Single Euploid Embryo Transfer: Fresh vs Frozen Debate

Amber R. Cooper, MD, MSCIAssistant Professor

Division of Reproductive Endocrinology & InfertilityWashington University in St. Louis

Kaylen Silverberg, M.D.Medical and Laboratory Director, Texas Fertility Center

Austin and San Antonio, TXClinical Associate Professor Dell Medical School

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

SHE

What we DO agree on….

1. Frozen embryo transfers are an important part of successful IVF clinics

2. Vitrification has improved ART success for both embryos and oocytes

3. Endometrial-Embryo synchrony is very important for successful implantation

4. PGS allows for eSET, reducing the risks associated with multiple gestation

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.

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.

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”

Shapiro et al 2010, comparing 236 matched cycles with elevated P4.

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Frozen-ThawedFresh

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.

Euploid (%) Aneuploid (%)0%

10%

20%

30%

40%

50%

60%

70%

80%

Day 5 Day 6 Day 7

Embryo developmental pace

Shapiro et al 2001.

Clinical Pregnancy Implantation0%

10%

20%

30%

40%

50%

60%

70%Day 5 BlastocystsDay 6 Blastocysts

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.

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

Fresh 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 FrozenIVF 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

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

An additive (or masking) effect?

Ovarian

Stimulation

In vitro culture

Extended Culture

Cryo-Preservation

…In subtle epigenetic effects…?

…Is cryo just one more piece…?

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 FrozenIVF 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 FrozenMaternal 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.

Fresh versus FrozenPerinatal 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

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 FrozenOther 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

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

-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)

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

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

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

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

SART TRENDS…

Trends in Fresh and FET outcomes

• SART registry• 2006-2011• Standard SART age groups

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

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!