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1. Is Frozen Embryo Transfer
better than Fresh?
Bruce Shapiro MD, PhD
Medical Director, Fertility Center of Las Vegas
Clinical Associate Professor, University of Nevada
School of Medicine

2. Disclosures
Research grants:
• Actavis
• Merck & Co.
Consulting/Speakers Bereaus:
• Merck
• TEVA
• Glycotope GMBH

3. 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.

4. 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
oocytes
Polge et al 1949, Zeilmaker et al 1984, Cohen et al 1985,
Chen 1986

5. 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.

6. Live Birth Rates at The Fertility
Center of Las Vegas
25
30
35
40
45
50
55
2004 2005 2006
Fresh - FCLV
FET - FCLV
Fresh - Nat Avg
FET - Nat Avg
Age <35
Live birth rate per transfer (%)

7. 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?

8. 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.

9. 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.

10. Advanced endometrial histology
Advanced endometrial histology has been
correlated with premature progesterone
elevation and implantation failure.
Nikas et al, 1999. Kolibianakis et al, 2002.

11. 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

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

13. Blastocysts

14. 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.

15. Embryo developmental pace
Shapiro et al 2001.
0%
10%
20%
30%
40%
50%
60%
70%
Clinical Pregnancy Implantation
Day 5 Blastocysts
Day 6 Blastocysts

16. 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?

17. 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.

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

19. 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.

20. 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.

21. 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.

22. 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

23. 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

24. 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.

25. 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.

26. 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.

27. Cryopreservation rescues cycles with
“premature luteinization”
Shapiro et al 2010, comparing 236 matched cycles with elevated P4.
0
10
20
30
40
50
60
70
80
90
Frozen-Thawed
Fresh

28. 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.

29. 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.

30. How does FET in young patients compare to
fresh donor cycles using young donors?
Results
• Similar implantation rates (65.9% vs
62.1%)
• Similar ongoing pregnancy rates
(79.7% vs 75.0%)
Shapiro et al 2010.

31. How does FET in young patients compare to
fresh donor cycles using young donors?
Shapiro et al 2010. Comparing 205 PTEC and donor cycles, egg
sources <35 years of age, double blastocyst transfer.
0
10
20
30
40
50
60
70
80
90
100
Autologous FET
Fresh Donor

32. How does FET in young patients compare to
fresh donor cycles using young donors?
• Conclusion: In the absence of cryodamage,
FET embryos can implant as readily as
those from fresh oocyte donor cycles.
Shapiro et al 2010.

33. 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.

34. 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.

35. Comparison of demographics and potential
confounders in matched
fresh and freeze-thaw transfers.
Fresh FET P value
Transfers 93 93
Patient age (y) * 33.8 33.8 NS
Age range (y) 23–45 22–45 NS
Day 5 blastulation * 23 (24.7) 23 (24.7) NS
Blast diameter (mm) * 192.5 192.6 NS
ICM (mm2) 4,047 3,939 NS
Troph cells 13.8 14.0 NS
eSET 23 19 NS
Genetic screening * 4 4 NS
Endometrium (mm) 10.1 9.1 0.0050
* Matching criterion
Shapiro et al 2013.

36. Comparison of matched
fresh and freeze-thaw transfers.

37. 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.

38. 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.

39. 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.

40. LH
surge
Ovulation Blastulation Embryo
implantation
window
Endometrial
Implantation
window
Oocyte/embryo development timeline
In natural menstrual cycle
P4 exposure
Follicular
phase

41. Trigger
injection
Oocyte
collection
Blastulation Embryo
implantation
window
Endometrial
implantation
window
Oocyte/embryo development timeline
Following ovarian stimulation
P4 exposure
Ovarian
stimulation

42. 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.

43. Randomized Trial: Fresh vs Frozen
in High Responders
• 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.

44. Results
Fresh FET P-value
Transfers 52 49
# Transferred 2.0 ± 0.1 1.9 ± 0.3 NS
Implantation rate 57% 65% NS
Clinical pregnancies
per transfer
65% 80% NS
Multiple preg rate (per
clinical preg) a
73.5% 59.0% NS
a Study halted for excessive multiple pregnancy rate

45. Randomized Trial: Fresh vs Frozen
in High Responders
• 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.

46. Clinical Pregnancy Rate According
to Presence of Supernumerary
Embryos
Supernumerary
blastocysts
Fresh
clinical
pregnancy
rate
FET
clinical
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

47. 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.

48. Results
Randomized 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.

49. Results
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.

50. Results

51. Fresh versus Frozen Risk Comparison
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

52. Fresh versus Frozen Risk Comparison
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.

53. Fresh versus Frozen Risk Comparison
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.
Maheshwari et al (2012)

54. Fresh versus Frozen Risk Comparison
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
Maheshwari et al 2012, Kalra et al 2011, Sullivan et al 2013,
Pinborg et al 2013

55. 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

56. Fresh versus Frozen Risk Comparison
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
Maheshwari et al 2012, Sullivan et al 2013

57. Trends in Fresh and FET outcomes
• SART registry
• 2006-2011
• Standard age groups

59. Trends in US National Average Live
Birth Rates

60. Trends in US National Average Live
Birth Rates

61. Trends in US National Average Live
Birth Rates

62. Trends in US National Average Live
Birth Rates

63. Trends in US National Average Live
Birth Rates

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

65. Trends in numbers of live births

66. Increasing asynchronous transfers in
fresh cycles with age
Retrospective study showing asynchrony factors
increasing with age
Shapiro et al, 2013

67. Live Birth Rates at The Fertility
Center of Las Vegas
30
35
40
45
50
55
60
65
70
2006 2007 2008 2009 2010 2011 2012
Fresh - FCLV
FET - FCLV
Fresh Nat Avg
FET - Nat Avg
Age <35
Live birth rate per transfer (%)

68. Average Ongoing Pregnancy Rates
at Fertility Center of Las Vegas

69. 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.

70. Thank you!