1. Freeze All The Paradigm Shift
SmartArt X Slides - Charo
4/18/2018
Said Daneshmand, MD
San Diego Fertility Center
Fertility Center of Las Vegas
1984: First live birth from a frozen embryo
1986: First pregnancy from frozen oocyte 1

2. Disclosures
Research grants:
Watson
Merck & Co.

3. SmartArt X Slides - Charo
Learning Objectives
4/18/2018
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. Motivation for fresh vs FET studies
In 2003 we changed our slow freezing protocols
In 2004 we noticed the pregnancy rates in our frozen cycles were slightly better than our fresh cycles
In 2005 the gap widened
In 2006 the difference stabilized

5. Live Birth Rates at The Fertility Center of Las Vegas
Age <35

6. 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” embryos were cryopreserved in a freeze all cycle and replaced in an FET cycle?

7. Ovarian Stimulation
Controlled ovarian stimulation (COS) with exogenous gonadotropins promotes development of multiple ovarian follicles
Follicles produce supraphysiologic levels of estradiol, progesterone, and other hormones
Estradiol and progesterone control endometrial development and maturation.

8. 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.

9. Advanced endometrial histology
Advanced endometrial histology has been correlated with:
Premature progesterone elevation
Implantation failure.
Nikas et al, Kolibianakis et al, 2002.

10. 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

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

12. Blastocysts

13. Embryo developmental pace
There is biological variation in embryonic developmental pace
Some 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, Daneshmand, et al 2001.

14. Embryo developmental pace;
a closer look
Why do day 5 blastocysts implant more readily than day 6 blastocysts in fresh IVF cycles?
An embryonic phenomenon?
An embryo-endometrium asynchrony effect?

15. Day 5 vs Day 6 Blastocysts
Do the day 5 and day 6 embryos have different implantation rates in FET cycles?
If day 5 and day 6 embryos have similar implantations rates in FET cycles in the absence of ovarian stimulation, shouldn’t they also have similar rates in donor oocyte cycles?

16. Day 5 vs Day 6 Blastocysts in Fresh, FET and Donor oocyte cycles
Retrospective study:
377 fresh autologous cycles
106 autologous FET cycles
56 fresh oocyte donation cycles
Shapiro, Daneshmand,2008

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

18. 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,Daneshmand et al 2008.

19. Day 5 vs Day 6 Blastocysts
The difference in implantation potential between day 5 and day 6 blastocysts in fresh cycles following stimulation vanishes in cycles without ovarian stimulation
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,Daneshmand et al 2008.

20. Degrees of Asynchrony Embryo developmental pace
Day 5 vs day 6 blastulation
Degree of expansion
Premature progesterone elevation
Shapiro,Daneshmand et al 2008.

21. 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,Daneshmand et al 2008

22. 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,Daneshmand et al, 2008, P<0.0001

23. 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, wouldn’t we be able to improve implantation rates if we cryopreserve all embryos in those cycles and transfer them into unstimulated FET cycles?
Also matched day of transfer and use of PGD.
Fresh vs frozen 2pn thaw
Bosch et al 2003, Shapiro,Daneshmand et al 2010.

24. 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,Daneshmand et al 2010.

25. 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,Daneshmand et al 2010.

26. Cryopreservation rescues cycles with “premature luteinization”
Shapiro et al Comparing 236 matched cycles with elevated P4.

27. Can FET in young patients be comparable to fresh donor cycles?
One of the advantages of donor oocyte cycles is the implantation of healthy embryos derived from young donors
The other advantage is the absence of an endometrium exposed to supraphysiolgic hormones of 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,Daneshmand et al 2010.

28. 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,Daneshmand et al 2010.

29. 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,Daneshmand et al 2010.

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

31. 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,Daneshmand et al 2010.

32. Could there be a embryo screening effect in FET cycles?
If we controlled for embryo morphology would there still be a difference between fresh and FET implantation rates
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,Daneshmand et al 2013.

33. 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,Daneshmand et al 2013.

34. 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
Shapiro,Daneshmand et al 2013.

35. What is the impact of reduced endometrial receptivity following ovarian stimulation?
Also matched use of PGD.
Fresh vs frozen 2pn thaw
Shapiro,Daneshmand et al, Comparing 186 cycles matched on maternal age, embryo morphology, and day of blastulation.

36. Is the reduced endometrial receptivity following ovarian stimulation associated with embryo developmental pace?
Also matched use of PGD.
Fresh vs frozen 2pn thaw
Shapiro,Daneshmand et al, Comparing 186 cycles matched on maternal age, embryo morphology, and day of blastulation.

37. 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,Daneshmand et al 2013.

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

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

40. 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,Daneshmand et al 2011.

41. 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,Daneshmand et al 2011.

42. a Study halted for excessive multiple pregnancy rate
Results
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 pregnancy rate (per clin preg) a
73.5%
59.0%
a Study halted for excessive multiple pregnancy rate

43. 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,Daneshmand et al 2011.

44. 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<0.0001

45. 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,Daneshmand et al 2011.

46. Randomized Trial: Fresh vs Frozen in Normal Responders
Results
Randomized Trial: Fresh vs Frozen in Normal Responders
55% clinical pregnancy rate in fresh transfers
84% clinical pregnancy rate in frozen transfers
Statistically significant difference (P=0.0013).
Shapiro,Daneshmand et al 2011.

47. 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.

48. Results

49. Shapiro, Daneshmand et al, 2011,2013
Strategy for avoiding negative effects of COS may be Cohort cryopreservation
Compared to fresh transfer after ovarian stimulation, frozen-thawed embryo transfer has been associated with:
Reduced risk of implantation failure, particularly with slowly developing embryos (e.g. day 6 blastocysts).
Shapiro, Daneshmand et al, 2011,2013

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

51. 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 (Shih et al (2008), Pinborg et al (2010), Pelkonen et al (2010), Nakashima (2012))
Reduced risk of low birthweight (Wang et al (2005), Pelkonen et al (2010), Kalra et al (2011), Maheshwari et al (2012), Nakashima et al (2012))
Reduced risk of small for gestational age (Imudia et al (2012), Pelkonen et al (2010), Maheshwari (2012))
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.

52. Perinatal Risk of Pre-Term Delivery
Fresh versus Frozen
Perinatal Risk of Pre-Term Delivery
Reduced risk of pre-term birth (Wang et al (2005), Pinborg et al (2010), Pelkonen et al (2010), Maheshwari et al (2012), Sullivan et al (2013), Pinborg (2013))
Reduced risk of pre-term low birthweight (Kalra et al (2011))
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. .

53. Fresh versus Frozen Other Perinatal Risks
Reduced risk of antepartum hemorrhage (Healy et al (2009), Maheshwari et al (2012))
Reduced risk of placenta previa (Maheshwari et al (2012))
Reduced risk of placental abruption (Healy et al (2009), Maheshwari et al (2012))
Reduced risk of perinatal mortality (Kalra et al (2011), Maheshwari et al (2012), Sullivan et al 2013))
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.

54. 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.

55. Trends in US National Average Success Rates

56. Trends in US National Average Success Rates

57. Trends in US National Average Success Rates

58. Trends in US National Average Success Rates

59. Trends in US National Average Success Rates

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

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

62. 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.

63. Thank you!