogueira,SergioRomero,LeenVanhoutte,DanielGdeMatosandJohanSmitz
10.Useofinvitromaturationinaclinicalsetting
Anne-MariaSuikkari
Micromanipulation
11.Equipmentandgeneraltechnicalaspectsofmicromanipulationofgametesandembryos 163 FrankLBarnes
12.Intracytoplasmicsperminjection:technicalaspects 171 GianpieroDPalermo,QueenieVNeri,TakumiTakeuchi,SimonJHongandZevRosenwaks
13.Assistedhatching 181 AnnaVeiga,IreneBoisoandItziarBelil
14.Humanembryobiopsyprocedures 191 AlanRThornhillandAlanHHandyside
DavidKGardnerandMichelleLane
17.Evaluationofembryoquality:newstrategiestofacilitatesingleembryotransfer
DennySakkasandDavidKGardner Cryopreservation
18.Thehumanoocyte:controlledratecooling
AndreaBoriniandGiovanniCoticchio 19.Thehumanoocyte:vitrification
LucindaLVeeckGosden,RosemaryBerrios,RichardBodine,RobertNClarkeandNikicaZaninovic
21.Thehumanembryo:vitrification
ZsoltPeterNagy,GáborVajta,Ching-ChienChangandHiltonKort
22.Managingthecryopreservedembryobank
PhillipMatson 23.Cryopreservationandstorageofspermatozoa
EileenAMcLaughlinandAllanAPacey
JosephPAlukal,DoloresJLambandLarryILipshultz
25.Ovariantissuecryopreservationandotherfertilitypreservationstrategies
ErkanBuyuk,OzgurOktem,MuratSonmezerandKutlukHOktay
DiagnosisofGeneticDiseaseinPreimplantationEmbryos
26.Severemalefactor:geneticconsequencesandrecommendationsforgenetictesting
IngeLiebaers,AndréVanSteirteghemandWillyLissens
27.Polarbodybiopsy
MarkusMontag,KatrinvanderVenandHansvanderVen
28.Clinicalapplicationofpolarbodybiopsy
YuryVerlinskyandAnverKuliev
29.Preimplantationgeneticdiagnosisforinfertility
SantiagoMunné
30.Geneticanalysisoftheembryo
YuralYaron,VeronicaGold,RonniGamzuandMiraMalcov
31.Proteomicanalysisoftheembryo
MandyKatz-Jaffe
Implantation
32.Embryonicandmaternaldialogueandtheanalysisofuterinereceptivity
FranciscoDomínguez,JoseAntonioHorcajadasandCarlosSimón
QualityManagementSystems
33.Qualitymanagementinreproductivemedicine
ChristophKeck,CeceliaSjöblom,RobertFischer,VeraBauklohandMichaelAlper
34.IndicationsforIVFtreatment:fromdiagnosistoprognosis
NickSMacklon,FrankJBroekmansandBartCJMFauser
35.Initialinvestigationofthepatient(femaleandmale)
BulentGulekli,TimJChildandSeangLinTan
36.Drugsusedforcontrolledovarianstimulation:clomiphenecitrate,aromataseinhibitors, metformin,gonadotropins,gonadotropin-releasinghormoneanalogs,and recombinantgonadotropins
ZeevShohamandColinMHowles
37.TheroleofFSHandLHinovulationinduction:currentconcepts
JuanBalasch
StimulationProtocols
38.EndocrinecharacteristicsofARTcycles
Jean-NoëlHuguesandIsabelleCédrin-Durnerin
39.TheuseofGnRHagonists
JudithAFHuirneandRoelSchats
40.GnRHantagonists
MichaelLudwig
41.MonitoringIVFcycles
MattsWiklandandTorbjörnHilljensjö
42.Oocytecollection
GabKovacs
43.Thelutealphase:lutealsupportprotocols
JamesPToner
44.Treatmentstrategiesinassistedreproductionforthelowresponderpatient
ArielWeissmanandColinMHowles
45.Repeatedimplantationfailure:thepreferredtherapeuticapproach
MarkADamarioandZevRosenwaks
TechnicalProceduresandOutcomes
46.UltrasoundinART
MarinkoMBiljan
47.Sperm-recoverytechniques:clinicalaspects
HermanTournayeandPatricioDonoso
48.Gameteintrafallopiantransfer(GIFT)andzygoteintrafallopiantransfer(ZIFT)
MachelleMSeibelandArielWeissman
49.Embryotransfer
LeifBungumandMonaBungum
50.Anesthesiaandin-vitrofertilization
EthanEHarow
51.Medicalconsiderationsofsingleembryotransfer
OutiHovatta
SpecialMedicalConditions
52.EndometriosisandART
AndyHuang,MarkHunterandAlanHDeCherney
53.PolycysticovariesandART
ThomasHTangandAdamHBalen
54.Prognostictestingforovarianreserve
FrankJBroekmans,BartCJMFauserandNickSMacklon
55.Managementofhydrosalpinx
AnnikaStrandell
ComplicationsofTreatment
56.Severeovarianhyperstimulationsyndrome
ZalmanLevineandDanielNavot
57.Theenvironmentandreproduction
KennethBarronandMachelleMSeibel
58.Bleeding,severepelvicinfection,andectopicpregnancy
RaoulOrvietoandZionBen-Rafael
59.Iatrogenicmultiplepregnancy:theriskofART
IsaacBlickstein
EggDonationandSurrogateMotherhood
60.Eggandembryodonation
MarkVSauerandMatthewACohen
61.Gestationalsurrogacy
PeterRBrinsden
FutureDirectionsandClinicalApplications
62.Humanembryonicstemcells
RachelEigesandBenjaminReubinoff
63.MicrofluidicsinART:currentprogressandfuturedirections
JasonESwain,ThomasBPool,ShuichiTakyamaandGaryDSmith
TheSupportTeam
64.TheevolvingroleoftheARTnurse:acontemporaryreview
JoanneLLibraro 65.PatientsupportintheARTprogram
SharonNCovington
66.Therelationshipbetweenstressandinvitrofertilizationoutcome
AndreaMechanickBraverman
EthicsandLegislation
67.Theimpactoflegislationandsocioeconomicfactorsintheaccesstoand globalpracticeofART
FernandoZegers-HochschildandKarlGNygren
68.RecentethicaldilemmasinART
FrançoiseShenfield
ListofContributors AshokAgarwal
CenterforReproductiveMedicine,Glickman UrologicalandKidneyInstitute andObstetrics–GynecologyandWomen’sHealth Institute ClevelandClinic Cleveland,Ohio,USA
MichaelAlper
BostonIVF Waltham,Massachusetts,USA
JosephPAlukal ScottDepartmentofUrology BaylorCollegeofMedicine Houston,Texas,USA
JanellArcher
ReproductiveServices
TheRoyalWomen’sHospitalandMelbourneIVF Melbourne,Victoria,Australia
HWGordonBaker UniversityofMelbourneDepartmentofObstetrics andGynaecology
TheRoyalWomen’sHospitalandMelbourneIVF Melbourne,Victoria,Australia
JuanBalasch
DepartmentofObstetricsandGynecology FacultyofMedicine HospitalClinic UniversityofBarcelona Barcelona,Spain
AdamHBalen
ReproductiveMedicineandSurgery LeedsGeneralInfirmary Leeds,UK
FrankLBarnes IVFLabs,LLC SaltLakeCity,Utah,USA
KennethBarron
DepartmentofObstetricsandGynecology UniversityofMassachusettsSchoolofMedicine Worcester,Massachusetts,USA
VeraBaukloh FertilityCenterHamburg Hamburg,Germany
ItziarBelil
ReproductiveMedicineService InstitutUniversitariDexeus Barcelona,Spain
ZionBen-Rafael
DepartmentofObstetricsandGynecology RabinMedicalCenter,PetahTikva andSacklerFacultyofMedicine TelAvivUniversity TelAviv,Israel
RosemaryBerrios
TheCenterforReproductiveMedicineandInfertility WeillMedicalCollegeofCornellUniversity NewYork,NewYork,USA
†MarinkoMBiljan
IsaacBlickstein
DepartmentofObstetricsandGynecology KaplanMedicalCenter Rehovot,Israel
RichardBodine
TheCenterforReproductiveMedicineandInfertility WeillMedicalCollegeofCornellUniversity NewYork,NewYork,USA
IreneBoiso
CentredeReproduccióAssistida ClinicaSagradaFamilia Barcelona,Spain
AndreaBorini TecnobiosProcreazione CentreforReproductiveHealth Bologna,Italy
HaroldBourne
ReproductiveServicesandMelbourneIVF TheRoyalWomen’sHospital Carlton,Victoria,Australia
AndreaMechanickBraverman PsychologicalandComplementaryCare ReproductiveMedicineAssociatesofNewJersey Morristown,NewJersey,USA
PeterRBrinsden BournHallClinic Bourn,Cambridge,UK
FrankJBroekmans
DepartmentofReproductionandGynaecology UniversityMedicalCentreUtrecht Utrecht,TheNetherlands
LeifBungum
ReproductiveMedicineCentre MalmoUniversityHospital Malmo,Sweden
MonaBungum
ReproductiveMedicineCentre MalmoUniversityHospital Malmo,Sweden
ErkanBuyuk DepartmentofObstetricsandGynecology AlbertEinsteinCollegeofMedicineofYeshiva University NewYork,NewYork,USA
IsabelleCédrin-Durnerin UniversityofParisXIII DivisionofReproductiveMedicine HôpitalJeanVerdier Bondy,France
Ching-ChienChang ReproductiveBiologyAssociates Atlanta,Georgia,USA
TimJChild OxfordFertilityUnit NuffieldDepartmentofObstetricsandGynaecology UniversityofOxford JohnRadcliffeHospital Oxford,UK
RobertNClarke TheCenterforReproductiveMedicineandInfertility WeillMedicalCollegeofCornellUniversity NewYork,NewYork,USA
JacquesCohen GalileoResearchLaboratories AnsoniaStation NewYork,NewYork,USA
MatthewACohen DepartmentofObstetricsandGynecology CollegeofPhysicians&Surgeons ColumbiaUniversity NewYork,NewYork,USA
GiovanniCoticchio TecnobiosProcreazione Bologna,Italy
SharonNCovington PsychologicalSupportServices ShadyGroveFertilityReproductiveScienceCenter Rockville,Maryland,USA
MarkADamario DepartmentofObstetrics,GynecologyandWomen’s Health UniversityofMinnesota Minneapolis,Minnesota,USA
AlanHDeCherney DepartmentofObstetricsandGynecology DavidGeffenSchoolofMedicine LosAngeles,California,USA
NavaDekel DepartmentofBiologicalRegulation TheWeizmannInstituteofScience Rehovot,Israel
DanielGdeMatos EMDSeronoReproductiveBiologyInstitute Rockland,Massachusetts,USA
FranciscoDomínguez FundaciónInstitutoValencianodeInfertilidad InstitutoUniversitarioIVI ValenciaUniversity Valencia,Spain
PatricioDomoso CentreforReproductiveMedicine ClinicaAlemanadeSantiago Santiago,Chile
DavidHEdgar ReproductiveServices
TheRoyalWomen’sHospitalandMelbourneIVF Melbourne,Victoria,Australia
RobertGEdwards DuckEndFarm DryDrayton Cambridge,UK
RachelEiges IVFUnit,DepartmentofObstetricsand Gynecology andGoldyneSavad InstituteofGeneTherapy HadassahUniversityHospital Jerusalem,Israel
JurisErenpreiss AndrologyLaboratory RigaStradinsUniversity Riga,Latvia
BartCJMFauser DepartmentofReproductiveMedicine UniversityMedicalCenterUtrecht Utrecht,TheNetherlands
RobertFischer FertilityCenterHamburg Hamburg,Germany
RonniGamzu
DepartmentofObstetricsandGynecology LisMaternityHospital TelAvivSouraskyMedicalCenter TelAviv,Israel
DavidKGardner DepartmentofZoology UniversityofMelbourne, Victoria,Australia
JohnGarrisi GalileoResearchLaboratoriesLLC NewYork,NewYork,USA
AntoniaGilligan AlphaEnvironmental,Inc. JerseyCity,NewJersey,USA
VeronicaGold
SaraRacineInVitroFertilizationUnit TelAvivSouraskyMedicalCenter TelAviv,Israel
IritGranot IVFUnit,DepartmentofObstetricsandGynecology KaplanMedicalCenter Rehovot,Israel
BulentGulekli DokuzEylulUniversitesi TipFakultesi KadinHastaliklariveDogumABD Balcova-Izmir,Turkey
AlanHHandyside TheLondonBridgeFertility,Gynaecologyand GeneticsCentre London,UK
EthanEHarow OutpatientSurgicalCenter EdithWolfsonMedicalCenter Holon,Israel
TorbjörnHillensjö FertilityCentreScandinavia Carlander’sHospital Göteborg,Sweden
SimonJHong AndrologyandAssistedFertilization CornellInstituteforReproductiveMedicine NewYork,NewYork,USA
JoseAntonioHorcajadas FundaciónInstitutoValencianodeInfertilidad InstitutoUniversitarioIVI ValenciaUniversity Valencia,Spain
OutiHovatta KarolinskaInstitute KarolinskaUniversityHospitalHuddinge Stockholm,Sweden
ColinMHowles GlobalMedicalAffairs MerckSeronoInternationalSA Geneva,Switzerland
AndyHuang DepartmentofObstetricsandGynecology DavidGeffenSchoolofMedicine LosAngeles,California,USA
Jean-NoëlHugues UniversityofParisXIII DivisionofReproductiveMedicine HôpitalJeanVerdier Bondy,France
JudithAFHuirne
DepartmentofObstetricsandGynecology DivisionofReproductionandFertilityInvestigation IVFCenter VrijeUniversiteitMedicalCenter Amsterdam,TheNetherlands
MarkHunter
DepartmentofObstetricsandGynecology DavidGeffenSchoolofMedicine LosAngeles,California,USA
MandyKatz-Jaffe
ColoradoCenterforReproductiveMedicine LoneTree,Colorado,USA
ChristophKeck DepartmentofObstetricsandGynecology UniversityofFreiburg Freiburg,Germany
HiltonKort ReproductiveBiologyAssociates Atlanta,Georgia,USA
GabKovacs MonashIVF Richmond,Victoria,Australia
AnverKuliev ReproductiveGeneticsInstitute Chicago,Illinois,USA
MasashigeKuwayama KatoLadies’Clinic Shinjuku Tokyo,Japan
DoloresJLamb ScottDepartmentofUrology BaylorCollegeofMedicine Houston,Texas,USA
MichelleLane
DepartmentofObstetricsandGynecology UniversityofAdelaide Adelaide,SouthAustralia andRepromed Dulwich,SouthAustralia,Australia
ZalmanLevine DivisionofReproductiveEndocrinologyand Infertility
NewYorkMedicalCollege FertilityInstituteofNewJerseyandNewYork Westwood,NewJersey,USA
JoanneLLibraro CenterforReproductiveMedicineandInfertility WeillMedicalCollege NewYork,NewYork,USA
IngeLiebaers CenterforMedicalGenetics UniversityHospitalVUB Brussels,Belgium
LarryILipshultz DivisionofMaleReproductiveMedicineandSurgery BaylorCollegeofMedicine ScottDepartmentofUrology Houston,Texas,USA
WillyLissens CenterforMedicalGenetics UniversityHospitalVUB Brussels,Belgium
MichaelLudwig CentreforReproductiveMedicineand GynaecologicalEndocrinology EndokrinologikumHamburg Hamburg,Germany
NickSMacklon DivisionofReproductiveMedicine DepartmentofObstetricsandGynecology ErasmusMedicalCenter Rotterdam,TheNetherlands
MiraMalcov SaraRacineInVitroFertilizationUnit TelAvivSouraskyMedicalCenter TelAviv,Israel
PhillipMatson HollywoodFertilityCentre HollywoodPrivateHospital MonashAvenue Nedlands,WesternAustralia,Australia
DavidHMcCulloh UniversityReproductiveAssociates,PC HasbrouckHeights,NewJersey,USA
EileenAMcLaughlin ARCCentreofExcellenceinBiotechnologyand Development SchoolofEnvironmentalandLifeSciences UniversityofNewcastle Callaghan,NewSouthWales,Australia
MarkusMontag DepartmentofGynaecologicalEndocrinologyand ReproductiveMedicine UniversityClinicsBonn Bonn,Germany
SantiagoMunné InstituteforReproductiveMedicineandScienceof SaintBarnabus Reprogenetics Livingston,NewJersey,USA
ZsoltPeterNagy
ReproductiveBiologyAssociates Atlanta,Georgia,USA
DanielNavot DivisionofReproductiveEndocrinologyand Infertility NewYorkMedicalCollege
FertilityInstituteofNewJerseyandNewYork Westwood,NewJersey,USA
QueenieVNeri
AndrologyandAssistedFertilization CornellInstituteforReproductiveMedicine NewYork,NewYork,USA
DanielaNogueira
FollicleBiologyLaboratory CenterforReproductiveMedicine VrijeUniversiteitBrussel Brussels,Belgium
KarlGNygren FertilityandIVFUnit SophiahemmetHospital Stockholm,Sweden
KutlukHOktay DepartmentofObstetrics&Gynecology NewYorkMedicalCollege,Valhalla andInstituteforFertilityPreservation CenterforHumanReproduction andMemorialSloanKetteringCancerCenter NewYork,NewYork,USA
OzgurOktem
TheCenterforReproductiveMedicineandInfertility WeillMedicalCollegeofCornellUniversity NewYork,NewYork,USA
RaoulOrvieto
DepartmentofObstetricsandGynecology RabinMedicalCenter,PetahTikva andSacklerFacultyofMedicine TelAvivUniversity TelAviv,Israel
AllanAPacey
AcademicUnitofReproductiveandDevelopmental Medicine
UniversityofSheffield Sheffield,UK
GianpieroDPalermo AndrologyandAssistedFertilization CornellInstituteforReproductiveMedicine NewYork,NewYork,USA
ThomasBPool FertilityCenterofSanAntonio SanAntonio,Texas,USA
BenjaminReubinoff IVFUnit,DepartmentofObstetricsand Gynecology andGoldyneSavadInstitute ofGeneTherapy HadassahUniversityHospital Jerusalem,Israel
LauraFRienzi
CentreforReproductiveMedicine ClinicaValleGiulia Rome,Italy
SergioRomero
FollicleBiologyLaboratory CenterforReproductiveMedicine VrijeUniversiteitBrussel Brussels,Belgium
ZevRosenwaks
TheCenterforReproductiveMedicine andInfertility WeillMedicalCollegeofCornellUniversity NewYork,NewYork,USA
DennySakkas DepartmentofObstetricsandGynecology YaleUniversitySchoolofMedicine NewHaven,Connecticut,USA
MarkVSauer DepartmentofObstetricsandGynecology CollegeofPhysicians&Surgeons ColumbiaUniversity NewYork,NewYork,USA
RoelSchats
DepartmentofObstetricsandGynecology DivisionofReproductionand FertilityInvestigation IVFCenter VrijeUniversiteitMedicalCenter Amsterdam,TheNetherlands
LynetteScott FertilityCenterofNewEngland Reading,Massachusetts,USA
MachelleMSeibel
DepartmentofObstetricsandGynecology UniversityofMassachusettsSchoolofMedicine Worcester,Massachusetts,USA
RakeshSharma
CenterforReproductiveMedicine,Glickman UrologicalandKidneyInstitute andObstetrics–GynecologyandWomen’sHealth Institute ClevelandClinic Cleveland,Ohio,USA
FrançoiseShenfield
ReproductiveMedicineUnit UniversityCollegeHospitalandMedicalSchool London,UK
ZeevShoham DepartmentofObstetricsandGynecology KaplanMedicalCenter Rehovot,Israel
KaylenSilverberg
TexasFertilityCenter AustinIVF Austin,Texas,USA
CarlosSimón
FundaciónInstitutoValencianodeInfertilidad InstitutoUniversitarioIVI ValenciaUniversity andCentrodeInvestigaciónPríncipeFelipe Valencia,Spain
CeciliaSjöblom NURTURE UniversityofNottingham Queen’sMedicalCentre Nottingham,UK
GaryDSmith DepartmentofObstetricsandGynecology andReproductiveMedicineProgram UniversityofMichigan AnnArbor,Michigan,USA
JohanSmitz
RadioimmunologyandReproductiveBiology CenterforReproductiveMedicine UniversityHospitalVUB Brussels,Belgium
MuratSonmezer DepartmentofObstetricsandGynecology SchoolofMedicine AnkaraUniversity Ankara,Turkey
AnnikaStrandell ReproductiveMedicine DepartmentofObstetricsandGynecology SahlgrenskaUniversityHospital Göteborg,Sweden
Anne-MariaSuikkari VäestöliittoFertilityClinics Helsinki,Finland
JasonESwain FertilityCenterofSanAntonio SanAntonio,Texas,USA
TakumiTakeuchi AndrologyandAssistedFertilization CornellInstituteforReproductiveMedicine NewYork,NewYork,USA
ShuichiTakyama DepartmentofObstetricsandGynecology UniversityofMichigan AnnArbor,Michigan,USA
SeangLinTan McGillReproductiveCenter RoyalVictoriaHospital DepartmentofObstetricsandGynecology McGillUniversity Montreal,Quebec,Canada
ThomasHTang
ReproductiveMedicineandSurgery LeedsGeneralInfirmary Leeds,UK
AlanRThornhill
TheLondonBridgeFertility,Gynaecologyand GeneticsCentre andDepartmentofObstetricsandGynecology UniversityCollegeLondon London,UK
JamesPToner
AtlantaCenterforReproductiveMedicine Woodstock,Georgia,USA
HermanTournaye CenterforReproductiveMedicine UniversityHospitaloftheDutchSpeakingBrussels FreeUniversity Brussels,Belgium
TomTurner TexasFertilityCenter AustinIVF Austin,Texas,USA
FilippoMUbaldi CentreforReproductiveMedicine ClinicaValleGiulia Rome,Italy
GáborVajta AcademicDirector PIVETMedicalCentre Perth,WesternAustralia Australia
HansvanderVen DepartmentofGynaecologicalEndocrinologyand ReproductiveMedicine UniversityofBonn Bonn,Germany
KatrinvanderVen DepartmentofGynaecologicalEndocrinologyand ReproductiveMedicine UniversityofBonn Bonn,Germany
LeenVanhoutte FollicleBiologyLaboratory CenterforReproductiveMedicine VrijeUniversiteitBrussel Brussels,Belgium
AndréVanSteirteghem CenterforMedicalGenetics UniversityHospitalVUB Brussels,Belgium
LucindaLVeeckGosden
TheCenterforReproductiveMedicineandInfertility WeillMedicalCollegeofCornellUniversity NewYork,NewYork,USA
AnnaVeiga ReproductiveMedicineService InstitutUniversitariDexeus BancdeLiniesCellulars CentredeMedicinaRegenerativadeBarcelona Barcelona,Spain
YuryVerlinsky ReproductiveGeneticsInstitute Chicago,Illinois,USA
ArielWeissman IVFUnit DepartmentofObstetricsandGynecology EdithWolfsonMedicalCenter Holon,Israel
MattsWikland FertilityCentreScandinavia Carlander’sHospital Göteborg,Sweden
YuralYaron PrenatalGeneticDiagnosisDivision GeneticInstitute TelAvivSouraskyMedicalCenter TelAviv,Israel
NikicaZaninovic
TheCenterforReproductiveMedicineandInfertility WeillMedicalCollegeofCornellUniversity NewYork,NewYork,USA
FernandoZegers-Hochschild UnitofReproductiveMedicine ClínicalasCondes Santiago,Chile
Introduction:thebeginningsof human invitro fertilization RobertGEdwards
Invitrofertilization(IVF)anditsderivativesinpreimplantationdiagnosis,stemcells,andtheethicsof assistedreproductioncontinuetoattractimmense attentionscientificallyandsocially.Allthesetopics wereintroducedby1970.Hardlyadaypasseswithout somepublicrecognitionofeventsrelatedtothisstudy, andclinicsspreadeverfurtherworldwide.Nowwe mustbeapproaching1.5millionIVFbirths,itistimeto celebratewhathasbeenachievedbysomanyinvestigators,clinical,scientific,andethical.Whilemuchofthis Introductioncoversthemassiveaccumulationof eventsbetween1960and2000,italsobrieflydiscussesnewperspectivesemerginginthe21stcentury. Freshadvancesalsoincreasecuriosityabouthow thesefieldsofstudybeganandhowtheirethical implicationswereaddressedinearlierdays.Asfor me,Iamstillstirredbyrecollectionsofthoseearly days.FoundationswerelaidinEdinburgh,London, andGlasgowinthe1950sandearly1960s.Discoveries madethenledtolaterdaysinCambridge,working therewithmanyPhDstudents.Italsoresultedinmy workingwithPatrickSteptoeinOldham.Ourjoint openingofBournHallin1980,whichbecamethe largestIVFclinicofitskindatthetime,signifiedthe endofthebeginningofassistedhumanconception andtheonsetofdedicatedappliedstudies.
Introduction Firstofall,Imustexpressinlimitedspacemytributestomyteachers,evenifinadequately.These includeinvestigatorsfromfar-offdayswhenthefundamentalfactsofreproductivecycles,surgicaltechniques,endocrinology,andgeneticswereelicitedby manyinvestigators.Thesefieldsbegantomoveinthe 20thcentury,andifonepioneerofthesetimesshould besaluted,itmustbeGregoryPincus.Famousforthe contraceptivepill,hewasadistinguishedembryologist,andpartofhisworkdealtwiththematurationof mammalianoocytesinvitro.Hewasthefirsttoshow howoocytesaspiratedfromtheirfollicleswould begintheirmaturationinvitro,andhowanumber
maturedandexpelledafirstpolarbody.Ibelievehis majorworkwasdoneinrabbits,wherehefoundthat the10–11-hourtimingsofmaturationinvitro accordedexactlywiththoseoccurringinvivoafteran ovulatorystimulustothefemalerabbit.
Pincusetalalsostudiedhumanoocytes. 1 Extractingoocytesfromexcisedovaries,theyidentifiedchromosomesinalargenumberofoocytesand interpretedthisasevidenceofthecompletionofmaturationinvitro.Manyoocytespossessedchromosomesafter12hours,theproportionremaining constantoverthenext30hoursandlonger.Twelve hourswastakenastheperiodofmaturation. Unfortunately,chromosomeswerenotclassifiedfor theirmeioticstage.Maturingoocyteswouldbe expectedtodisplaydiakinesisormetaphase-Ichromosomepairs.Fullymatureoocyteswoulddisplay metaphase-IIchromosomes,signifyingtheywerefully ripeandreadyforfertilization.Nevertheless,itiswell knownthatoocytescanundergoatresiaintheovary involvingtheformationofmetaphase-IIchromosomes inmanyofthem.TheseoocytescomplicatedPincus’ estimates,evenincontrols,andwerethesourceofhis errorwhichledlaterworkerstoinseminatehuman oocytes12hoursaftercollectionandculturein vitro. 2,3 Workonhumanfertilizationinvitro,and indeedcomparablestudiesinanimals,remainedin abeyancefromthenandformanyyears.
ProgressinanimalIVFhadalsobeenslow.After manyrelativelyunsuccessfulattemptsinseveral speciesinthe1950sand1960s,avirtualdogmaarose thatspermatozoahadtospendseveralhoursinthe femalereproductivetractbeforeacquiringthepotential tobindtothezonapellucidaandachievefertilization. Inthelate1960sAustinandChangindependently identifiedtheneedforspermcapacitation,identified byadelayinfertilizationafterspermatozoahad enteredthefemalereproductivetract.4,5 Thisdiscoverywastakenbymanyinvestigatorsasthereasonfor thefailuretoachievefertilizationinvitro,andwhy spermatozoahadtobeexposedtosecretionsofthe femalereproductivetract.Atthesametime,Chang
reportedthatrabbiteggsthathadfullymaturedin vitrofailedtoproducenormalblastocysts,noneof themimplantingnormally.6
ModernbeginningsofhumanIVF, preimplantationgeneticdiagnosis,and embryostemcells MyPhDbeganattheInstituteofAnimalGenetics, EdinburghUniversity,in1952,encouragedby ProfessorConradWaddington,theinventorofepigenesis,andsupervisedbyDrAlanBeatty.Atthetime, capacitationwasgaininginsignificance.Mychosen topicwasthegeneticcontrolofearlymammalian embryology,specificallythegrowthofpreimplantationmouseembryoswithalteredchromosomecomplements.Achievingtheseaimsincludedaneedto exposemousespermatozoatoX-rays,ultravioletlight, andvariouschemicalsinvitro.Thiswoulddestroy theirchromatinandpreventthemfrommakingany geneticcontributiontotheembryo,hopefullywithout impairingtheircapacitytofertilizeeggsinvivo. Resultingembryoswouldbecomegynogenetichaploids.Later,myworkchangedtoexposingovulated mouseoocytestocolchicineinvivo,inorderto destroytheirsecondmeioticspindleinvivo.This treatmentfreedallchromosomesfromtheirattachmenttothemeioticspindle,andtheythenbecame extrudedfromtheeggintotinyartificialpolarbodies. Thefertilizingspermatozoonthusenteredanempty egg,whichresultedintheformationofandrogenetic haploidembryoswithnogeneticcontributionfrom thematernalside.Forthreeyears,myworkwasconcentratedinthemousehouse,workingatmidnightto identifymousefemalesinestrusbyvaginalsmears, collectingepididymalspermatozoafrommales,and practisingartificialinseminationwithsamplesof treatedspermatozoa.Thisresearchwassuccessful,as mouseembryoswereidentifiedwithhaploid,triploid, tetraploid,andaneuploidchromosomes.Moreover, thewidescientifictalentintheInstitutewasaperfect placeforfreshcollaborativestudies.Forexample, JulioSirlinandIappliedtheuseofradioactiveDNA andRNAprecursorstothestudyofspermatogenesis, spermiogenesis,fertilization,andembryogenesis,and gainedknowledgeunavailableelsewhere.
Anevengreaterfortunebeckoned.AllenGates, newlyarrivedfromtheUnitedStates,broughtcommercialsamplesofOrganon’spregnantmares’serum (PMS)richinfollicle-stimulatinghormone(FSH),and humanchorionicgonadotropin(hCG)withitsstrong luteinizinghormone(LH)activitytoinduceestrus andovulationinimmaturefemalemice.Working withMervynRunner,7 hehadusedlowdosesofeach hormoneatanintervalof48hourstoinduceoocyte maturation,mating,andovulationinimmature mousefemales.Henowwishedtomeasuretheviabilityof3-dayembryosfromimmaturemicebytransferringthemtoanadulthosttogrowtoterm.8 Iwasmore
interestedinstimulatingadultmicewiththese gonadotropinstoinduceestrusandovulationatpredictabletimesofday.Thiswouldhelpmyresearch, andIwasbynowwearyoftakingmousevaginal smearsatmidnight.Myfuturewife,RuthFowler,and Iteameduptotestthisnewapproachtosuperovulatingadultmice.Wechosepregnantmares’serumto inducemultifolliculationandhCGtotriggerovulation,varyingdosesandtimesfromthoseutilizedby AllenGates.PMSbecameobsoleteforhumanstudies sometimelater,butitsimpacthasstayedwithme fromthatmoment,evenuntiltoday.
OpinioninthosedayswasthatexogenoushormonessuchasPMSandhCGwouldstimulatefollicle growthandovulationinimmaturefemalemammals, butnotinadultsbecausetheywouldinteractbadly withanadult’sreproductivecycles.Infact,they workedwonderfullywell.Dosesof1–3IUofPMS inducedthegrowthofnumerousfollicles,andsimilar dosesofhCG42hourslaterinvokedestrusandovulationafurther6hourslaterinalmostallofthem. Often,70ormoreovulatedoocytescrowdedthe ampulla,mostofthembeingfertilizedanddevelopingtoblastocysts. 9 Oocytematuration,ovulation, mating,andfertilizationwereeachcloselytimedin alladults,anotherhighlyunusualaspectofstimulation.10 Diakinesiswasidentifiedasthegerminalvesicleregressed,withmetaphaseIalittlelaterand metaphaseII,expulsionofthefirstpolarbody,and ovulationat11.5–12hoursafterhCG.Multiplefertilizationledtomultipleimplantationandfetalgrowth tofullterm,justassimilartreatmentsinanovulatory womenresultedinquintupletsandotherhigh-order multiplepregnanciesafewyearslater.Yearsafterwards,germinalvesiclebreakdownanddiakinesis weretoproveequallydecisiveinidentifyingmeiosis andovulationinhumanoocytesinvivoandinvitro. Evenastheseresultsweregained,RuthandI departedin1957fromEdinburghtotheCalifornia InstituteofTechnology,whereIswitchedinto immunologyandreproduction,atopicthatwasto dominatemylifeforfiveorsixyearsonmyreturnto theUK.
TheInstituteatEdinburghhadgivenmeanexcellentbasisingenetics,butequallyinreproduction.I hadgainedconsiderableknowledgeaboutthe endocrinecontrolofestruscycles,ovulation,spermatozoa,andthemalereproductivetract,artificial insemination,andthestagesofembryogrowthinthe oviductanduterus,superovulationanditsconsequences,andtheuseofradiolabeledcompounds. Waddingtonhadalsobeendeeplyinterestedinethics andinrelationshipsbetweenscienceandreligion, andinstilledthesetopicsinhisstudents.Ihadbeen essentiallytrainedinreproduction,genetics,andscientificethics,andallofthisknowledgewastoprove ofimmensevalueinmylatercareer.Avisittothe CaliforniaInstituteofTechnologywidenedmyhorizonsintothemolecularbiologyofDNAandthegene, afieldtheninitsinfancy.
AfterayearinCalifornia,Londonbeckonedme,to theNationalInstituteforMedicalResearchworking withDrsAlanParkesandColin(Bunny)Austin.Iwas fortunateindeedtohavetwosuchexcellentcolleagues.Aftertwointenseyearsinimmunology,my curiosityreturnedtomaturatingoocytesandfertilizationinvitro.Sincetheymaturedsoregularlyandeasilyinvivo,itshouldbeeasytostimulatematuration inmouseoocytesinvitrobyusinggonadotropins.In fact,tomyimmensesurprise,whenliberatedfrom theirfolliclesintoculturemedium,oocytesmatured immediatelyinvastnumbersinallgroups,with exactlythesametimingasthosematuringinvivofollowinganinjectionofhCG.Addinghormonesmade nodifference.Rabbit,hamster,andratoocytesalso maturedwithin12hours,eachattheirownspeciesspecificrates.Buttomysurprise,oocytesfromcows, sheep,andrhesusmonkeys,andtheoccasional baboon,didnotmatureinvitrowithin12hours. Theirgerminalvesiclespersistedunmoved,arrested inthestageknownasdiffusediplotene.Whyhadthey notrespondedlikethoseofrats,mice,andrabbits? Howwouldhumanoocytesrespond?Auniqueopportunityemergedtocollectpiecesofhumanovary,and toaspiratehumanoocytesfromtheiroccasionalfollicles.Igraspeditwithalacrity.
Movingtohumanstudies MollyRosewasalocalgynecologistintheEdgware andDistrictHospitalwhodeliveredtwoofourdaughters.Sheagreedtosendmeslithersorwedgesof ovariessuchasthoseremovedfrompatientswith polycysticdisease,asrecommendedbySteinand Leventhal,orwithmyomataorotherdisorders demandingsurgery.Stein–Leventhalwedgeswerethe bestsourceofoocytes,withtheirnumeroussmall graafianfollicleslinedupinacontinuousrimjust belowtheovariansurface.Thoughsampleswererare, theyprovidedenoughoocytestostartwith.These oocytesrespondedjustastheoocytesfromcows, sheep,andpigs,theirgerminalvesiclespersistingand diakinesisbeingabsentafter12hoursinvitro.
Thiswasdisappointing,andespeciallysoforme, sinceTjioandLevan,andFord,hadidentified46 diploidchromosomesinhumans,whilestudiesby teamsinEdinburgh(Scotland)andFrancehadmadeit clearthatmanyhumanbeingswereheteroploid.This wasmysubject,becausechromosomalvariationsmostly aroseduringmeiosisandthiswouldbeeasilyassessed inmaturingoocytesatdiakinesis.Variousgroupsalso discoveredmonosomyordisomyinmanymenand women.SomewomenwereXOorXXX;somemenwere XYYandXYYY.Trisomy21provedtobethemostcommoncauseofDown’ssyndrome,andothertrisomies weredetected.Allthisnewinformationremindedmeof mychromosomestudiesintheEdinburghmice.
Forhumanstudies,Iwouldhavetoobtaindiakinesis andmetaphaseIinhumanoocytes,andthencontinue thisanalysistometaphaseIIwhentheoocyteswould
befullymature,readyforfertilization.Despitebeing disappointedatcurrentfailurewithhumanoocytes,it wastimetowritemyfindingsfor Nature in1962.11 Therewassomuchtowriteregardingtheanimalwork, anddescribingthenewideasthentakingshapeinmy mind.IhadheardInstitutelecturesoninfertility,and realizedthatfertilizinghumanoocytes invitro and replacingembryosintothemothercouldhelptoalleviatethiscondition.Itcouldalsobepossibletotype embryosforgeneticdiseaseswhenafamilialdispositionwasidentified.Piecesoftissue,oroneortwoblastomeres,wouldhavetobeexcisedfromblastocystsor cleavingembryos,butthisdidnotseemtobetoodifficult.Therewerefewgeneticmarkersavailableforthis purposeintheearly1960s,butitmightbepossibleto sexembryosbytheirXXorXYchromosomecomplementbyassessingmitosesincellsexcisedfrommorulaeorblastocysts.Choosingfemaleembryosfortransfer wouldavertthebirthofboyswithvarioussex-linked disorderssuchashemophilia.Clearly,Iwasbecoming totallycommittedtohumanIVFandembryotransfer. Whilelookinginthelibraryforanynewlypublished papersrelevanttomyproposed Nature manuscript,I discoveredthoseearlierpapersofPincusandhiscolleaguesdescribedabove.Theyhadapparentlysucceeded30yearsearlierinmaturinghumanoocytes culturedfor12hours,whereIhadfailed.My Nature paper11 becameverydifferentfromthatoriginally intended,eventhoughitretainedenoughforpublication.ThoseresultsofPincusetalhadtoberepeated. Aftertryinghard,Ifailedcompletelytorepeatthem, despiteinfusingintactovariesinvitrowithgonadotropinsolutions,usingdifferentculturemediato inducematuration,andusingjointculturesofmaturing mouseoocytesandnewlyreleasedhumanoocytes. Addinghormonestoculturemediaalsofailed.Itbegan toseemthatmenstrualcycleshadaffectedoocytephysiologyinadifferentmannerthaninnonmenstruating mammalianspecies.Finally,anotherlineofinquiry emergedaftertwoyearsoffruitlessresearchonthepreciousfewhumanoocytesavailable.Perhapsthetiming ofmaturationinmiceandrabbitsdifferedfromthatof thoseoocytesobtainedfromcows,baboons,and humans.EvenasmydaysinLondonwereending, MollyRosesentaslitherofhumanovary.Thefew oocyteswereplacedinculturejustasbefore.Theirgerminalvesiclesremainedstaticfor12hoursasIalready knew,andthenafter20hoursinvitro.Threeoocytes remained,andIwaitedtoexaminethemuntiltheyhad beeninvitrofor24hours.Thefirstcontainedagerminalvesicle,sodidthesecond.Therewasoneleftand oneonly.Itsimageunderthemicroscopewaselectrifying.Igazeddownatchromosomesindiakinesis,andat aregressinggerminalvesicle.Thechromosomeswere superbexamplesofhumandiakinesiswiththeirclassicalchiasmata.Atlast,IwasonthewaytohumanIVF,to completionofthematurationprogramandtheonsetof studiesonfertilizationinvitro.
ThiswasthestepIhadwaitedfor,amarkerthat Pincushadmissed.Henevercheckedfordiakinesis,
andapparentlyconfusedatreticoocytes,whichcontainedchromosomes,withmaturingoocytes.Endless humanstudieswereopening.Itwaseasynow,even onthebasisofoneoocyteindiakinesis,tocalculate thetimingofthefinalstagesofmaturationbecause thepost-diakinesisstagesofmaturationwerenottoo differentfromnormalmitoticcyclesinsomaticcells. Thiscalculationprovidedmewithanestimateof about36hoursforfullmaturation,whichwouldbe themomentforinsemination.Allthesegapsin knowledgehadtobefilled.Butnow,myresearchprogramwasstretchingfarintothefuture.Atthiswonderfulmoment,JohnPaul,anoutstandingcell biologist,invitedmetojoinhimandRobinColeat GlasgowUniversitytostudydifferentiationinearly mammalianembryos.Thiswasexciting,toworkin biochemistrywithaleadingcellbiologist.Hehad heardthatIwasexperimentingwithveryearly embryos,tryingtogrowcelllinesfromthem.Healso wantedtogrowstemcellsfrommammalianembryos andstudytheminvitro.Thisbeganoneofmymost memorable12monthsofresearch.John’slaboratory hadfacilitiesunknownoutside,withCO2 incubators, numerouscelllinesinconstantcultivation,cryopreservationfacilities,andtheuseofmediadroplets heldunderliquidparaffin.Wedecidedtostartwith rabbits.Celllinesdidnotgroweasilyfromcleaving rabbitembryos.Incontrast,stemcellsmigratedoutin massivenumbersfromculturesofrabbitblastocysts, formingmuscle,nerves,phagocytes,bloodislands, andothertissuesinvitro.12 Stemcellsweredifferentiatinginvitrointovirtuallyallthetissuesofthebody. Incontrast,dissectingtheinnercellmassfromblastocystsandculturingitintactorasdisaggregatedcells producedlinesofcellswhichdividedanddivided, withouteverdifferentiating.Onelineoftheseembryonicstemcellsexpressedspecificenzymes,diploid chromosomes,andafibroblasticstructureasitgrew over200andmoregenerations.Anotherwasepithelioidandhaddifferentenzymesbutwassimilarin otherrespects.Theabilitytomakewhole-embryoculturesproducingdifferentiatingcellswasnowcombinedwitheverlastinglinesofundifferentiatedstem cellswhichreplicatedovermanyyearswithout changing.Ideasofusingstemcellsforgraftingtoovercomeorgandamageinrecipientsbegantoemerge.My thoughtsreturnedconstantlytogrowingstemcells fromhumanembryostorepairdefectsintissuesof childrenandadults.
AlmostatmylastmomentinGlasgow,withthis newsetofideasinmymind,apieceofexcisedovary yieldedseveraloocytes.Beingplacedinvitro,twoof themhadreachedmetaphaseIIandexpelledapolar bodyat37hours.Thisshowedthatanothertargeton theroadtohumanIVFhadbeenachievedasthe wholepatternofoocytematurationcontinuedto emergebutwithincreasingclarity. CambridgeUniversity,mynextandfinalhabitation, isanastonishingplace.Lookingbackonthosedays,it seemsthatthePhysiologicalLaboratorywasnotthe
idealplacetosettleinthataugustuniversity. Nevertheless,amixtureofimmunologyandreproductionremainedmydominantthemesasIrejoinedAlan ParkesandBunnyAustinthere.Ihadtodoimmunologytoobtainagranttosupportmyfamily,butthoughts ofhumanoocytesandembryoswereneverfaraway. Onepossiblemodelofthehumansituationwasthecow andotheragriculturalspecies,andlargenumbersof cow,pig,andsheepoocyteswereavailablefromovaries giventomebythelocalslaughterhouse.Eachspecies haditsowntiming,allofthemlongerthan12hours.13 Pigoocyteswereclosesttohumans,requiring37hours. Ineachspecies,maturationtimingsinvitrowere exactlythesameasthosearisinginvivoinresponseto anhCGinjection.Thismademesuspectthatawoman ovulated36–37hoursafteraninjectionofhCG.Human oocytesalsotrickledin,improvingmyprovisional timingsofmaturation,andoneortwoofthemwere inseminated,butwithoutsignsoffertilization.
Moreoocyteswereurgentlyneededtoconcludethe timingsofoocytemeiosis.SurgeonsinJohnsHopkins Hospital,Baltimore,performedtheStein–Leventhal operation,whichwouldallowmetocollectovarian tissue,aspirateoocytesfromtheirfollicles,andretain theremainingovariantissuesforpathologyifnecessary.IhadalreadymetVictorMcKusick,whoworked inJohnsHopkins,atmanyconferences.Iaskedforhis supportformyrequesttoworkwiththehospitalgynecologistsforsixweeks.Hefoundasourceoffunds, madelaboratoryspaceavailable,and,awonderful invitation,introducedmetoHowardandGeorgeanna Jones.ThissignificantmomentwasequaltomymeetingwithMollyRose.TheJonesesprovedtobesuperb andunstintingintheirsupport.Sufficientwedgesand otherovarianfragmentswereavailabletocompletemy maturationprograminhumanoocytes.Withinthree weeks,everystageofmeiosiswasclassifiedand timed. 14 Wealsoundertookpreliminarystudieson inseminatinghumanoocytesthathadmaturedin vitro,tryingtoachievespermcapacitationbyusing differentmediaoraddingfragmentsofampullatothe cultures,andevenattemptingfertilizationinrhesus monkeyoviducts.Twonucleiwerefoundinsome inseminatedeggs,resemblingpronuclei,butsperm tailswerenotidentifiedsonoclaimscouldbemade.15 Duringthosesixweeks,however,oocytematuration wasfullytimedat37hours,permittingmenowtopredictwithcertaintythatwomenwouldovulateat37 hoursafteranhCGinjection.
Asimplemeansofaccesstothehumanovarywas nowessentialinordertoidentifyhumanovarianfolliclesinvivoandtoaspiratethem36hoursafterhCG, justbeforefollicularrupture.Whocouldprovidethis? Andhowaboutspermcapacitation?Onlyinhamsters hadfertilizationinvitrobeenachieved,usinginvivo maturedoocytesandepididymalspermatozoa.16 Imet VictorLewis,mythirdclinicalcolleague,andwe noticedwhatseemedtobeanaphaseIIinsome inseminatedeggs.Again,nospermtailswereseen withintheeggs.
Anattempttoachievehumancapacitation,in ChapelHill,NorthCarolina,UnitedStates,working withRobertMcGaugheyandhiscolleagues,also failed. 17 Asmallintrauterinechamberlinedwith porousmembranewasfilledwithwashedhuman spermatozoa,sealed,andinsertedovernightintothe uterusofhumanvolunteersatmid-cycle.Molecules enteringitcouldreactwiththespermatozoa.No maturedhumaneggswerefertilized.Laterevidence indicatedthatthechambercontainedinflammatory proteins,perhapsexplainingthefailure.
Decisivestepstoclinicalhumanin vitrofertilization BackintheUnitedKingdom,myintentiontoconceive humanchildreninvitrohadgrownevenstronger.So manymedicaladvantagescouldflowfromit.Asmall numberofhumanembryoshadbeenflushedfrom humanoviductsoruteriaftersexualintercourse,providingslenderinformationontheseearlieststagesofhuman embryology.Itwastimetoattainhumanfertilizationin vitro,inordertomoveclosetoworkingwithinfertile patients.Ethicalissuesandmoraldecisionswould emerge,oneaftertheother,infullpublicview.Matters suchascloningandsexingembryos,theriskofabnormalitiesinthechildren,theclinicaluseofembryostem cells,theethicsofoocytedonationandsurrogatepregnancy,andtherighttoinitiatehumanembryoniclifein vitrowouldneverbeveryfaraway.Theseissueswereall acceptable,sinceIwasconfidentthatstudiesofhuman conceptionwereessentialforfuturemedicine,andcorrectethically,medically,andscientifically.Theincreasingknowledgeofgeneticsandembryologycouldassist manypatientsifIcouldachievehumanfertilizationand growembryosforreplacementintotheirmothers.
FewhumanoocyteswereavailableintheUnited Kingdom.Despitethisscarcity,oneortwoofthose maturedandfertilizedinvitropossessedtwonuclei afterinsemination.Buttherewerenoobvioussperm tails.Idevisedacowmodelforhumanfertilization, usinginvitromaturedoocytesandinseminationin vitrowithselectedsamplesofhighlyactivewashed bullspermatozoaextractedfromneatsemen.Itwasa pleasuretoseesomefertilizedbovineeggs,with spermtailsandcharacteristicpronuclei,especially usingspermatozoafromoneparticularbull.Herewas amodelforhumanIVF,andapreludetoaseriesof eventswhichimpliedthatmattersinmyresearch weresuddenlychanging.Acolleaguehadstressedthat formalinfixativeswereneededtodetectspermtailsin eggs.BarryBavisterjoinedourteamtostudyforhis PhDanddesignedamediumofhighpH,whichgave excellentfertilizationratesinhamsters.Wedecidedto collaboratebyusingitfortrialsonhumanfertilization invitro.Finally,whilebrowsinginthelibraryofthe PhysiologicalLaboratory,Ireadapaperin TheLancet whichinstantlycaughtmyattention.WrittenbyDrP.C. SteptoeoftheOldhamandDistrictGeneralHospital,18
itdescribedlaparoscopy,withitsnarrowtelescope andinstrumentsandtheminuteabdominalincisions. Hecouldvisualizetheampullaandplacesmall amountsofmediumthere,inanoperationlasting30 minutesorlessandmaybeevenwithoutusinganesthesia.ThisisexactlywhatIwanted,becauseaccessto theampullawasequivalenttogainingaccesstoovarianfollicles.Despiteadvicetothecontraryfromseveralmedicalcolleagues,Itelephonedhimabout collaborationandstressedtheuncertaintyinachievingfertilizationinvitro.Herespondedmostpositively,justasMolly,HowardandGeorgeanna,and Victorhaddone.Wedecidedtogettogether.
Lastbutbynomeansleast,MollyRosesentasmall pieceofovarytoCambridge.Itsdozenormoreoocytes werematuredinvitrofor37hours,whenBarryandI addedwashedspermatozoasuspendedinhismedium. Weexaminedthemafewhourslater.Toourdelight, spermatozoawerepushingthroughthezonapellucida, intoseveraloftheeggs.Maternalandpaternalpronucleiwereformingbeautifully.Wesawpolarbodiesand spermtailswithintheeggs.Thateveningin1969,we watchedindelightvirtuallyallthestagesofhumanfertilizationinvitro(Fig1).Onefertilizedegghadfragments,asChanghadforecastfromhisworkonoocyte maturationandfertilizationinvitroofrabbiteggs.This evidencestrengthenedtheneedtoabandonoocyte maturationinvitroandreplaceitbystimulatingmaturationbymeansofexogenoushormones.Our1969 paperin Nature surprisedaworldunaccustomedtothe ideaofhumanfertilizationinvitro.19
IncrediblyfruitfuldaysfollowedinourCambridge laboratory.RichardGardner,anotherPhDcandidate, andIexcisedsmallpiecesoftrophectodermfrom rabbitblastocystsandsexedthembystainingthesex chromatinbody.Thoseclassifiedasfemaleweretransferredintoadultfemalesandwereallcorrectlysexed atterm.Thisworktransferredmytheoreticalideasofa fewyearsearlierintothepracticeofpreimplantation diagnosisofinheriteddisease,inthiscaseforsexlinkeddiseases. 20 AlanHenderson,acytogeneticist, andIanalyzedchiasmataduringdiakinesisinmouse andhumaneggs,andexplainedthehighfrequencies ofDown’ssyndromeinoffspringofoldermothersasa consequenceofmeioticerrorsarisinginoocytes formedlastinthefetalovary,whichwerethenovulatedlastatlatermaternalages. 21 DaveSharpe,a lawyerfromWashington,joinedforcestowriteanarticlein Nature22 ontheethicsofinvitrofertilization,the firsteverpaperinthefield.Ifollowedthisupwitha detailedanalysisofethicsandlawinIVFcoveringscientificpossibilities,oocytedonation,surrogacyby embryotransfer,andothermatters.22 Sothefirstethicalpaperswerewrittenbyscientistsandlawyersand notbyphilosophers,ethicists,orpoliticians.
TheOldhamyears PatrickandIbeganourcollaborationsixmonthslater intheOldhamandDistrictGeneralHospital,almost
Fig1 Acompositepictureofthestagesoffertilizationofthehumanegg.Upperleft:aneggwithafirstpolarbodyandspermatozoa attachedtotheouterzonapellucida.Uppercentral:spermatozoaaremigratingthroughthezonapellucida.Upperright:aspermatazoonwithatailbeatingoutsidethezonapellucidaisattachingtotheoocytevitellinemembrane.Lowerleft:aspermatozooninthe ooplasm,withenlargingheadanddistinctmid-pieceandtail.Lowercentral:furtherdevelopmentofthespermheadintheooplasm. Lowerright:apronucleateeggwithtwopronucleiandpolarbodies.Noticethatthepronucleiareapparentlyalignedwiththepolar bodies,althoughmoredimensionsmustbescoredtoensurethatpolarityhasbeenestablishedinallaxes.
200milesnorthofCambridge.Hehadworkedclosely withtwopioneers,PalmerinParis23 andFragenheim inGermany.24 Heimprovedthepneumoperitoneumto gainworkingspaceintheabdominalcavity,andused carbonfiberstopasscoldlightintotheabdomenfrom anexternalsource.25 Bynow,Patrickwaswaitingin thewings,readytobeginclinicalIVFindistant Oldham.Wehadalongtalkaboutethicsandfound ourstancestobeverysimilar.Workstartedinthe OldhamandDistrictGeneralHospitalandmovedlater toKershaw’sHospital,setupbymyassistants,especiallyJeanPurdy.Weknewtheroutine.Itwasbased onmyEdinburghexperienceswithmice.PieroDonini fromSeronoLaboratoriesinRomehadpurifiedurinaryhumanmenopausalgonadotropins(hMG)asa sourceofFSH,andtheproductwasusedclinicallyto stimulatefolliclegrowthinanovulatorywomenby BrunoLunenfeld.26 ItremovedtheneedforPMS,so avoidingtheuseofnonhumanhormones.Weused low-dosagelevelsinpatients,i.e.2–3vials(atotalof 150–225IU)givenondays3and5,and5000–7000IU ofhCGonday10.Initially,thetimingofoocytematurationinvitrowasconfirmed,byperforminglaparoscopiccollectionsofoocytesfromovarianfolliclesat 28hoursafterhCGtocheckthattheywerein metaphaseI.27 Wethenmovedto36hourstoaspirate
maturemetaphaseIIoocytesforfertilization.Those beautifuloocytesweresurroundedbymassesofviscouscumuluscellsandwerematuringexactlyaspredicted.Wewitnessedfollicularruptureat37hours throughthelaparoscope.Folliclescouldbeclassified fromtheirappearanceasovulatoryornonovulatory, thisdiagnosisbeingconfirmedlaterbyassayingseveralsteroidsintheaspiratedfollicularfluids(Fig2). Itwasapleasureandanewdutytomeetthe patientssearchingforhelptoalleviatetheirinfertility. Wedidourbest,drivingfromCambridgetoOldham andarrivingatnoontopreparethesmalllaboratory there.PatrickhadstimulatedthepatientswithhMG andhCG,andheandhisteamledbyMurielHarris arrivedtoprepareforsurgery.Patrick’slaparoscopy wassuperb.Ovarianstimulation,eventhoughmild, producedfiveorsixmaturefolliclesperpatient,and ripeoocytescameinasteadystreamintomyculture mediumforinseminationandovernightincubation. Thenextmorning,theformationoftwopronucleiand spermtailsindicatedfertilizationhadoccurred,even insimplemedia,nowwithanear-neutralpH. Complexculturemedia,Ham’sF10andothers,each withaddedserumorserumalbumin,sustainedearly andlatercleavages,28 and,evenmorefascinating,the gradualappearanceofmorulaeandthenlight,
Fig2 Eightsteroidswereassayedinfluidsextractedfromhumanfolliclesaspirated36–37hoursafterhumanchorionicgonadotropin (hCG).Thefollicleshadbeenclassifiedasovulatingornonovulatingbylaparoscopicexaminationinvivo.Datawereanalyzedbyclusteranalysis,whichgroupsfollicleswithsimilarfeatures.Theupperillustrationshowsdatacollectedduringthenaturalmenstrualcycle. Notethattwosharplyseparatedgroupsoffollicleswereidentified,eachwithverylowlevelsofwithin-groupvariance.Attemptingto combinethetwogroupsresultedinamassiveincreaseofwithin-groupvariation,indicatingthattwosharplydifferentgroupshadbeen identified.Thesedifferentgroupsaccordedexactlywiththetwogroupsidentifiedbymeansofsteroidassays.Thelowerfigureshowsthe sameanalysisduringstimulatedcyclesonfluidscollectedat36–37hoursafterhCG.Withthisformofstimulation,folliclegrowthdisplaysconsiderablevariationwithingroups.Attemptstocombineallthegroupsresultinamoderatelylargeincreaseinvariation.This evidencesuggeststhatfolliclesvaryconsiderablyintheirstateofdevelopmentinsimulatedcyclesusinghumanmenopausal gonadotropin(hMG)andhCG.
translucentblastocysts(Fig3).29 Herewasmyreward –growingembryoswasnowroutine,andexaminationsofmanyofthemconvincedmethatthetimehad cometoreplacethemintothemothers’uteri.Ihad becomehighlyfamiliarwiththeteratologicprinciples ofembryonicdevelopment,andknewmanyteratologists.
TheonlyworryIhadwasthechanceofchromosomal monosomyortrisomy,onthebasisofourmousestudies,buttheseconditionscouldbedetectedlaterin gestationbyamniocentesis.Ourhumanstudieshad surpassedworkonallanimals,apointrubbedineven morewhenwegrewblastocyststoday9afterthey
hadhatchedfromtheirzonapellucida(Fig4).30 This beautifullyexpandedblastocysthadalargeembryonicdiscwhichwasshoutingthatitwasapotential sourceofembryonicstemcells.
Whenhumanblastocystsbecameavailable,we triedtosexthemusingthesexchromatinbodyasin rabbits.Unfortunately,theyfailedtoexpresseither sexchromatinorthemaleYbodysowewereunable tosexthemasfemaleormaleembryos.Human preimplantationgeneticdiagnosiswouldhavetowait alittlelonger.
Duringtheseyearstherewereveryfewplauditsfor us,asmanypeoplespokeagainstIVF.Criticismwas mostlyaimedatme,asusualwhenscientistsbring newchallengestosociety.Criticismcamenotonly fromthePopeandarchbishops,butalsofromscientistswhoshouldhaveknownbetter,includingJames Watson(whotestifiedtoaUSSenateCommitteethat manyabnormalbabieswouldbeborn),andMax Perutz,whosupportedhim.Thesescientistcritics knewvirtuallynothingaboutmyfield,sowho advisedthemtomakesuchridiculouscharges? Cloningfootballteamsorintelligentsiawasalways raisedbyethicists,whichclearlydominatedtheir thoughtsratherthantheintensehopesofourinfertile patients.Yetonetheologian,GordonDunstan,who becameaclosefriend,knewallaboutIVFfromus, andwroteanexcellentbookonitsethics.Hewasfar aheadofalmosteveryscientistinmyfieldofstudy. Ourpatientsalsogaveustheirstaunchsupport,and sodidtheOldhamEthicalCommittee,BunnyAustin backhomeinCambridge,andElliottPhilip,acolleagueofPatrick’s.
Growingembryosbecameroutine,sowedecidedto transferoneeachtoseveralpatients.Hereagainwe wereinuntestedwaters.Transferringembryosviathe cervicalcanal,theobviousroutetotheuterus,was virtuallyanewanduntestedmethod.Wewouldhave todoourbest.Fromnowon,weworkedwithpatients whohadseriouslydistortedtubesornonewhatsoever.Thisstepwasessential,sincenoonewould havebelievedwehadestablishedatest-tubebabyina womanwithnearnormaltubes.Thishadtobeaconditionofourinitialwork.Curiously,itledmanypeopletomakethebigmistakeofbelievingthatwe startedIVFtobypassoccludedoviducts.Yetwe alreadyknewthatembryoscouldbeobtainedformen witholigozoospermiaorantibodiestotheirgametes, andforwomeninvariousstagesofendometriosis.
Oneendocrinologicalproblemdidworryme. StimulationwithhMGandhCGshortenedthesucceedinglutealphase,toaveryshorttimeforembryos toimplantbeforetheonsetofmenstruation.Levelsof urinarypregnanediolalsodeclinedsoonafteroocyte collection.Thisconditionwasnotaresultoftheaspirationofgranulosaandcumuluscells,andlutealsupportwouldbeneeded,preferablyprogesterone.Csapo etalstressedhowthishormonewasproducedbythe ovariesforthefirst8–10weeksbeforetheplacenta
tookoverthisfunction.31 Injectionsofprogesteronein oilgivenoverthatlongperiodoftimeseemedunacceptablesinceitwouldbeextremelyuncomfortable forpatients.Whilemullingoverthisproblem,my attentionturnedtothoseearlierendocrinologistswho believedthatexogenoushormoneswoulddistortthe reproductivecycle,althoughIdoubttheyevenknew anythingaboutadeficientlutealphase.
ThisishowweunknowinglymadeourbiggestmistakeinearlyIVFdays.OurchoiceofPrimulotdepot, aprogestogen,meantitshouldbegivenevery5days tosustainpregnancies,sinceitwassupposedtosave threatenedabortions.So,webeganembryotransfers topatientsinstimulatedcycles,givingthisluteal phasesupport.Eventhoughourworkwasslowedby havingtowaittoseeifpregnanciesaroseinonegroup ofpatientsbeforestimulatingthenext,enough patientshadaccumulatedafter2–3years.Noneofour patientswaspregnant,anddisasterloomed.Ourcriticswereevenmorevociferousastheyearspassed, andmutualsupportbetweenPatrickandmehadto pullusthrough.
Twentyormoredifferentfactorscouldhavecaused ourfailure,e.g.cervicalembryotransfers,abnormal embryos,toxicculturedishesorcatheters,inadequate lutealsupport,incompatibilitybetweenpatients’ cyclesandthatimposedbyhMGandhCG,inherent weaknessinhumanimplantation,andmanyothers. Wehadtogleaneveryscrapofinformationfrom ourfailures.IknewKenBagshaweinLondon,who wasworkingwithimprovedassaymethodsfor gonadotropichormones.Heofferedtomeasureblood samplestakenfromourpatientsovertheimplantationperiodusinghisnewhCG- β assay.Hetelephoned:threeormoreofourpatientspreviously undiagnosedhadactuallyproducedshort-livedrises ofhCG- β overthisperiod.Everythingchangedwith thisinformation.Wehadestablishedpregnancies afterall,buttheyhadabortedveryearly.Wecalled thembiochemicalpregnancies,atermthatstillsticks today.Ithadtakenusalmostthreeyearstoidentify thecauseofourfailure,andthefingerofsuspicion pointedstraightatPrimulot.Iknewitwasluteolytic, butitwasapparentlyalsoanabortifacient,andour ethicaldecisiontouseithadcausedmuchheartache, immenselossofworkandtime,anddespairforsome ofourpatients.Thesocialpressureshadbeen immense,withcriticsclaimingourembryosweredud andourwholeprogramwasawasteoftime;butwe hadcomethroughitandnowknewexactlywhatto donext.
WeaccordinglyreducedlevelsofPrimulotdepot, andutilizedhCGandprogesteroneaslutealaids. Suspicionswerealsoemergingthathumanembryos wereverypooratimplanting.Wehadreplacedsingle embryosintomostofourpatients,rarelytwo. Increasinglywebegantowonderifmoreshouldbe replaced,aswhenwereplacedtwoinaprogram involvingtransfersofoocytesandspermatozoainto
Fig3 SuccessivestagesofhumanpreimplantationdevelopmentinvitroinacompositeillustrationmadeinOldhamin1971.Upper left:4-cellstageshowingthecrossedblastomerestypicalofmostmammals.Uppermiddle:8-cellstageshowingtheevenoutlineof blastomeresandasmallpieceofcumulusadherenttothezonapellucida.Upperright:a16–32-cellstage,showingtheonsetofcompactionoftheouterblastomeres.Often,blastocelicfluidcanbeseenaccumulatingbetweenindividualcellstogivea“stripey”appearancetotheembryo.Lowerleftandmiddle:twolivingblastocystsshowingadistinctinnercellmass,single-celledtrophectoderm, blastoceliccavity,andthinningzonapellucida.Lowerright:afixedpreparationofahumanblastocystat5days,showingmorethan100 even–sizednucleiandmanymitoses.
Fig4 Ahatchedhumanblastocystafter9daysinculture. Noticethedistinctembryonicdiscandthepossiblebilaminar structureofthemembrane.Theblastocysthasexpandedconsiderably,asshownbycomparingitsdiameterwiththatoftheshed zonapellucida.Thezonacontainsdyingandnecroticcellsand itsdiameterprovidesanestimateoftheoriginaloocyteend embryodiameters.
theampullasothatfertilizationcouldoccurinvivo. ThisprocedurewaslatercalledGIFT(gameteintrafallopiantransfer)byRicardoAsch.Wenowsuspected thatsingleembryotransferscouldproducea15–20% chanceofestablishingpregnancy,justasourfirstclinicalpregnancyaroseafterthetransferofasingleblastocystinapatientstimulatedwithhMGandhCG. 32 Thencamefantasticnews–ahumanembryofertilizedandgrowninvitrohadproducedapregnancy. Everythingseemedfine,evenwithultrasound images.Mycultureprotocolsweresatisfactoryafter all.Patrickrang:hefearedthepregnancywasectopic andhehadtoremoveitsometimeafter10gestational weeks.Everynewapproachwetestedseemedtobe endingindisaster,yetwewouldnotstop,sincethe workitselfseemedhighlyethical,andconceivinga childforourpatientswasperhapsthemostwonderfulthinganyonecoulddoforthem.Inanycase, ectopicpregnanciesarenowknowntobearegular featurewithassistedconception.
Isensedthatwewereenteringthefinalphaseofour Oldhamwork,sevenyearsafteritbegan.Wehadto speedup,partlybecausePatrickwasclosetoretiring
Fig5 Thefirstattemptsatgameteintrafallopiantransfer(GIFT) werecalledoocyterecoverywithtubalinsemination(ORTI).In thistreatmentcycle,usinghumanmenopausalgonadotropin (hMG)andhumanchorionicgonadotropin(hCG),includingadditionalinjectionsofhCGforlutealsupport,asinglepreovulatory oocyteand1.6millionspermweretransferredintotheampulla. ODGH,OldhamandDistrictGeneralHospital;LMP,lastmenstrualperiod;RTMindicatesstagesofthemenstrualcycle.
fromtheNationalHealthService.Fourstimulation protocolsweretestedinanattempttoavoidproblems withthelutealphase:hMGandhCG;clomiphene, hMG,andhCGtogainabetterlutealphase;bromocryptine,hMG,andhCGbecausesomepatientshadhigh prolactinconcentrations;andhCGaloneatmid-cycle. Wealsotestedwhatcametobeknownasgamete intrafallopiantransfer(GIFT),callingitORTI(oocyte recoverywithtubalinsemination,bytransferringone ortwoeggsandspermatozoatotheampulla)(Fig5). Natural-cycleIVFwasintroduced,basedoncollections ofurinesamplesatregularintervalseighttimesdaily, tomeasureexactlytheonsetoftheLHsurge,usinga modifiedHiGonavisassay(Fig6).Cryopreservation wasalsointroduced,byfreezingoocytesandembryos thatlookedtobeingoodconditionwhenthawed.A recipientwasgivenadonoreggfertilizedbyherhusband’sspermatozoa,butpregnancydidnotoccur. LesleyandJohnBrowncameasthesecondentrants fornatural-cycleIVF.Lesleyhadnooviducts.Heregg wasaspiratedinafewmomentsandinseminated simplyandefficiently.Theembryogrewbeautifully andwastransferredanhourorsoafteritbecame8-cell. Theirpositivepregnancytestafewdaysaftertransfer wasanothermilestone–surelynothingcouldnowpreventtheirembryodevelopingtofullterminanormal reproductivecycle,butthoseninemonthslastedavery longtime.Threemorepregnancieswereestablished usingnatural-cycleIVFasweabandonedtheother approaches.Atriploidembryodiedinutero–more
badluck.Athirdpregnancywaslostthroughprematurelaboronamountainwalkingholiday,twoweeks afterthemother’samniocentesis. 32,33 Itwasalovely, well-developedboy.LouiseBrown’sbirth,andthen Alistair’s,provedtoawaitingworldthatscienceand medicinehadenteredhumanconception.Ourcritics declaredthatthebirthswereafake,andadvised againstattendingourpresentationonthewholeofthe OldhamworkattheRoyalCollegeofObstetriciansand Gynaecologists.
IVFworldwide TheOldhamperiodwasover.Goodfacilitieswere nowneeded,withspaceforalargeIVFclinic.Bourn HallwasanoldJacobeanhouseinlovelygrounds nearCambridge(Fig7).FacilitiesonofferforIVFin Cambridgewerefartoosmall,sowepurchasedit mostlywithventurecapital.Itwasessentialtoconceive100or1000IVFbabiestoensurethatthe methodwassafeandeffectiveclinically.The immensedelaysinestablishingBournHalldelayed ourworkbytwoyearsafterLouise’sbirth.Finally,on minimalfinance,BournHallopenedinSeptember 1980onashoestring,supportedbyourowncashand loans.Thedelaygavetherestoftheworldachanceto joininIVF.AlexLopatadeliveredanIVFbabyin Australia,andoneortwootherswerebornelsewhere. Natural-cycleIVFwaschoseninitiallyatBournHall sinceithadprovedsuccessfulinOldham,andwe becameexpertsinit.Pregnanciesflowed,at15%per cycle.AnAustralianteamofAlanTrounsonandCarl WoodannouncedtheestablishmentofseveralIVF pregnanciesafterstimulationbyclomipheneandhCG andreplacingtwoorthreeembryos, 34 sotheyhad movedaheadofusduringthedelayedopeningof BournHall.Ourowneffortnowexpandedprodigiously.ThousandsofpatientsqueuedforIVF.Simon Fishel,JacquesCohen,andCarolFehillyjoinedthe embryologyteamamongyoungertrainees,andnew cliniciansjoinedPatrickandJohnWebster.Patients andpregnanciesincreasedrapidly,andtheworldwas leftstandingfarbehind.HowardandGeorgeanna JonesbeganinNorfolkusinggonadotropinsforovarianstimulation.JeanCohenbeganinParis,Wilfred FeichtingerandPeterKemeterinVienna,Klaus DiedrichandHansvanderVenninBonn,Lars HambergerandMattsWiklandinSweden,andAndre vanSteirteghemandPaulDevroeyinBrussels.IVF wasnowtrulyinternational.
TheopeningofBournHallhadnotdeterredour critics.Theyputupafiercerearguardactionagainst IVF,alongsideLIFE,SPUC(SocietyfortheUnborn Child),individualgynecologists,andothers.
ObjectionsraisedagainstIVFincludedlowratesof pregnancy(noonementionedthesimilarlowrates ofpregnancywithnaturalconception),thepossibilitiesofoocyteandembryodonation,surrogatemothers,unmarriedparents,one-sexparents,embryo
