Fresh or frozen embryos – which are better

Is Frozen Embryo Transferbetter than Fresh?

Bruce Shapiro MD, PhDMedical Director, Fertility Center of Las Vegas

Clinical Associate Professor, University of Nevada School of Medicine

Disclosures

Research grants:• Actavis• Merck & Co.

Consulting/Speakers Bereaus:• Merck• TEVA• Glycotope GMBH

Learning Objectives

• Review indicators of embryo-endometrium asynchrony in fresh autologous cycles

• Review the effects of ovarian stimulation on perinatal outcome and maternal risks

• Review the use of embryo cohort cryopreservation to circumvent such risks.

Early history of freezing human embryos or gametes

• 1949 – First human gamete cryopreservation (sperm vitrification)

• 1984 - First live birth with FET• 1985 – First pregnancies with thawed

blastocysts• 1986 - First live birth with thawed

oocytesPolge et al 1949, Zeilmaker et al 1984, Cohen et al 1985, Chen 1986

Motivation for fresh vs FET studies

• In 2004 we noticed the pregnancy rates in our FET cycles were as good as those in our fresh cycles

• In 2005-2006, our live birth rates with FET began to exceed those with fresh transfer.

Live Birth Rates at The Fertility Center of Las Vegas

2004 2005 200625

30

35

40

45

50

55

Fresh - FCLVFET - FCLVFresh - Nat AvgFET - Nat Avg

Age <35

Live birth rate per transfer (%)

Rationale for investigation of FET cycles and implantation potential

• If supernumerary “second-best” frozen embryos implanted more readily than fresh primary embryos, then could further improvement be realized if “best” primary embryos were cryopreserved in a freeze all cycle and replaced in an FET cycle?

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.

Endometrial Changes

• Mature pinopodes appear 1-2 days earlier in cycles with COS and are less numerous

• Pinopode function not yet confirmed, but generally believed to have role in implantation and the endometrial receptive phase

• Progesterone receptor down-regulated 1-2 days earlier in cycles with COS.

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 premature progesterone elevation and implantation failure.

Nikas et al, 1999. Kolibianakis et al, 2002.

Gene expression profiles

• Gene expression profiles are different between natural cycles and cycles of COS consistent with dysregulation of gene expression in hyperstimulated cycles

• Many genes associated with the implantation window on hCG +7 were delayed by 2 days

• This is consistent with histological and biochemical discrepancies found previously in other studies.

Horcajadas et al, 2007

Overall effect of ovarian stimulation on the endometrium

• Following COS, the endometrium is “histologically advanced, biochemically different, and genomically dysregulated.”

Horcajadas et al, 2007.

Blastocysts

Embryo developmental pace

• There is biological variation in embryonic developmental pace

• Some embryos form expanded blastocysts on day 5 of development, others on day 6

• Day 5 blastocysts implant more readily than day 6 blastocysts in fresh IVF cycles following ovarian stimulation.

Shapiro et al 2001.

Embryo developmental pace

Shapiro et al 2001.

Clinical Pregnancy Implantation0%

10%

20%

30%

40%

50%

60%

70%Day 5 BlastocystsDay 6 Blastocysts

Questions raised by the effect of embryo developmental pace on IVF outcome

• Why do fresh day 5 blastocysts implant more readily than fresh day 6 blastocysts?

• Do day 5 and day 6 blastocysts have different implantation rates in FET cycles?

• If day 5 and day 6 blastocysts have similar implantations rates in FET cycles in the absence of COS, should they also have similar rates in donor oocyte cycles?

Studied Day 5 and Day 6 blastocyst transfers in Fresh, FET 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.

Day 5 vs Day 6 Blastocysts

• Similar aneuploidy rates • Similar implantation potential in frozen-

thawed cycles• Frozen-thawed day 6 blastocysts

transferred in cycles without ovarian stimulation implant more readily than fresh day 6 blastocysts in cycles with ovarian stimulation.

Kroener et al 2012. Murata et al 2005. Richter et al 2006. Shapiro et al 2008.

Day 5 vs Day 6 Blastocysts

• Conclusion: The different implantation potential between day 5 and day 6 blastocysts is consistent with advanced endometrial development in cycles of ovarian stimulation, so that slower embryos are less likely to implant because the endometrial receptive phase ends prematurely.

Richter et al 2006. Shapiro et al 2008.

Study: Are there degrees of asynchrony?

• Retrospective analysis• 361 fresh blastocyst transfers• 25 independent variables potentially

affecting IVF success• Outcome measure of clinical pregnancy• Multiple logistic regression modeling• Validated against a second set of 219

blastocyst transfers

Shapiro et al 2008.

Model of synchrony factors in fresh autologous cycles

Day of Blastulation

P4 Level

Blastocyst Diameter

Fresh Model

5 Low Large 80%5 Low Small 54%5 High Large 62%5 High Small 33%6 Low Large 68%6 Low Small 38%6 High Large 46%6 High Small 20%

Shapiro et al 2008

Comparison of FET Results with Fresh Transfer Model

Day of Blastulation

P4 Level

Blastocyst Diameter

Fresh Model

FET Results

5 Low Large 80% 88%5 Low Small 54% 76%5 High Large 62% 87%5 High Small 33% 85%6 Low Large 68% 78%6 Low Small 38% 69%6 High Large 46% 77% 6 High Small 20% 73%

Shapiro et al 2008, Shapiro et al 2013 P<0.0001

Fresh versus frozen in cycles with “premature luteinization”

• If premature elevation of progesterone at the time of the hCG trigger is associated with decreased implantation rates, could we improve implantation rates if we cryopreserved all embryos and transferred them in FET cycles?

Bosch et al 2003, Shapiro et al 2010.

Retrospective study of fresh versus frozen in cycles with “premature luteinization”

• 118 fresh transfers matched to 118 freeze-all cycles, all in cycles with P4>1.0 on day of trigger

• Matched on maternal age and number of bipronuclear oocytes produced

• Similar numbers of transferred blastocysts

Shapiro et al 2010.

Retrospective study of fresh versus frozen in cycles with “premature luteinization”

Results

• Cancellation rate greater with FET• Pregnancy, implantation, ongoing

pregnancy per transfer, and ongoing pregnancy per retrieval all greater with FET

• Pregnancy loss rate lower after FET.

Shapiro et al 2010.

Cryopreservation rescues cycles with “premature luteinization”

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

Cance

llatio

n Rate

Pregna

ncy/T

ransfe

r

Loss

/Preg

nanc

y

Ongoin

g/Tran

sfer

Ongoin

g/Retr

ieval

0102030405060708090

Frozen-ThawedFresh

Can FET in young patients be comparable to fresh donor cycles?

• One advantage of donor oocyte cycles is the transfer of healthy embryos derived from young donors

• Another advantage is the 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?

Shapiro et al 2010.

How does FET in young patients compare to fresh donor cycles using young donors?

• Compared 205 autologous FET and fresh oocyte donation cycles

• Autologous patients and oocyte donors <35 years of age in oocyte retrieval cycle

Shapiro et al 2010.


Results

• Similar implantation rates (65.9% vs 62.1%)

• Similar ongoing pregnancy rates (79.7% vs 75.0%)

Shapiro et al 2010.


Shapiro et al 2010. Comparing 205 PTEC and donor cycles, egg sources <35 years of age, double blastocyst transfer.

0

20

40

60

80

100

Autologous FETFresh Donor


• Conclusion: In the absence of cryodamage, FET embryos can implant as readily as those from fresh oocyte donor cycles.

Shapiro et al 2010.

Could there be a embryo screening effect in FET cycles?

• If we controlled for embryo morphology, would fresh and FET implantation rates still differ?

• Could the difference in implantation and pregnancy rates between fresh and FET cycles be due to a screening effect so that only the morphologically best appearing embryos remain after thaw for transfer?

Shapiro et al 2013.

What is the nature of the reduced endometrial receptivity following ovarian stimulation?

• A matched-cohort study compared 93 fresh and 93 frozen-thawed single-blastocyst transfers, matched for patient age, embryo morphology, and day of blastulation.

• Fresh transfers had significantly lower ongoing pregnancy rate than FET with day 6 blastocysts, but not with day 5 blastocysts.

Shapiro et al 2013.

Comparison of demographics and potential confounders in matched

fresh and freeze-thaw transfers. Fresh FET P value

Transfers 93 93Patient age (y) * 33.8 33.8 NSAge range (y) 23–45 22–45 NSDay 5 blastulation * 23 (24.7) 23 (24.7) NSBlast diameter (mm) * 192.5 192.6 NSICM (mm2) 4,047 3,939 NSTroph cells 13.8 14.0 NSeSET 23 19 NSGenetic screening * 4 4 NSEndometrium (mm) 10.1 9.1 0.0050

* Matching criterionShapiro et al 2013.

Comparison of matchedfresh and freeze-thaw transfers.

What is the impact of reduced endometrial receptivity following ovarian stimulation?

Shapiro et al, 2013. Comparing 186 cycles matched on maternal age, embryo morphology, and day of blastulation.

Is the reduced endometrial receptivity following ovarian stimulation associated with

embryo developmental pace?

Shapiro et al, 2013. Comparing 186 cycles matched on maternal age, embryo morphology, and day of blastulation.

What is the nature of the reduced endometrial receptivity following ovarian stimulation?

• Conclusion: COS reduces implantation of slowly-developing embryos, consistent with the embryo-endometrium asynchrony hypothesis.

Shapiro et al 2013.

LHsurge

Ovulation Blastulation Embryoimplantation

window

EndometrialImplantation

window

Oocyte/embryo development timelineIn natural menstrual cycle

P4 exposure

Follicularphase

Triggerinjection

Oocytecollection

Blastulation Embryoimplantation

window

Endometrialimplantation

window

Oocyte/embryo development timelineFollowing ovarian stimulation

P4 exposure

Ovarianstimulation

Randomized Trial: Fresh vs Frozen in High Responders

• Randomized trial comparing fresh and frozen embryo transfers in 101 HIGH responders (>15 antral follicles) age 18-40 years.

Shapiro et al 2011.


• 65% clinical pregnancy rate in fresh transfers• 80% clinical pregnancy rate in frozen transfers• Difference not statistically significant (P=0.1109).

Shapiro et al 2011.

Results

Fresh FET P-valueTransfers 52 49# Transferred 2.0 ± 0.1 1.9 ± 0.3 NSImplantation rate 57% 65% NSClinical pregnanciesper transfer

65% 80% NS

Multiple preg rate (per clinical preg) a

73.5% 59.0% NS

a Study halted for excessive multiple pregnancy rate


• However, significantly worse embryo morphology was observed in the frozen embryo transfer group.

• Post-hoc analysis showed superior ongoing pregnancy rate after frozen-thawed embryo transfer when controlling for embryo morphology.

Shapiro et al 2011.

Clinical Pregnancy Rate According to Presence of Supernumerary Embryos

Supernumerary blastocysts

Freshclinical pregnancy rate

FETclinical pregnancy rate

Present 33/43 (77%) 23/24 (96%)

Not Present 1/9 (11%) 16/25 (64%)P<0.0001 when comparing fresh and FET in logistic regression, while adjusting for presence of supernumerary embryos as a marker of embryo quality

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.

ResultsRandomized Trial: Fresh vs Frozen in Normal

Responders

• 54.7% clinical pregnancy rate in fresh transfers

• 84.0% clinical pregnancy rate in frozen transfers

• Statistically significant difference (P=0.0013).

Shapiro et al 2011.

ResultsFresh 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.

Results

Fresh versus Frozen Risk ComparisonIVF 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 Risk ComparisonMaternal 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 Frozen Risk ComparisonPerinatal 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.

Maheshwari et al (2012)

Fresh versus Frozen Risk ComparisonPerinatal 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 Frozen Risk ComparisonOther 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

Trends in Fresh and FET outcomes

• SART registry• 2006-2011• Standard age groups

Trends in US National Average Live Birth Rates





Ratio of FET cycles to fresh cycle starts, 2006-2012

Trends in numbers of live births

Increasing asynchronous transfers in fresh cycles with age

Retrospective study showing asynchrony factors increasing with age

Shapiro et al, 2013

Live Birth Rates at The Fertility Center of Las Vegas

2006 2007 2008 2009 2010 2011 2012303540455055606570

Fresh - FCLVFET - FCLVFresh Nat AvgFET - Nat Avg

Age <35

Live birth rate per transfer (%)

Average Ongoing Pregnancy Rates at Fertility Center of Las Vegas

Conclusions

• Ovarian stimulation impairs endometrial receptivity, particularly through embryo-endometrium asynchrony

• Embryo cohort cryopreservation circumvents the compromised endometrium

• Frozen-thawed embryo transfer may be associated with certain reduced maternal and perinatal risks, when compared to fresh autologous transfers.

Thank you!

Art & Photos

Fresh or frozen embryos – which are better