ART and Adverse Pregnancy Outcome Catherine Racowsky, PhD, HCLD [email protected] Department of Obstetrics and Gynecology Brigham and Women’s Hospital

ART andART andAdverse Pregnancy OutcomeAdverse Pregnancy Outcome

Catherine Racowsky, PhD, HCLDCatherine Racowsky, PhD, [email protected]

Department of Obstetrics and GynecologyDepartment of Obstetrics and GynecologyBrigham and Women’s HospitalBrigham and Women’s Hospital

Harvard Medical School, Boston MAHarvard Medical School, Boston MA

2nd Congress of Current Opinion in Reproductive Medicine and Assisted Reproductive Technologies

Cesme, Turkey: April 19, 2008

Lecture OutlineLecture Outline

What we are doing when we perform ARTWhat we are doing when we perform ART

Consider challenges relevant to the topic of adverse Consider challenges relevant to the topic of adverse

outcomes and ARToutcomes and ART

Review possible causes of adverse outcomesReview possible causes of adverse outcomes

Discuss current knowledge regarding risks of adverse Discuss current knowledge regarding risks of adverse

outcomesoutcomes

Summarize risks and causes of adverse outcomesSummarize risks and causes of adverse outcomes

Address gaps in our knowledgeAddress gaps in our knowledge

The Goals of ARTThe Goals of ART

To minimize the risk of multiple gestations

To optimize pregnancy rates

To produce healthy, genetically normal,singleton full-term deliveries

ICSI IVF

AssistedHatching

PGD

Gamete Handling & Evaluation

Insemination

Zygote Identification

Micro-Manipulations

Embryo Growth

The ART of ARTThe ART of ARTOvarian Stimulation

Oocyte Collection Sperm Collection

The Critical Questions are …The Critical Questions are …

Are we doing harm when treating Are we doing harm when treating infertility patients with ART?infertility patients with ART?

Do the ART treatments Do the ART treatments per seper se cause cause adverse outcomes?adverse outcomes?

ChallengesChallenges

Accurate assessment of risksAccurate assessment of risks Study design issues:Study design issues:

Methodologies (retrospective, prospective, multicenter, Methodologies (retrospective, prospective, multicenter, meta-analyses)meta-analyses)

Size of datasets (power analyses & validity)Size of datasets (power analyses & validity) Comparative group issues (1yr versus 5yrs of infertility)Comparative group issues (1yr versus 5yrs of infertility) Typically, a lack of appropriate controlsTypically, a lack of appropriate controls

Distinguishing among the possible causesDistinguishing among the possible causes Genetic causes associated with sub-fertilityGenetic causes associated with sub-fertility Ovarian stimulationOvarian stimulation In vitro technologies per seIn vitro technologies per se

Study Design Issues Study Design Issues

Unit of Analysis?Unit of Analysis? Couple?Couple? Woman? Man?Woman? Man? Which cycle?Which cycle? Which pregnancy?Which pregnancy?

Analysis (assessment of correlations)Analysis (assessment of correlations) What groups (Singletons? Twins? Triplets?)What groups (Singletons? Twins? Triplets?) What correlative outcomes?What correlative outcomes? Which statistical tests?Which statistical tests?

Possible Causes of Adverse Possible Causes of Adverse OutcomesOutcomes

Possible Causes of Adverse Possible Causes of Adverse OutcomesOutcomes

Oocyte

Sperm Zygote Embryos Transfer

OA or NOA GENETICS

Age Environment

EnvironmentAge

GENETICS Ovarian Stimulation

Culture ConditionsSystemMedia

Gas PhaseDuration

Manipulations

Assisted HatchingBlastomere Bx

IVF

ICSI

Number & Qualityof Embryos

Endometrial ReceptivityPlacentationMaternal HealthUterine EnvironmentGestational Order

Moore and Persaud. The developing human, clinically oriented embryology. 1998

Possible Causes of Adverse OutcomesPossible Causes of Adverse Outcomes

Causes of Adverse OutcomesCauses of Adverse Outcomes Possible causes:Possible causes:

Ovarian stimulation-related affectsOvarian stimulation-related affects Culture-induced phenomena Culture-induced phenomena Unidentified contributions from parents of originUnidentified contributions from parents of origin

Known causes:Known causes: Identifiable contributions from parents of originIdentifiable contributions from parents of origin Multiple gestationsMultiple gestations

Possible Causes of Adverse OutcomesPossible Causes of Adverse Outcomes“Ovarian Stimulation”“Ovarian Stimulation”

LL Day of Menstrual Cycle1 3 5 7 9 11 13 15

Cohort ofCohort ofGrowingGrowingFolliclesFollicles

Egg RetrievalEgg Retrieval36 h post hCG36 h post hCG

AtresiaAtresia

Est

roge

n

ExogenousExogenousFSHFSH

RecruitmentRecruitmentSelectionSelection

DominanceDominance

DFDF

DFDF

DFDF

NN N-1 N-1N-1 N-1N-1

Possible Causes of Adverse OutcomesPossible Causes of Adverse OutcomesIs Ovarian Stimulation a Stressor?Is Ovarian Stimulation a Stressor?

Urinary gonadotropins to adult CD1 miceUrinary gonadotropins to adult CD1 mice Lower levels of:Lower levels of:

VEGF120VEGF120 VEGF receptors (flt-1 and flk-1 mRNA)VEGF receptors (flt-1 and flk-1 mRNA)

Reduced size of:Reduced size of: EmbryoEmbryo Implantation siteImplantation site

Delayed implantationDelayed implantation Prolonged gestational periodProlonged gestational period

Both urinary hFSH and hCG contributed to the adverse effects

Sibug et al., 2005Sibug et al., 2005

Possible Causes of Adverse OutcomesPossible Causes of Adverse OutcomesIs Ovarian Stimulation a Stressor?Is Ovarian Stimulation a Stressor?

Cryopreserved versus Fresh OutcomesCryopreserved versus Fresh Outcomes Lower incidence of pre-term deliveries following Lower incidence of pre-term deliveries following

transfer of frozen-thawed embryos transfer of frozen-thawed embryos versusversus fresh fresh embryos embryos (Wennerholm, ‘97; Bergh ’99)(Wennerholm, ‘97; Bergh ’99)

Possible Impact on Placentation DevelopmentPossible Impact on Placentation Development

Possible Causes of Adverse OutcomesPossible Causes of Adverse Outcomes“Culture-Induced Effects”“Culture-Induced Effects”

Does in vitro culture modulate genetic expressionDoes in vitro culture modulate genetic expressionand/or effect post-natal development?and/or effect post-natal development?

OvarianStimulationRegimen

OocyteQuality

Culture System

EmbryoTransfer

LutealSupport

Impact of Uterine Receptivity“Quality” of Maternal System

Possible Causes of Adverse OutcomesPossible Causes of Adverse Outcomes“Culture-Induced Effects”: The Dishes“Culture-Induced Effects”: The Dishes

Organ dishes

Microdrop system

Oil overlay

Medium drops

Embryonic density? Co-culture with granulosa cells? Endometrial co-culture?

Possible Causes of Adverse OutcomesPossible Causes of Adverse Outcomes“Culture-Induced Effects”: Other Variables“Culture-Induced Effects”: Other Variables

Other “contact” materials

Gas phase O2 tension: kinetics of development & birth weight

(Thompson et al ’90)

Commercial culture media vary widely in complexity Simple: HTF Complex: G series P1 Global

Several studies have shown media effects on development: Differential allocation to ICM and TE (Van Soom et al ’97) Large calf syndrome (Walker et al ’96)

Imprinting defects (e.g. H19 gene in mouse; Doherty et al ‘00)

Possible Causes of Adverse OutcomesPossible Causes of Adverse OutcomesCulture Media FormulationsCulture Media Formulations

0

200

400

600

800

Viable singleton pregnancies at 12 weeks gestation (day 3 ET)

Orasanu et al ’06 RBMOnline 12:590-598

hC

G o

n d

ay

15

po s

t-E

T

(mI U

/ ml)

P1 IVF-500 G1.2 G1.3N = 281 81 171 153

a

b

a,cdP<0.05

Possible Causes of Adverse OutcomesPossible Causes of Adverse Outcomes“Culture-Induced Effects: Day of Transfer”“Culture-Induced Effects: Day of Transfer”

After Day 5 transfer:After Day 5 transfer: Increased incidence of monozygotic twins Increased incidence of monozygotic twins (Behr et al ’00; Menezo et al ’02)(Behr et al ’00; Menezo et al ’02) Increased incidence of monochorionic twins (Skiadas et al ’08)(Skiadas et al ’08) Increased incidence of male neonates? Increased incidence of male neonates? (Menezo et al ’99; Kausche et al ‘01)(Menezo et al ’99; Kausche et al ‘01)

Egg

Ret

rieva

l

Days Post-Fertilization 1 2 3 4 50

Day of Embryo Transfer

Possible Causes of Adverse OutcomesPossible Causes of Adverse Outcomes “Imprinting Defects in ART Babies”

Angelman’s Syndrome (ch 15)Angelman’s Syndrome (ch 15)Incidence of this rare subtype estimated at 1/300,0003 isolated cases reported among ICSI births1 case had a fertile fatherAll had epigenetic defect with loss of methylation of maternal allele

Beckwith-Weidemann’s Syndrome (ch 11)Beckwith-Weidemann’s Syndrome (ch 11)Baseline risk of 1/15,0003 BWS Registry studies found incidences of 3/65, 6/143 and 6/149 RR estimate of BWS children being ART-conceived from 4 to 6-foldAll cases due to imprinting defect

Clinical evidence is suggestive but not sufficient to conclude Clinical evidence is suggestive but not sufficient to conclude that ART techniques may increase frequenciesthat ART techniques may increase frequencies

http://www.laeknabladid.is/media/tolublod/1313

Possible Causes of Adverse OutcomesPossible Causes of Adverse OutcomesUnidentified Causes from OocyteUnidentified Causes from Oocyte

OI/IUI Treated WomenOI/IUI Treated Women Infertile Group: Donor spermInfertile Group: Donor sperm Fertile Group: Donor spermFertile Group: Donor sperm

Infertile women had LBW neonates than the Infertile women had LBW neonates than the fertile groupfertile group (Gaudoin ’03)(Gaudoin ’03)

Possible Causes of Adverse OutcomesPossible Causes of Adverse OutcomesUnidentified Causes from SpermUnidentified Causes from Sperm

Birth defects in ICSI versus spontaneously conceived infantsBirth defects in ICSI versus spontaneously conceived infants

Stu

dy

Re f

er e

n ce

Odds Ratio

Morin et al ’89

Hansen et al ’02

Isaksson et al ’02

Koivurova et al ’02

Ericson et al ’01

Dhont et al ’99

Westergaard et al ’99

Pooled Estimate

.1 .5 1 10 50

Known Causes of Adverse OutcomesKnown Causes of Adverse Outcomes“Parents of Origin”“Parents of Origin”

CF mutations (CBAVD), Yq11 micro deletionsCF mutations (CBAVD), Yq11 micro deletions DOR: poor embryo quality DOR: poor embryo quality Aneuploidy-related variablesAneuploidy-related variables

010203040506070

20-34 35-39 40-45

Aneuploidy

Implantation

Munne et al ’01,’04,‘06Munne et al ’01,’04,‘06

Maternal Age (y)

FemaleFemale

%

0

5

10

15

20

“0” 0-4 5-10 11-20 >20

Aneuploidy

Yoshida et al ’95Yoshida et al ’95

Sperm Concentration (x106/ml)

MaleMale

%

Known Cause of Adverse OutcomesKnown Cause of Adverse OutcomesMultiple GestationsMultiple Gestations

28.4%Twins 67.2%

Singletons

4.4%Triplets Plus

2005 US Pregnancies by 2005 US Pregnancies by MultiplicityMultiplicity

Increased risk of pre-term Increased risk of pre-term deliverydeliveryAssociated risks of prematurityAssociated risks of prematurityObstetrical complicationsObstetrical complications

SARTCORS Data Reporting System, 2005

ASRM/SART GuidelinesASRM/SART Guidelinesfor Number of Embryos to Transferfor Number of Embryos to Transfer

September, 2006September, 2006

ART programs should be aggressively moving towards SET in select patient groups who have good prognosis

< 35y 1 or 2 embryos

35-37y: 2 or 3 embryos

38-40y: 3 or 4 embryos

>40y: < 5 embryos

Most FavorableMost Favorable

Least FavorableLeast Favorable

SART Practice Committee Report

Risks ofRisks ofAdverse OutcomesAdverse Outcomes

Risk of Adverse Outcomes in SingletonsRisk of Adverse Outcomes in Singletons

AntenatalAntenatal

# IVF SpontOutcome Studies % % OR (95% CI)

Gestational diabetes 4 6.8 4.7 2.0 (1.4, 3.0)

Placenta previa 6 2.4 0.9 2.9 (1.5, 5.4)

Preeclampsia 8 10.3 3.8 1.6 (1.2, 2.0)

Preterm delivery after 5 10.3 5.6 2.1 (1.7, 2.7)spontaneous labor

Vaginal bleeding 7 16.6 2.9 2.5 (1.9, 3.3)

Jackson et al ’04 BMJ 103:551-63

Studies included cohort, matched cohort or external comparisons


PerinatalPerinatal


Perinatal mortality 8 2.0 0.66 2.19 (1.61, 2.98)

Preterm delivery 14 11.5 5.3 1.95 (1.73, 2.20)

Low birth weight 10 9.5 3.8 1.77 (1.40, 2.22)

Very low birth weight 8 2.5 0.99 2.70 (2.31, 3.14)



L & D/NeonatalL & D/Neonatal


Labor induction 7 21.9 19.6 1.2 (1.0, 1.3)

Spontaneous labor 7 49.0 61.3 0.6 (0.5, 0.7)

Caesarian delivery 14 26.7 19.4 2.1 (1.7, 2.6)

Elective 7 11.4 6.7 1.9 (1.5, 2.5)

Emergent 7 8.0 5.9 1.5 (1.1, 2.0)

NICU admits 5 17.8 7.8 1.6 (1.3, 2.0)

Neonatal deaths 7 0.6 0.3 2.0 (1.2, 3.4)


Risk of Adverse OutcomesRisk of Adverse Outcomes

TwinsTwins


Perinatal mortality 6 2.3 4.3 0.58 (0.4, 0.8)

Preterm delivery <37 wk 9 50.0 46.0 1.10 (1.0, 1.1)

Preterm delivery <32 wk 3 6.8 7.1 0.95 (0.8, 1.2)

Low birth weight 5 55.0 53.0 1.0 (1.0, 1.1)

Very low birth weight 5 6.7 7.6 0.89 (0.7, 1.1)

Small for gestational age 4 24.0 20.0 1.3 (0.97, 1.7)

Studies included cohort, matched cohort or external comparisons


Risk of Adverse OutcomesRisk of Adverse Outcomes

Compared with non-assisted singleton pregnancies, Compared with non-assisted singleton pregnancies, ART singleton pregnancies have significantly worse ART singleton pregnancies have significantly worse outcomes for: outcomes for:

AntenatalAntenatal PerinatalPerinatal Neonatal and most L&D variablesNeonatal and most L&D variables

Most odds ratios are >2Most odds ratios are >2 Only one of these ART-related adverse outcomes for Only one of these ART-related adverse outcomes for

singletons is also evident for twinssingletons is also evident for twins

ConclusionsConclusions


Parents of origin: sub-fertility?Parents of origin: sub-fertility? Ovarian stimulation?Ovarian stimulation? Technology?Technology?

The Etiology?The Etiology?


Danish Study Danish Study (Westergaard et al ’99)(Westergaard et al ’99)

1298 ART patients1298 ART patients 1298 non-treated controls1298 non-treated controls

The Etiology: Sub-fertility?The Etiology: Sub-fertility?

Outcome OR (95% CI)

< 37 weeks 1.41 (1.02, 1.94)

< 1500g 3.84 (1.43, 10.3)

< 2500g 1.50 (1.08, 2.10)

Sub-fertility may be involved


Belgian Cohort StudyBelgian Cohort Study 12,021 ovarian stimulation, no ART12,021 ovarian stimulation, no ART 12,021 controls12,021 controls

The Etiology: Ovarian StimulationThe Etiology: Ovarian Stimulation

Outcome OR (95% CI)

< 1500g 3.21 (2.31, 4.47)

1500-2500g 1.86 (1.65, 2.10)

< 32 weeks 1.89 (1.69, 2.12)

Ovarian stimulation may be involved although …..


Comparison of :Comparison of :Infertile Infertile versusversus fertile women, both fertile women, both without without

treatment, showed the infertile treatment, showed the infertile group had:group had: Increased risk of VLBW, OR = 1.5 Increased risk of VLBW, OR = 1.5 (McElrath ’97)(McElrath ’97)

Number of smaller studies with conflicting findingsNumber of smaller studies with conflicting findings

The Etiology??The Etiology??

Throws the etiology back onto “sub-fertility” issues …..

Risks Associated with ICSIRisks Associated with ICSI

Outcome # Studies OR (95% CI)

Major birth defects 15 1.32 (1.20, 1.45)

All infants (singletons and multiples) 17 1.36 (1.28, 1.45)

Singletons only 15 1.31 (1.17, 1.46)

Hansen et al ’02 NEJM 346:725-30

• Results do not appear to be related to the ICSI-procedure itself

• ICSI babies are at a small, but increased risk for chromosomal abnormalities, mostly from paternal inheritance

Lie ’05 Int J Epid 34:696-701

ICSI vs IVFICSI vs IVF

Reviewed in Van Steirteghem et al ’02 Hum Reprod Update;8:111-6

Risk of Adverse Infant Neuro-DevelopmentRisk of Adverse Infant Neuro-Development

Most studies are reassuring, but methodological problems prevailMost studies are reassuring, but methodological problems prevail

An increased RR has been observed for:An increased RR has been observed for:

Cerebral palsy overall (OR 3.7; 2.8 in singletons)Cerebral palsy overall (OR 3.7; 2.8 in singletons)

Developmental delay (OR 4.0)Developmental delay (OR 4.0)Stromberg et al ’02 Lancet 359;461-5Stromberg et al ’02 Lancet 359;461-5

BUT, this appears to be mostly due to premature birthBUT, this appears to be mostly due to premature birth (Hvidtjorn et al ’06 Pediatrics;118:475-82)(Hvidtjorn et al ’06 Pediatrics;118:475-82)

HOWEVER, in vitro-derived mice exhibit specific behavioral alterations in anxiety/locomotor activity and spatial HOWEVER, in vitro-derived mice exhibit specific behavioral alterations in anxiety/locomotor activity and spatial memory memory (Ecker et al ’06 PNAS;101:1595-1600)(Ecker et al ’06 PNAS;101:1595-1600)

Meeting schedule:Meeting schedule:

The first meeting was in October, 2006The first meeting was in October, 2006

We currently strive to meet for 1.5 hrs monthlyWe currently strive to meet for 1.5 hrs monthly

After primary mission is established, we will meet as necessary regarding new issues and new technologies and policies etc.After primary mission is established, we will meet as necessary regarding new issues and new technologies and policies etc.

SummarySummary Meta-analyses reveal worse perinatal outcomes for Meta-analyses reveal worse perinatal outcomes for

ART singletons ART singletons versusversus non-ART singletons. non-ART singletons.

Conversely, IVF twins seem to be at no higher risk Conversely, IVF twins seem to be at no higher risk than spontaneous twins.than spontaneous twins.

The etiology for these adverse outcomes in The etiology for these adverse outcomes in singletons is unknown but may be related to:singletons is unknown but may be related to: The infertility per seThe infertility per se

The ovarian stimulation The ovarian stimulation

The lab technologyThe lab technology

Summary Summary (cont.)(cont.) Slightly higher risk of malformations and chromosomal Slightly higher risk of malformations and chromosomal

abnormalities in ICSI babies, mostly related to parents abnormalities in ICSI babies, mostly related to parents of originof origin

Psycho-motor development is normal, neuro-Psycho-motor development is normal, neuro-developmental outcome may be influenced by neonatal developmental outcome may be influenced by neonatal problemsproblems

An increased incidence of very rare disorders remains An increased incidence of very rare disorders remains possible (etiology unknown, but may be lab-related)possible (etiology unknown, but may be lab-related)

Recommended that patients are counseled about Recommended that patients are counseled about potential risks, their possible etiologies and our current potential risks, their possible etiologies and our current knowledgeknowledge base base

Gaps in Our KnowledgeGaps in Our Knowledge Etiologies of many of the adverse outcomes remain to be Etiologies of many of the adverse outcomes remain to be

resolvedresolved

Challenges remain regarding teasing out infertility factors Challenges remain regarding teasing out infertility factors versusversus treatment-related issues treatment-related issues

(e.g. ART for tubal ligation (e.g. ART for tubal ligation versusversus disease-related reasons disease-related reasons))

Absence of linkage of lab technologies with gestational Absence of linkage of lab technologies with gestational complications, birth, infant & child health outcomescomplications, birth, infant & child health outcomes: : Culture mediaCulture media ICSI, AH, PGDICSI, AH, PGD Prolonged embryo cultureProlonged embryo culture Frozen Frozen versusversus fresh transfers fresh transfers

Barker DJ. The developmental origins of adult disease. Eur J Epid ’03;8(8):733-6

Barker HypothesisBarker HypothesisA baby's nourishment before birth and during infancy, as manifest in patterns of fetal and infant growth, "programmes" the development of risk factors such as raised blood pressure and glucose intolerance that are key determinants of coronary heart disease.

Female Health

Ov Stim

Male Health Lab Effects

UterineReceptivity

Documents

ART and Adverse Pregnancy Outcome Catherine Racowsky, PhD, HCLD [email protected] Department of Obstetrics and Gynecology Brigham and Women’s Hospital