1. ART and Adverse Pregnancy Outcome
Catherine Racowsky, PhD, HCLD
Department of Obstetrics and Gynecology
Brigham and Women’s Hospital
Harvard Medical School, Boston MA
2nd Congress of Current Opinion in Reproductive Medicine and Assisted Reproductive Technologies
Cesme, Turkey: April 19, 2008

2. Lecture Outline What we are doing when we perform ART
Consider challenges relevant to the topic of adverse outcomes and ART
Review possible causes of adverse outcomes
Discuss current knowledge regarding risks of adverse outcomes
Summarize risks and causes of adverse outcomes
Address gaps in our knowledge

3. The Goals of ART To minimize the risk of multiple gestations
To optimize pregnancy rates
To produce healthy, genetically normal,
singleton full-term deliveries

4. The ART of ART ICSI IVF Assisted Hatching PGD
Gamete Handling & Evaluation
Insemination
Zygote Identification
Micro-
Manipulations
Embryo Growth
The ART of ART
Ovarian Stimulation
Oocyte Collection Sperm Collection

5. The Critical Questions are …
Are we doing harm when treating infertility patients with ART?
Do the ART treatments per se cause adverse outcomes?

6. Challenges Accurate assessment of risks
Study design issues:
Methodologies (retrospective, prospective, multicenter, meta-analyses)
Size of datasets (power analyses & validity)
Comparative group issues (1yr versus 5yrs of infertility)
Typically, a lack of appropriate controls
Distinguishing among the possible causes
Genetic causes associated with sub-fertility
Ovarian stimulation
In vitro technologies per se

7. Study Design Issues Unit of Analysis?
Couple?
Woman? Man?
Which cycle?
Which pregnancy?
Analysis (assessment of correlations)
What groups (Singletons? Twins? Triplets?)
What correlative outcomes?
Which statistical tests?

8. Possible Causes of Adverse Outcomes

9. Possible Causes of Adverse Outcomes
Oocyte
Sperm
Zygote Embryos Transfer
OA or NOA
GENETICS
Age
Environment
Ovarian Stimulation
Culture Conditions
System
Media
Gas Phase
Duration
Manipulations
Assisted Hatching
Blastomere Bx
IVF
ICSI
Number & Quality
of Embryos

10. Possible Causes of Adverse Outcomes
Endometrial Receptivity
Placentation
Maternal Health
Uterine Environment
Gestational Order
Moore and Persaud. The developing human, clinically oriented embryology. 1998

11. Causes of Adverse Outcomes
Possible causes:
Ovarian stimulation-related affects
Culture-induced phenomena
Unidentified contributions from parents of origin
Known causes:
Identifiable contributions from parents of origin
Multiple gestations

12. Possible Causes of Adverse Outcomes “Ovarian Stimulation”
LL Day of Menstrual Cycle
Cohort of
Growing
Follicles
Egg Retrieval
36 h post hCG
Atresia
Estrogen
Exogenous
FSH
Recruitment
Selection
Dominance DF N
N-1

13. Possible Causes of Adverse Outcomes Is Ovarian Stimulation a Stressor?
Urinary gonadotropins to adult CD1 mice
Lower levels of:
VEGF120
VEGF receptors (flt-1 and flk-1 mRNA)
Reduced size of:
Embryo
Implantation site
Delayed implantation
Prolonged gestational period
Both urinary hFSH and hCG contributed to the adverse effects
Sibug et al., 2005

14. Possible Causes of Adverse Outcomes Is Ovarian Stimulation a Stressor?
Possible Impact on Placentation Development
Cryopreserved versus Fresh Outcomes
Lower incidence of pre-term deliveries following transfer of frozen-thawed embryos versus fresh embryos (Wennerholm, ‘97; Bergh ’99)

15. Possible Causes of Adverse Outcomes “Culture-Induced Effects”
Does in vitro culture modulate genetic expression
and/or effect post-natal development?
Impact of Uterine Receptivity
“Quality” of Maternal System
Ovarian
Stimulation
Regimen
Oocyte
Quality
Culture
System
Embryo
Transfer
Luteal
Support

16. Possible Causes of Adverse Outcomes “Culture-Induced Effects”: The Dishes
Organ dishes
Microdrop system
Oil overlay
Medium drops
Embryonic density?
Co-culture with granulosa cells?
Endometrial co-culture?

17. Possible Causes of Adverse Outcomes “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
P 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)

18. Possible Causes of Adverse Outcomes Culture Media Formulations
Viable singleton pregnancies at 12 weeks gestation (day 3 ET)
Orasanu et al ’06 RBMOnline 12:
hCG on day 15 post-ET (mIU/ml)
P IVF G G1.3
N = a b
a,c d
P<0.05

19. Possible Causes of Adverse Outcomes “Culture-Induced Effects: Day of Transfer”
Egg Retrieval
Days Post-Fertilization
Day of Embryo Transfer
After Day 5 transfer:
Increased incidence of monozygotic twins (Behr et al ’00; Menezo et al ’02)
Increased incidence of monochorionic twins (Skiadas et al ’08)
Increased incidence of male neonates? (Menezo et al ’99; Kausche et al ‘01)

20. Possible Causes of Adverse Outcomes
“Imprinting Defects in ART Babies”
Angelman’s Syndrome (ch 15)
Incidence of this rare subtype estimated at 1/300,000
3 isolated cases reported among ICSI births
1 case had a fertile father
All had epigenetic defect with loss of methylation of maternal allele
Beckwith-Weidemann’s Syndrome (ch 11)
Baseline risk of 1/15,000
3 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-fold
All cases due to imprinting defect
Clinical evidence is suggestive but not sufficient to conclude that ART techniques may increase frequencies

21. Possible Causes of Adverse Outcomes Unidentified Causes from Oocyte
OI/IUI Treated Women
Infertile Group: Donor sperm
Fertile Group: Donor sperm
Infertile women had LBW neonates than the fertile group (Gaudoin ’03)

22. Possible Causes of Adverse Outcomes Unidentified Causes from Sperm
Birth defects in ICSI versus spontaneously conceived infants
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
Study Reference
Odds Ratio

23. Known Causes of Adverse Outcomes “Parents of Origin”
CF mutations (CBAVD), Yq11 micro deletions
DOR: poor embryo quality
Aneuploidy-related variables
Aneuploidy
Implantation
Munne et al ’01,’04,‘06
Maternal Age (y)
Female %
“0” >20
Yoshida et al ’95
Sperm Concentration (x106/ml)
Male

24. Known Cause of Adverse Outcomes Multiple Gestations
4.4%
Triplets Plus
2005 US Pregnancies by Multiplicity
Increased risk of pre-term delivery
Associated risks of prematurity
Obstetrical complications
28.4%
Twins
67.2%
Singletons
SARTCORS Data Reporting System, 2005

25. for Number of Embryos to Transfer
ASRM/SART Guidelines
for Number of Embryos to Transfer
September, 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 Favorable
Least Favorable
SART Practice Committee Report

26. Risks of
Adverse Outcomes

27. Risk of Adverse Outcomes in Singletons
Antenatal
# IVF Spont
Outcome Studies % % OR (95% CI)
Gestational diabetes (1.4, 3.0)
Placenta previa (1.5, 5.4)
Preeclampsia (1.2, 2.0)
Preterm delivery after (1.7, 2.7)
spontaneous labor
Vaginal bleeding (1.9, 3.3)
Studies included cohort, matched cohort or external comparisons
Jackson et al ’04 BMJ 103:551-63

28. Risk of Adverse Outcomes in Singletons
Perinatal
# IVF Spont
Outcome Studies % % OR (95% CI)
Perinatal mortality (1.61, 2.98)
Preterm delivery (1.73, 2.20)
Low birth weight (1.40, 2.22)
Very low birth weight (2.31, 3.14)
Jackson et al ’04 BMJ 103:551-63

29. Risk of Adverse Outcomes in Singletons
L & D/Neonatal
# IVF Spont
Outcome Studies % % OR (95% CI)
Labor induction (1.0, 1.3)
Spontaneous labor (0.5, 0.7)
Caesarian delivery (1.7, 2.6)
Elective (1.5, 2.5)
Emergent (1.1, 2.0)
NICU admits (1.3, 2.0)
Neonatal deaths (1.2, 3.4)
Jackson et al ’04 BMJ 103:551-63

30. Risk of Adverse Outcomes
Twins
# IVF Spont
Outcome Studies % % OR (95% CI)
Perinatal mortality (0.4, 0.8)
Preterm delivery <37 wk (1.0, 1.1)
Preterm delivery <32 wk (0.8, 1.2)
Low birth weight (1.0, 1.1)
Very low birth weight (0.7, 1.1)
Small for gestational age (0.97, 1.7)
Studies included cohort, matched cohort or external comparisons
Jackson et al ’04 BMJ 103:551-63

31. Risk of Adverse Outcomes
Conclusions
Compared with non-assisted singleton pregnancies, ART singleton pregnancies have significantly worse outcomes for:
Antenatal
Perinatal
Neonatal and most L&D variables
Most odds ratios are >2
Only one of these ART-related adverse outcomes for singletons is also evident for twins

32. Risk of Adverse Outcomes in Singletons
The Etiology?
Parents of origin: sub-fertility?
Ovarian stimulation?
Technology?

33. Risk of Adverse Outcomes in Singletons
The Etiology: Sub-fertility?
Danish Study (Westergaard et al ’99)
1298 ART patients
1298 non-treated controls
Outcome OR (95% CI)
< 37 weeks (1.02, 1.94)
< 1500g (1.43, 10.3)
< 2500g (1.08, 2.10)
Sub-fertility may be involved

34. Risk of Adverse Outcomes in Singletons
The Etiology: Ovarian Stimulation
Belgian Cohort Study
12,021 ovarian stimulation, no ART
12,021 controls
Outcome OR (95% CI)
< 1500g (2.31, 4.47)
g (1.65, 2.10)
< 32 weeks (1.69, 2.12)
Ovarian stimulation may be involved although …..

35. Risk of Adverse Outcomes in Singletons
The Etiology??
Comparison of :
Infertile versus fertile women, both without treatment, showed the infertile group had:
Increased risk of VLBW, OR = 1.5 (McElrath ’97)
Number of smaller studies with conflicting findings
Throws the etiology back onto “sub-fertility” issues …..

36. Risks Associated with ICSI
Outcome # Studies OR (95% CI)
Major birth defects (1.20, 1.45)
All infants (singletons and multiples) (1.28, 1.45)
Singletons only (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:
ICSI vs IVF
Reviewed in Van Steirteghem et al ’02 Hum Reprod Update;8:111-6

37. Risk of Adverse Infant Neuro-Development
Most studies are reassuring, but methodological problems prevail
An increased RR has been observed for:
Cerebral palsy overall (OR 3.7; 2.8 in singletons)
Developmental delay (OR 4.0)
Stromberg et al ’02 Lancet 359;461-5
BUT, this appears to be mostly due to premature birth (Hvidtjorn et al ’06 Pediatrics;118:475-82)
HOWEVER, in vitro-derived mice exhibit specific behavioral alterations in anxiety/locomotor activity and spatial memory (Ecker et al ’06 PNAS;101: )
Meeting schedule:
The first meeting was in October, 2006
We 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.

38. Summary
Meta-analyses reveal worse perinatal outcomes for ART singletons versus non-ART singletons.
Conversely, IVF twins seem to be at no higher risk than spontaneous twins.
The etiology for these adverse outcomes in singletons is unknown but may be related to:
The infertility per se
The ovarian stimulation
The lab technology

39. Summary (cont.)
Slightly higher risk of malformations and chromosomal abnormalities in ICSI babies, mostly related to parents of origin
Psycho-motor development is normal, neuro- developmental outcome may be influenced by neonatal problems
An increased incidence of very rare disorders remains possible (etiology unknown, but may be lab-related)
Recommended that patients are counseled about potential risks, their possible etiologies and our current knowledge base

40. Gaps in Our Knowledge
Etiologies of many of the adverse outcomes remain to be resolved
Challenges remain regarding teasing out infertility factors versus treatment-related issues
(e.g. ART for tubal ligation versus disease-related reasons)
Absence of linkage of lab technologies with gestational complications, birth, infant & child health outcomes:
Culture media
ICSI, AH, PGD
Prolonged embryo culture
Frozen versus fresh transfers

41. Barker Hypothesis
A 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.
Ov Stim
Male Health
Lab Effects
Uterine
Receptivity
Female Health
Barker DJ. The developmental origins of adult disease. Eur J Epid ’03;8(8):733-6