1. Is Frozen Embryo Transfer better than Fresh?
SmartArt X Slides - Charo
4/11/2017
Bruce Shapiro MD, PhD
Medical Director, Fertility Center of Las Vegas
Clinical Associate Professor, University of Nevada School of Medicine
45 minutes long with 15 minutes for Q&A 1

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

3. SmartArt X Slides - Charo
Learning Objectives
4/11/2017
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
SmartArt X Slides - Charo
Early history of freezing human embryos or gametes
4/11/2017
1949 – First human gamete cryopreservation (sperm vitrification)
First live birth with FET
1985 – First pregnancies with thawed blastocysts
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 , our live birth rates with FET began to exceed those with fresh transfer.

6. Live Birth Rates at The Fertility Center of Las Vegas
Live birth rate per transfer (%)
Age <35

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, Nikas et al, Develioglu et al, Horcajadas et al 2007.

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

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. 377 fresh autologous cycles 106 autologous FET 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.

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 Murata et al Richter et al 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 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%
Small
54%
High
62%
33% 6
68%
38%
46%
20%
Low P4 level = P4< 1.0 ng/ml
Large blastocyst diameter = Diameter >190 um on day 5 or >205 um on day 6.
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%
Small
54%
76%
High
62%
87%
33%
85% 6
68%
78%
38%
69%
46%
77%
20%
73%
Low P4 level = P4< 1.0 ng/ml
Large blastocyst diameter = Diameter >190 um on day 5 or >205 um on day 6.
Shapiro et al 2008, Shapiro et al 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?
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw
Bosch et al 2003, Shapiro et al 2010.

25. Matched on maternal age and number of bipronuclear oocytes produced
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
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw
Shapiro et al 2010.

26. Cancellation rate greater with FET
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.
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw
Shapiro et al 2010.

27. 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.

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?
69 non-donor FET using PTEC
136 fresh oocyte donation cycles
Shapiro et al 2010.

29. Compared 205 autologous FET and fresh oocyte donation cycles
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
69 non-donor FET using PTEC
136 fresh oocyte donation cycles
Shapiro et al 2010.

30. Similar implantation rates (65.9% vs 62.1%)
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%)
69 non-donor FET using PTEC
136 fresh oocyte donation cycles
Shapiro et al 2010.

31. How does FET in young patients compare to fresh donor cycles using young donors?
Shapiro et al Comparing 205 PTEC and donor cycles, egg sources <35 years of age, double blastocyst transfer.

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.
69 non-donor FET using PTEC
136 fresh oocyte donation 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?
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw
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.
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw
Shapiro et al 2013.

35. Comparison of demographics and potential confounders in matched
fresh and freeze-thaw transfers.
Fresh FET P value
Transfers
Patient age (y) * NS
Age range (y) 23– –45 NS
Day 5 blastulation * 23 (24.7) 23 (24.7) NS
Blast diameter (mm) * NS
ICM (mm2) 4, , NS
Troph cells NS
eSET NS
Genetic screening * NS
Endometrium (mm)
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw
* Matching criterion
Shapiro et al 2013.

36. fresh and freeze-thaw transfers.
Comparison of matched
fresh and freeze-thaw transfers.
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw

37. What is the impact of reduced endometrial receptivity following ovarian stimulation?
Also matched use of PGD.
Fresh vs frozen 2pn thaw
Shapiro et al, 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?
Also matched use of PGD.
Fresh vs frozen 2pn thaw
Shapiro et al, 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.
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw
Shapiro et al 2013.

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

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

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 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 a Study halted for excessive multiple pregnancy rate Fresh FET
P-value
Transfers 52 49
# Transferred
2.0 ± 0.1
1.9 ± 0.3 NS
Implantation rate
57%
65%
Clinical pregnancies
per transfer
80%
Multiple preg rate (per clinical preg) a
73.5%
59.0%
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
Present
33/43 (77%)
23/24 (96%)
Not Present
1/9 (11%)
16/25 (64%)
P< 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 years
Shapiro et al 2011.

48. Randomized Trial: Fresh vs Frozen in Normal Responders
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 Implantation rate 37/95 = 38.9% 63/89 = 70.8%
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, Ishihara et al, Shapiro et al, 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.
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)

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
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

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
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

57. Trends in Fresh and FET outcomes
SART registry
Standard 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.

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
Live birth rate per transfer (%)
Age <35

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!