Single Euploid Embryo Transfer: Fresh vs Frozen Debate SmartArt X Slides - Charo 4/19/2017 Amber R. Cooper, MD, MSCI Assistant Professor Division of Reproductive Endocrinology & Infertility Washington University in St. Louis Kaylen Silverberg, M.D. Medical and Laboratory Director, Texas Fertility Center Austin and San Antonio, TX Clinical Associate Professor Dell Medical School 20-30 minute talk + 15 minutes for questions 1

Disclosures AC: Research Support: Beckman Coulter, Inc Advisory Board: Celmatix, Inc. KS: Research grants: Actavis; Finox Consulting/Speakers Bureaus: Good Start Genetics, Illumina, Serono, Myriad Genetics, Actavis, Abbvie

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What we DO agree on…. Frozen embryo transfers are an important part of successful IVF clinics Vitrification has improved ART success for both embryos and oocytes Endometrial-Embryo synchrony is very important for successful implantation PGS allows for eSET, reducing the risks associated with multiple gestation

FRESH OR FROZEN EMBRYO TRANSFER??? Where we differ… What is the best FIRST line approach if you have a SINGLE EUPLOID embryo… (assuming time/shipping barrier removed) FRESH OR FROZEN EMBRYO TRANSFER???

Dr. SILVERBERG: “The Trouble with Fresh…”

Problems with Fresh eSET… Effects of ovarian stimulation Endometrial changes (advanced histology) Premature luteinization Dyssynchrony between embryo and endometrium Prevents use of euploid blasts that are not vitrifiable until Day 6 or 7 Only available to programs with onsite PGS

Ovarian Stimulation Controlled ovarian stimulation (COS) with exogenous FSH promotes development of multiple ovarian follicles Multiple follicles produce supraphysiologic levels of estradiol, progesterone, and other hormones These hormones affect and control endometrial development, maturation, and uterine contractile activity, and can alter the window of implantation COS can lead to premature luteinization, lessening the likelihood of implantation

Endometrial Changes Pinopodes appear 1-2 days earlier in cycles with COS and are less numerous Pinopode function not yet confirmed, but theoretically have role in implantation and endometrial receptivity Progesterone receptor down-regulated 1-2 days earlier in cycles with COS. All of these factors are consistent with advanced endometrial histology. …when compared to natural cycles. Mirkin et al, 2004. Nikas et al, 1999. Develioglu et al, 1999. Horcajadas et al 2007.

Advanced endometrial histology Advanced endometrial histology has been correlated with implantation failure in fresh cycles. Nikas et al, 1999. Kolibianakis et al, 2002.

Overall effect of ovarian stimulation on the endometrium Following COS, the endometrium is “histologically advanced, biochemically different, and genomically dysregulated.” Horcajadas et al, 2007.

Premature Luteinization Adversely Affects Pregnancy Rates in IVF cycles

And it Does so by Adversely Affecting the Endometrium, not the Embryo Retrospective evaluation (n=96) Embryos frozen due to premature luteinization and subsequently transferred had higher pregnancy rate than did embryos frozen from cycles without premature luteinization Silverberg K, et al. Fertil Steril 1994;61:508

And Freezing of Embryos with Subsequent FET can Salvage the Cycle Retrospective cohort study, n=236 cycles All with Premature luteinization 118 with fresh ET, 118 with freeze all and FET Freeze-all resulted in better outcomes: Implantation rates (56.8 vs. 26.9%, P<0.05) Ongoing pregnancy rates/transfer (70.5 vs. 35.2%, P<0.05) Shapiro B, et al. Fertil Steril 2010;93:636-41

Cryopreservation rescues cycles with “premature luteinization” The cancellation rates did not differ significantly (P=0.093). All other rates shown differed significantly. The difference in pregnancy losses was mainly in biochemical pregnancy losses, which differed significantly (4/81 in FET vs 15/62 in fresh transfer, per pregnancy, P=0.0010). Shapiro et al 2010, comparing 236 matched cycles with elevated P4.

Embryo developmental pace There is biological variation in embryonic development Some embryos form expanded blastocysts on day 5 of development, others on day 6 or 7 Day 5 and 6 blastocysts are more likely to be chromosomally normal than day 7 blastocysts in fresh IVF cycles following ovarian stimulation. Silverberg K et al, 2015.

Embryonic Euploidy Nguyen J, Silverberg K, et al, 2014.

Embryo developmental pace Shapiro et al 2001.

Therefore: Some (Day 6, Day 7) euploid blastocysts won’t even be available for fresh ET, as fresh ET must occur on Day 5 In addition: Vitrification allows for embryo batching/banking, and is available to labs that do not have onsite PGS capability MOST IMPORTANTLY: Vitrification with subsequent FET allows for TRULY optimal embryo selection that you CAN’T achieve with fresh ET

Vitrification with FET Affords Pregnancy Rates At Least As Good As Fresh ET

Pregnancy Rates in Fresh, Frozen, and donor oocyte cycles Retrospective study: 377 fresh autologous cycles 106 autologous FET cycles 56 fresh oocyte donation cycles Shapiro et al 2008.

Contrasting patterns of clinical pregnancy rates in fresh and FET Shapiro et al 2008.

Can FET in young patients be comparable to fresh donor cycles? Advantages of donor oocyte cycles: transfer of healthy embryos derived from young oocytes Absence of an endometrium exposed to supraphysiolgic hormone levels resulting from COS Therefore, shouldn’t the implantation and pregnancy rates of young patients in FET cycles rival those of donor oocyte cycles? 69 non-donor FET using PTEC 136 fresh oocyte donation cycles Shapiro et al 2010.

Autologous FET (136) vs. fresh (69) oocyte donation cycles How does FET in young patients compare to fresh donor cycles using young donors? Autologous FET (136) vs. fresh (69) oocyte donation cycles All patients and donors <35 yo Implantation rates (65.9% vs 62.1%) Ongoing pregnancy rates (79.7% vs 75.0%). 69 non-donor FET using PTEC 136 fresh oocyte donation cycles Shapiro et al 2010.

The passenger was on time, but the train left early

Randomized Trial: Fresh vs Frozen in Normal Responders Randomized trial comparing fresh and frozen embryo transfers in 103 NORMAL responders (8-15 antral follicles) age 18-40 years. Shapiro et al 2011.

Randomized Trial: Fresh vs Frozen in Normal Responders Cryo P-value Implantation rate 37/95 = 38.9% 63/89 = 70.8% <0.0001 Clinical pregnancy rate per transfer * 29/53 = 54.7% 42/50 = 84.0% 0.0013 Ongoing pregnancy rate per transfer 27/53 = 50.9% 39/50 = 78.0% 0.0072 * The study was halted at this interim stopping point because the P-value was less than 0.03, per the pre-defined stopping rule.

Fresh versus Frozen IVF Outcomes When compared to fresh transfer, embryo cryopreservation followed by FET has been associated with: Reduced risk of implantation failure in normal responders Reduced risk of implantation failure following premature luteinization Reduced risk of IVF failure per retrieval Shapiro et al 2011, Shapiro et al 2010, Roque et al 2012

Preliminary Conclusions -Why Vit/Thaw is UNQUESTIONABLY SUPERIOR… Avoid potential adverse effects of COS on endometrium Avoid effects of premature luteinization No need for in house PGS lab More euploid embryos available for use Better perinatal/neonatal outcome (as you will see)

Dr. Cooper: “The Trouble with Frozen…”

So let me get this straight…

WHAT DID YOU JUST HEAR? Endometrial-Embryo synchrony is very important Pay attention to method and degree of COH Early progesterone elevations may be detrimental Good quality day 5 blasts have much better implantation rates than day 6 in fresh cycles; Those that are slower growing may do better in a frozen cycle

So….SLOW DOWN THE TRAIN !! (or at least put it on the right schedule)

Maybe we should focus on WHY there is dyssynchrony Too much drug? Hyperstimulation? Type of stimulation? Advanced follicular development, delayed trigger? Do we need a more physiologic trigger? Improved monitoring of the luteal phase?

Do we agree on which FET approach is best? Timing of embryo cryopreservation? 2PN, CD2/3, CD5/6 Slow-freezing vs vitrification? Medicated or natural cycle ET? Leuprolide? Recommended number of embryos to transfer? Culture media? Timing and delivery method of progesterone? So, how do we really look at outcomes critically? And as Dr. Scott says…

Slow-freezing vs Vitrification: Vitrification data still limited 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 Yet the majority of practice is vitrified blasts 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

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

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

Other issues with FET Additional cost Additional time Disposition of excess embryos Optimal length of storage for vitrification? The lab is critical… What about transferring embryos between centers?

And do all patients really need and/or benefit from aneuploid screening? I concede….some may May improve eSET in some patients But…well selected patients may benefit from eSET without AS (at least in our clinic) Much of success relies on TE biopsy and advanced genetic testing Are all abnormals abnormal? 10-12% may not be What about the 10+% that don’t have blastocysts develop?

Risks to Mom and Baby…

Fresh versus Frozen IVF Outcomes When compared to fresh transfer, embryo cohort cryopreservation followed by frozen-thawed transfer has been associated with: Reduced risk of implantation failure in normal responders Reduced risk of implantation failure following premature progesterone elevation Reduced risk of IVF failure per retrieval Shapiro et al 2011, Shapiro et al 2010, Roque et al 2012

Fresh versus Frozen Maternal Risks When compared to fresh transfer, frozen-thawed transfer has been associated with: Reduced risk of late-onset OHSS Reduced risk of ectopic pregnancy Reduced risk of pre-eclampsia. ASRM Practice Committee 2008, Ng et al, 1998. Ishihara et al, 2011. Shapiro et al, 2012. Maheshwari et al 2012, Imudia 2013.

Perinatal Risks related to Birthweight Fresh versus Frozen Perinatal Risks related to Birthweight When compared to fresh transfer, frozen-thawed transfer has been associated with: Greater mean birthweight Reduced risk of low birthweight Reduced risk of small for gestational age. Compared with fresh transfer: Wang (2005) reported reduced risk of low birthweight and prematurity. Shih (2008) reported greater birthweight with frozen embryos. Healy (2009) reported reduced risk of antepartum haemorrhage and placental abruption. Pinborg (2010) reported greater birthweight and reduced risks of prematurity and low birthweight. Henningsen (2011) reported greater birthweight with fresh than with FET. Kalra (2011) reported reduced perinatal morbidity. Kalra (2011) (registry study) reported reduced risks of low birthweight, low birthweight at term, pre-term low birthweight. Imudia (2012) reported reduced risk of SGA and pre-eclampsia. Maheshwari (2012) reported reduced risks of antepartum haemorrhage, prematurity, SGA, low birthweight, perinatal mortality, placenta previa, and placental abruption. Nakashima (2012) reported greater birthweight and reduced risk of low birthweight Sullivan (2013) reported reduced risks of prematurity and perinatal death. Pinborg (2013) reported reduced risk of prematurity. Maheshwari et al (2012)

Perinatal Risk of Pre-Term Delivery Fresh versus Frozen Perinatal Risk of Pre-Term Delivery When compared to fresh transfer, frozen-thawed transfer has been associated with: Reduced risk of pre-term birth Reduced risk of pre-term low birthweight Compared with fresh transfer: Wang (2005) reported reduced risk of low birthweight and prematurity. Shih (2008) reported greater birthweight with frozen embryos. Healy (2009) reported reduced risk of antepartum haemorrhage and placental abruption. Pinborg (2010) reported greater birthweight and reduced risks of prematurity and low birthweight. Henningsen (2011) reported greater birthweight with fresh than with FET. Kalra (2011) reported reduced perinatal morbidity. Kalra (2011) (registry study) reported reduced risks of low birthweight, low birthweight at term, pre-term low birthweight. Imudia (2012) reported reduced risk of SGA and pre-eclampsia. Maheshwari (2012) reported reduced risks of antepartum haemorrhage, prematurity, SGA, low birthweight, perinatal mortality, placenta previa, and placental abruption. Nakashima (2012) reported greater birthweight and reduced risk of low birthweight Sullivan (2013) reported reduced risks of prematurity and perinatal death. Pinborg (2013) reported reduced risk of prematurity. . Maheshwari et al 2012, Kalra et al 2011, Sullivan et al 2013, Pinborg et al 2013

Risks Associated with Pre-Term Delivery Inability to regulate body temperature Respiratory distress or apnea Visual issues, including retinopathy Feeding problems, digestive issues Prolonged hospitalization Intellectual disabilities Low birthweight Hearing loss Jaundice Bleeding in the brain Infection Cerebral palsy Neonatal death

Fresh versus Frozen Other Perinatal Risks When compared to fresh transfer, frozen-thawed transfer has been associated with: Reduced risk of antepartum hemorrhage Reduced risk of placenta previa Reduced risk of placental abruption Reduced risk of perinatal mortality Compared with fresh transfer: Wang (2005) reported reduced risk of low birthweight and prematurity. Shih (2008) reported greater birthweight with frozen embryos. Healy (2009) reported reduced risk of antepartum haemorrhage and placental abruption. Pinborg (2010) reported greater birthweight and reduced risks of prematurity and low birthweight. Henningsen (2011) reported greater birthweight with fresh than with FET. Kalra (2011) reported reduced perinatal morbidity. Kalra (2011) (registry study) reported reduced risks of low birthweight, low birthweight at term, pre-term low birthweight. Imudia (2012) reported reduced risk of SGA and pre-eclampsia. Maheshwari (2012) reported reduced risks of antepartum haemorrhage, prematurity, SGA, low birthweight, perinatal mortality, placenta previa, and placental abruption. Nakashima (2012) reported greater birthweight and reduced risk of low birthweight Sullivan (2013) reported reduced risks of prematurity and perinatal death. Pinborg (2013) reported reduced risk of prematurity. Maheshwari et al 2012, Sullivan et al 2013

Risks to Mom and Baby…

Fresh vs Frozen: Perinatal risks IF you have euploid blasts to work with, IF they are recovered, IF they survive, and IF they are of optimal quality 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 SGA, LBW and PTD often better with FET -most data with slow-freezing

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 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. O’Neill KE and Cooper AR. Fertil Steril 2010; 94(4); S19

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) 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 Sandin S et al, JAMA 2013

SART TRENDS…

Trends in Fresh and FET outcomes SART registry 2006-2011 Standard SART age groups Compared with fresh transfer: Wang (2005) reported reduced risk of low birthweight and prematurity. Shih (2008) reported greater birthweight with frozen embryos. Healy (2009) reported reduced risk of antepartum haemorrhage and placental abruption. Pinborg (2010) reported greater birthweight and reduced risks of prematurity and low birthweight. Henningsen (2011) reported greater birthweight with fresh than with FET. Kalra (2011) reported reduced perinatal morbidity. Kalra (2011) (registry study) reported reduced risks of low birthweight, low birthweight at term, pre-term low birthweight. Imudia (2012) reported reduced risk of SGA and pre-eclampsia. Maheshwari (2012) reported reduced risks of antepartum haemorrhage, prematurity, SGA, low birthweight, perinatal mortality, placenta previa, and placental abruption. Nakashima (2012) reported greater birthweight and reduced risk of low birthweight Sullivan (2013) reported reduced risks of prematurity and perinatal death. Pinborg (2013) reported reduced risk of prematurity.

Trends in US National Average Live Birth Rates

Trends in US National Average Live Birth Rates

Trends in US National Average Live Birth Rates

Trends in US National Average Live Birth Rates

Trends in US National Average Live Birth Rates

Trends in numbers of live births Values calculated from SART national reports. Fresh live births are products of live birth per cycle and number of cycles. FET live births are product of live birth per transfer and number of transfers.

BUT… …I expect the 38+ year old who HAS blastocysts to freeze to do better than her age matched friends

BUT… It goes back to what is our FIRST line therapy for a single euploid blastocyst… There is a place for FET There is room for improvement in fresh cycles We need more research on vitrified blasts (and oocytes) We need more research on extended culture (would outcomes look as good with day 2/3 freeze?) Genetic testing is good but not perfect If time and money weren’t an issue… And if only humans were efficient reproducers…

Summary of arguments Fresh Most cryopreservation done at blast stage, so arguments against blast transfer are potentially valid; success is based on predominantly blast transfers Hypothetical additional epigenetic concerns Increased cost of cryopreservation and subsequent FET

Summary of arguments Frozen Cryopreservation can be done at MC stage with cleavage stage or blast transfer Only hypothetical epigenetic concerns to date Lower costs from: Increased implantation and pregnancy rate Savings in elimination of multiples Avoiding FETs of non-viable embryos Fewer cycles required for pregnancy

Summary of arguments Fresh Loss of embryos from freeze/thaw PGS and the transfer of euploid embryos may negate any lowered implantation rates in fresh cycles Larger babies therefore higher cesarean section rates and potentially long term health consequences of faster/larger growth Convenience from completing retrieval and transfer in one cycle (TIME is important to aging women!)

Summary of arguments Frozen Avoids wasting good embryos by not transferring into a suboptimal endometrium Higher implantation rate increases success with single embryo transfer Lower risk of preterm birth and low birth weight babies Convenience of scheduling subsequent FET Opportunity to freeze and wait for PGS results “Aging” women prefer better results

Thank you!