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

Disclosures Research grants: Watson Merck & Co.

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.

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

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

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?

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.

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 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 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.

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?

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?

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

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

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 2006. Shapiro,Daneshmand et al 2008.

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

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

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, 2008,2013 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, 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.

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.

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.

Cryopreservation rescues cycles with “premature luteinization” Shapiro et al 2010. Comparing 236 matched cycles with elevated P4.

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.

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.

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.

How does FET in young patients compare to fresh donor cycles using young donors? Shapiro,Daneshmand et al 2010. Comparing 205 PTEC and donor cycles, egg sources <35 years of age, double blastocyst transfer.

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.

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.

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.

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 Also matched day of transfer and use of PGD. Fresh vs frozen 2pn thaw Shapiro,Daneshmand et al 2013.

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, 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? Also matched use of PGD. Fresh vs frozen 2pn thaw Shapiro,Daneshmand 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. Also matched day of transfer and use of PGD. Fresh vs frozen 2pn thaw Shapiro,Daneshmand et al 2013.

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

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

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,Daneshmand et al 2011.

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.

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

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.

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

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,Daneshmand et al 2011.

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.

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.

Results

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

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, 1998. Ishihara et al, 2011. Shapiro,Daneshmand et al, 2012.) Reduced risk of pre-eclampsia (Imudia et al (2012), Maheshwari et al (2012))

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.

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. .

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.

Trends in Fresh and FET outcomes SART registry 2006-2011 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.

Trends in US National Average Success Rates

Trends in US National Average Success Rates

Trends in US National Average Success Rates

Trends in US National Average Success Rates

Trends in US National Average Success Rates

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

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!