The Debate: Fresh vs Frozen Transfer The Cold, Hard Truth Amber R. Cooper, MD, MSCI Assistant Professor Division of Reproductive Endocrinology & Infertility Department of Obstetrics & Gynecology

Disclosures Scientific advisory board Celmatix Grant funding: K12HD063086-01, UL1TR000488 Industry research support: Beckman Coulter, Inc

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What we DO agree on…. Frozen embryo transfers are an important part of successful IVF clinics Vitrification has improved freezing success with both embryos and oocytes Endometrial-Embryo synchrony is very important for successful implantation Absolute risks of both approaches are overall quite small

FRESH OR FROZEN EMBRYO TRANSFER??? Where we differ… What is the best first line approach… FRESH OR FROZEN EMBRYO TRANSFER???

What is the best FET cycle approach? Timing of embryo cryopreservation? 2PN, CD2/3, CD5/6 Slow-freezing vs vitrification? Transfer in a medicated or natural cycle? Use of leuprolide? Recommended number of embryos to transfer Culture media? Frequency of SET? Timing of progesterone start?

Slow-freezing vs Vitrification: Perinatal risk No perinatal risks D3 embryos Liu SY, Hum Reprod 2013 Shi W, Fertil Steril 2012 No perinatal risks D5/6 embryos Takahashi K, Fertil Steril 2005 (no diff congenital defects) Wikland M, Hum Reprod 2010 (vit blastocyst vs D3 slow) The majority of perinatal risk data is from slow freezing and cleavage stage embryos About 4000 babies first couple studies

Is extended embryo culture ideal? Yes, for improving implantation and pregnancy rates, and minimizing higher order multiples BUT D5/6 transfer as compared to D3 increases the risk of Preterm Delivery by 30-40% US: Kalra S et al. Obstet Gynecol 2012; SART data, 69,039 LB, fresh ET Canada: Dar S et al. Hum Reprod 2013; 12,712 LB Sweden: Kallen B et al. Fertil Steril 2010; 13,873 LB D5/6 vs D3 also with increased absolute birthweight and incidence of LGA China: Zhu J et al. Hum Reprod 2014; 2,929 singletons, fresh, 2009-2012 Makinen S, Hum Reprod 2013 2/3 CD3; SART 2004-2006 Fresh, no diff in LBW % China study-difference pt bias, initial tx d3 tfr; day 5/6 if past implantation failure

Fresh vs Frozen: Perinatal risks Numerous cohort and meta analyses suggest outcomes better with FET as compared to fresh (SGA, LBW, PTD) but still worst than spontaneous Pelkonen 2010, Pinborg 2010, Sazonova 2013, Wennerholm 2009, Maheshwari 2012, Pinborg 2012 No difference in birth defects in fresh vs FET Meta-analysis: Maheshwari A, Fertil Steril 2012; OR 1.1 (0.8-1.4) Hansen M Obstet Gynecol 2012, Davies NEJM 2012, Jalliday JL Hum Reprod 2010 -most data with slow-freezing SGA, LBW and PTD often better with FET

Fresh vs Frozen: Perinatal risks BUT, Numerous studies suggest increased risk of LGA and macrosomia with FET vs fresh or spontaneous Pelkonen 2010, Pinborg 2010, Sazonova 2012 Wennerholm 2013: Nordic cohort, CoNARTaS group: 6647 FET, 42242 fresh, 288542 spontaneous singletons-increased risk LGA, macrosomia, and perinatal mortality (49% increase) Wennerholm 2013, most slow freeze, day 2, txfr natural cycle Perinatal risk: stillbirth> or equal 28 wks to neonatal death Adj for mat age, parity, year of birth, gender, country

Perinatal risks: LGA Is the epigenetic regulation of the placenta altered? Nelissen 2011, Vergouw 2012 Denmark study FET vs fresh, sibling subgroup 2-4 fold higher incidence of LGA in FET siblings; Pinborg 2014 Is it in vitro culture media/methods? Young 1998 (cattle/sheep), Dumoulin 2010, Nelissen 2012 In mice imprinting changes with in vitro culture more pronounced with freezing; Market-Velker 2010 Is it the extended culture issue? Makinen 2013 An endometrial synchrony issue? Wilmut I 1981; D3 sheep embryos transferred to advanced lining caused 40% increased fetal size Pinborg 2014, >99% FET slow frozen CD2

Perinatal risks: LGA More recent vitrification data Kato et al, 2012; Japan (2006-2008 cycles) 4092 vitrified SET vs 2531 fresh SET Transfers D2 and D5 LGA crude OR 1.51 (1.32-1.74), aOR 1.23 (0.88-1.72) after adjustments including blastocyst culture D3 fresh vs D3 vitrified-no difference perinatal outcomes in general Liu SY et al 2013, Shi W et al 2012

And for SGA…? Fresh may have increased risk of SGA as compared to frozen Helmerhorst 2004, Jackson 2004, McDonald 2009 Though underlying infertility etiology and supraphysiologic levels of hormone may be more to blame… Cooper A 2011 Fertil Steril 1200 vs 460 F vs I; infertile had smaller neonates and more LBW than fertile at birth and in the first trimester; Within infertiliy subgroups no differences

Wash U fresh vs frozen sibling study CRL did not differ between the groups (difference 1.3 mm, 95% CI -1.1 to 3.7) No difference in birthweight between fresh & FET singleton siblings (difference 129 grams, 95% CI -155 to 412) FET singletons had a significantly shorter gestation than their fresh IVF siblings, delivering on average 14 days earlier (95% CI 3-25 days; p=0.01) Fresh (n=23) Frozen (n=23) p value Crown Rump Length (millimeters) 8.3 ± .8 7.3 ± 1.0 p =.86 Birthweight (grams) 3280 ± 100 3248 ± 158 p = .87 Gestational Age at Birth (days) 252 ± 3 241 ± 4 p = .01 O’Neill KE and Cooper AR. Fertil Steril 2010; 94(4); S19 Primary outcome were size (measured in the 1st trimester with CRL in mm and at birth with weight in grams) and gestational length measured in days.

Perinatal risks: Neurodevelopment? Swedish study, births 1982-2007 (2.5 million infants, 1.2% by IVF) IVF vs spont, ICSI vs no ISCI, fresh vs frozen No increased risk of autism in fresh vs frozen, ICSI vs no, only with surgically extracted sperm aRR MR if fresh + ICSI 1.47 (1.03-2.09), ICSI + frozen + preterm birth 3.47 (1.22-9.90) When adjusting for singletons only ICSI + frozen remained significant for MR; 2.36 (1.04-5.36) Sandin S et al, JAMA 2013 Rate per 100,000 person years. AR small Evolving theories of epigenetics in brain development Incidence of autism and MR 1-1.5% Risk of MR in IVF vs spont disappeared w singletons only Blastocyst data available for 2002+, subanalysis no difference though smaller numbers

The forest through the trees… Subtle differences at birth may have health consequences that manifest as adults Barker hypothesis Grace and Sinclair 2009 (animal review) Ceelen M, 2008; higher BP/FBG 8-18 y/o ART kids Hart and Norman 2013; higher BP/FBG in ART adults We need to sort out the underlying etiologies

An additive (or masking) effect? Ovarian Stimulation …In subtle epigenetic effects…? In vitro maturation Extended Culture …Is cryo just one more piece…? Cryo- Preservation

Other issues with FET Additional cost Disposition of excess embryos Optimal length of storage for vitrification?