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One and done: how many oocytes are needed to complete a family with one stimulation cycle?
P-76 IMPACT OF INCUBATION TEMPERATURE ON MOUSE EMBRYO DEVELOPMENT. A. Youssef,a,b M. Kandil,b A. Makhlouf,b D. Habib,b S. Mesiano,a J. Liu,a A. Ahmady.c aDepartments of Reproductive Biology, Case Western Reserve University, Department of Obstetrics and Gynecology, University Hospitals of Cleveland, Cleveland, Ohio, USA; bDepartment of Obstetrics and Gynecology, Women Health Hospital, Assiut University, Assiut, Egypt; cDepartment of Obstetrics & Gynecology, University of Southern California, Los Angeles, California. BACKGROUND: Incubation temperature is a critical factor affecting the development of the preimplantation embryo in in vitro culture. Temperature can influence media pH. Consequently, effects of incubation temperature on embryo development are confounded by effects on media pH. To address this problem we examined the effect of temperature on mouse embryo development at a fixed pH. OBJECTIVE: The purpose of this study was to determine the optimum temperature for mouse embryo development. MATERIALS AND METHODS: Cryopreserved one cell mouse embryos (n¼484) were thawed in G-MOPS PLUS (Vitrolife) media under oil for tissue culture (SAGE) then cultured for 5 days at pH 7.3 in media at temperature 36C (n¼100), 37C (n¼100), 37.5C (n¼100), 38C (n¼100) and 39C (n¼84) in a CO2 incubator. Embryos were cultured in groups of 5 to 10 in 50uL drops of medium under paraffin oil (Vitrolife). Incubator temperature and media pH were measured daily and the CO2 level was adjusted to maintain the pH within the assigned range ( 0.1). Embryo development was monitored daily and scored morphologically for five days. Our primary endpoints were rates of blastocyst and hatching embryos. Chi-Square test was used for comparison between the 5 groups and P value ˂ 0.05 was considered significant. RESULTS:
Day 5 Hatching Blastocyst Morula Compacting Morula 10 Cells 8 Cells 2 Cells Degeneration
37 c 37.5 c 38 c 39 c 36 c (n¼100) (n¼100) (n¼100) (n¼100) (n¼84) 22 46 20 1 2 2 7
51 42 1 2 2 2
76 16 4 1 3
69 20 5 1 5
14 18 8 44
The number of hatching embryos at temperature 37.5C was significantly higher than other temperatures (P < 0.001).The total number of hatching and blastocyst together at temperature 37C (n¼93) was significantly higher than at other temperatures (P < 0.001) but no significant difference between the highest value at degree 37C (n¼93) and the value at degree 37.5C (n¼92). A significantly increase number of embryos degenerated at temperature 39C (n¼44) (P < 0.01). CONCLUSIONS: Culture of mouse embryos at 37.5C (at pH 7.3) is optimal for embryo development to the hatching alone and post-hatching to the blastocyst stage. Media temperature 39C increased the number degenerating embryos and is not optimal for mouse embryo development. FINANCIAL SUPPORT: None. P-77 EVALUATING CHANGE IN DAY OF EMBRYO TRANSFER OVER TIME IN A RETROSPECTIVE DATABASE STUDY OF 60,253 TRANSFERS OVER 5 YEARS. K. S. Richter,a G. L. Mottla,b B. Hayward,c M. C. Mahony.c aShady Grove Fertility Center, Rockville, MD, USA; bShady Grove Fertility Center, Annapolis, MD, USA; cEMD Serono, Inc., Rockland, MA, USA. BACKGROUND: Outcomes of in vitro fertilization (IVF) are dependent not only on patient characteristics but also on both clinical strategy and laboratory processes, the latter including the day of transfer decision.
FERTILITY & STERILITYÒ
OBJECTIVE: To evaluate the change in day of embryo transfer over time in IVF cycles by women in the US. MATERIALS AND METHODS: Non-randomized, observational, retrospective cohort analysis of all autologous IVF cycles with fresh embryo transfer between July 2009 and June 2014 within a large US clinical dataset including patients from all 50 states. RESULTS: There were 24,046 Day 3 transfers and 36,207 Day 5/6 transfers. Female age at the time of treatment was significantly younger for Day 5/6 transfers compared to Day 3 transfers (34.8 versus 36.4 years). Patients having Day 5/6 transfers had higher antral follicle counts (16.1 versus 11.6), more oocytes retrieved (17.5 versus 9.6) and more embryos available for transfer or cryopreservation (4.3 versus 2.7) than patients getting Day 3 transfers. Day 5/6 transfers were more likely to be associated with a diagnosis of ovulation disorders (14.0% versus 8.5%) and less likely to be associated with diminished ovarian reserve (17.0% versus 21.5%) (p<0.0001 for all comparisons, Student’s t-test or chi-square as appropriate). The percentages of Day 3 versus Day 5/6 transfers shifted gradually and steadily over the 5 years of the study, from 49.7% Day 3 and 50.3% Day 5/ 6 in the third quarter of 2009, to 27.9% Day 3 and 72.1% Day 5/6 in the second quarter of 2014 (Figure 1, p<0.0001, Cochran-Armitage Trend test). CONCLUSIONS: From 2009 through 2014 there was a significant shift away from Day 3 transfer in favor of Day 5/6 transfer, likely due to multiple related factors. During this time, improvements in commercially available media and greater experience have increased confidence in laboratories’ abilities to culture embryos to the blastocyst stage. Blastocyst transfers are associated with higher implantation rates because of an improved ability to select the most viable embryos, and this has allowed greater emphasis on single embryo transfer in an effort to reduce multiple pregnancy risks. Factors including age, diagnoses, and ovarian reserve that are associated with larger cohorts of oocytes and embryos also increase the likelihood of Day 5/6 transfer. Further analyses of the impact of diagnosis, age, and other factors on transfer day will be presented. SUPPORT: Study supported by EMD Serono, Inc., Rockland, MA, USA (a business of Merck KGaA, Darmstadt, Germany).
P-78 ONE AND DONE: HOW MANY OOCYTES ARE NEEDED TO COMPLETE A FAMILY WITH ONE STIMULATION CYCLE? D. A. Vaughan,a A. Leung,a N. Resetkova,b,c A. Penzias,b,c D. Sakkas,b M. Alper.b aTufts Medical Center, Boston, MA; bBoston IVF, Boston, MA; cBeth Israel Deaconess Medical Center, Boston, MA. BACKGROUND: Data are conflicting regarding the optimum number of oocytes needed during an IVF cycle. With more aggressive stimulation, there are concerns regarding ovarian hyperstimulation syndrome (OHSS), with oocyte number known to directly correlate with risk1. In addition, some have suggested that controlled ovarian hyperstimulation leads to reduced implantation rates, possibly related to diminished uterine receptivity2. However, one needs to take into account risks/issues associated with each fresh stimulation cycle: surgical/anesthesia risks, cost and, not least, dropout rates and emotional stress. OBJECTIVE: We sought to ascertain the optimum number of oocytes retrieved in a single IVF stimulation cycle in order to complete a family (achieve R 2 live births). STUDY DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Using a validated database from a single, large, university-affiliated IVF center, we reviewed all homologous cycles with oocyte retrieval from January 1st 2012-December 31st 2013. To achieve homogeneous sampling, we included only patients % 38 years old, undergoing their first IVF cycle. We compared patients in whom moderate numbers of oocytes (10-14) were retrieved, with those with high oocyte yield (R 15). Our primary outcomes were cumulative live birth rate (LBR, both fresh/frozen cycles) and rate of family completion, as previously defined. RESULTS: During our study period, with patients meeting our inclusion criteria, we had 398 cycles in which R15 oocytes were retrieved and 292 cycles in which 10-14 oocytes were retrieved (Table 1). In total, 262/398 (66%) and 138/292 (47%) achieved at least one live birth. Amongst our cohort, 77/ 398 (19%) and 33/292 (11%) achieved R 2 live births. Of note, 116/398
e45
TABLE 1. Cohort characteristics.
Retrievals from 2012-2013 % 38 years old, 1st Cycle Fresh ET cases Frozen ET cases Retrievals leading to a Live Birth Retrievals leading to R 2 Live Birth Patients with R4 frozen embryos remaining Expected no. patients to achieve a 2nd child from their remaining frozen embryos Total Retrievals expected for Live Birth Total Retrievals expected for R 2 Live Births
R 15 Oocytes n (%)
10-14 Oocytes n (%)
398
292
345 (87%) 261 (66%) 262 (66%) 77 (19%) 116 (29%) 62 (53%)
230 (79%) 145 (27%) 138 (47%) 33 (11%) 37 (13%) 14 (38%)
0.007 0.001 0.001 0.004 0.001 0.001
284 (71%) 139 (35%)
151 (52%) 47 (16%)
0.001 0.001
(29%) and 37/292 (13%) still have R4 frozen embryos at the time of this study. Applying our frozen embryo transfer LBR from our cohort, we would expect at least 62/116 (53%) and 14/37 (38%) patients would have R1 further live birth. Thus, overall, we would expect 139 patients (35%) in whom R15 oocytes were retrieved to achieve R2 live births (thereby completing their family), compared with 47 patients (16%) in the 10-14 oocyte group (p¼0.001). CONCLUSION: There is no clear evidence that oocyte quality decreases with high embryo yield. Ultimately, more oocytes retrieved result in more embryos frozen and a higher cumulative LBR. With GnRH agonist trigger, coasting and/or freeze all cycles the risk of OHSS is almost eliminated. We believe the overall risks to the patient to be lower by reducing the need for fewer stimulated cycles. FINANCIAL SUPPORT: No direct financial support for this study. Other disclosures listed below. Dr. Alan Penzias: OvaScience Advisory Board, Reprosource Board of Directors, Recipient of Ferring Honoraria Dr. Michael Alper: Consultant for Ferring, EMD Serono, Reprosource and Good Start Genetics Dr. Denny Sakkas: Origio Board Advisor, Recipient of a grant from Ferring References: 1. Delvigne A, Vandromme J, Barlow P, Lejeune B, Leroy F. Are there predictive criteria of complicated ovarian hyperstimulation in IVF? Hum Reprod 1991 6:959–62.
e46
PCRS Abstracts
p (Fishers exact)
2. Simon C, Cano F, Valbuena D, Remohı J, Pellicer A. Clinical evidence for a detrimental effect on uterine receptivity of high serum estradiol levels in high and normal responder patients. Hum Reprod 1995;10: 2432–4.
Figure 1. Change in proportion of cycles with Day 3 versus Day 5/6 transfer between 2009 and 2014 (p<0.0001, Cochran-Armitage Trend test).
Vol. 105, No. 2, Supplement, February 2016
Day 5 Hatching Blastocyst Morula Compacting Morula 10 Cells 8 Cells 2 Cells Degeneration
37 c 37.5 c 38 c 39 c 36 c (n¼100) (n¼100) (n¼100) (n¼100) (n¼84) 22 46 20 1 2 2 7
51 42 1 2 2 2
76 16 4 1 3
69 20 5 1 5
14 18 8 44
The number of hatching embryos at temperature 37.5C was significantly higher than other temperatures (P < 0.001).The total number of hatching and blastocyst together at temperature 37C (n¼93) was significantly higher than at other temperatures (P < 0.001) but no significant difference between the highest value at degree 37C (n¼93) and the value at degree 37.5C (n¼92). A significantly increase number of embryos degenerated at temperature 39C (n¼44) (P < 0.01). CONCLUSIONS: Culture of mouse embryos at 37.5C (at pH 7.3) is optimal for embryo development to the hatching alone and post-hatching to the blastocyst stage. Media temperature 39C increased the number degenerating embryos and is not optimal for mouse embryo development. FINANCIAL SUPPORT: None. P-77 EVALUATING CHANGE IN DAY OF EMBRYO TRANSFER OVER TIME IN A RETROSPECTIVE DATABASE STUDY OF 60,253 TRANSFERS OVER 5 YEARS. K. S. Richter,a G. L. Mottla,b B. Hayward,c M. C. Mahony.c aShady Grove Fertility Center, Rockville, MD, USA; bShady Grove Fertility Center, Annapolis, MD, USA; cEMD Serono, Inc., Rockland, MA, USA. BACKGROUND: Outcomes of in vitro fertilization (IVF) are dependent not only on patient characteristics but also on both clinical strategy and laboratory processes, the latter including the day of transfer decision.
FERTILITY & STERILITYÒ
OBJECTIVE: To evaluate the change in day of embryo transfer over time in IVF cycles by women in the US. MATERIALS AND METHODS: Non-randomized, observational, retrospective cohort analysis of all autologous IVF cycles with fresh embryo transfer between July 2009 and June 2014 within a large US clinical dataset including patients from all 50 states. RESULTS: There were 24,046 Day 3 transfers and 36,207 Day 5/6 transfers. Female age at the time of treatment was significantly younger for Day 5/6 transfers compared to Day 3 transfers (34.8 versus 36.4 years). Patients having Day 5/6 transfers had higher antral follicle counts (16.1 versus 11.6), more oocytes retrieved (17.5 versus 9.6) and more embryos available for transfer or cryopreservation (4.3 versus 2.7) than patients getting Day 3 transfers. Day 5/6 transfers were more likely to be associated with a diagnosis of ovulation disorders (14.0% versus 8.5%) and less likely to be associated with diminished ovarian reserve (17.0% versus 21.5%) (p<0.0001 for all comparisons, Student’s t-test or chi-square as appropriate). The percentages of Day 3 versus Day 5/6 transfers shifted gradually and steadily over the 5 years of the study, from 49.7% Day 3 and 50.3% Day 5/ 6 in the third quarter of 2009, to 27.9% Day 3 and 72.1% Day 5/6 in the second quarter of 2014 (Figure 1, p<0.0001, Cochran-Armitage Trend test). CONCLUSIONS: From 2009 through 2014 there was a significant shift away from Day 3 transfer in favor of Day 5/6 transfer, likely due to multiple related factors. During this time, improvements in commercially available media and greater experience have increased confidence in laboratories’ abilities to culture embryos to the blastocyst stage. Blastocyst transfers are associated with higher implantation rates because of an improved ability to select the most viable embryos, and this has allowed greater emphasis on single embryo transfer in an effort to reduce multiple pregnancy risks. Factors including age, diagnoses, and ovarian reserve that are associated with larger cohorts of oocytes and embryos also increase the likelihood of Day 5/6 transfer. Further analyses of the impact of diagnosis, age, and other factors on transfer day will be presented. SUPPORT: Study supported by EMD Serono, Inc., Rockland, MA, USA (a business of Merck KGaA, Darmstadt, Germany).
P-78 ONE AND DONE: HOW MANY OOCYTES ARE NEEDED TO COMPLETE A FAMILY WITH ONE STIMULATION CYCLE? D. A. Vaughan,a A. Leung,a N. Resetkova,b,c A. Penzias,b,c D. Sakkas,b M. Alper.b aTufts Medical Center, Boston, MA; bBoston IVF, Boston, MA; cBeth Israel Deaconess Medical Center, Boston, MA. BACKGROUND: Data are conflicting regarding the optimum number of oocytes needed during an IVF cycle. With more aggressive stimulation, there are concerns regarding ovarian hyperstimulation syndrome (OHSS), with oocyte number known to directly correlate with risk1. In addition, some have suggested that controlled ovarian hyperstimulation leads to reduced implantation rates, possibly related to diminished uterine receptivity2. However, one needs to take into account risks/issues associated with each fresh stimulation cycle: surgical/anesthesia risks, cost and, not least, dropout rates and emotional stress. OBJECTIVE: We sought to ascertain the optimum number of oocytes retrieved in a single IVF stimulation cycle in order to complete a family (achieve R 2 live births). STUDY DESIGN: Retrospective cohort study. MATERIALS AND METHODS: Using a validated database from a single, large, university-affiliated IVF center, we reviewed all homologous cycles with oocyte retrieval from January 1st 2012-December 31st 2013. To achieve homogeneous sampling, we included only patients % 38 years old, undergoing their first IVF cycle. We compared patients in whom moderate numbers of oocytes (10-14) were retrieved, with those with high oocyte yield (R 15). Our primary outcomes were cumulative live birth rate (LBR, both fresh/frozen cycles) and rate of family completion, as previously defined. RESULTS: During our study period, with patients meeting our inclusion criteria, we had 398 cycles in which R15 oocytes were retrieved and 292 cycles in which 10-14 oocytes were retrieved (Table 1). In total, 262/398 (66%) and 138/292 (47%) achieved at least one live birth. Amongst our cohort, 77/ 398 (19%) and 33/292 (11%) achieved R 2 live births. Of note, 116/398
e45
TABLE 1. Cohort characteristics.
Retrievals from 2012-2013 % 38 years old, 1st Cycle Fresh ET cases Frozen ET cases Retrievals leading to a Live Birth Retrievals leading to R 2 Live Birth Patients with R4 frozen embryos remaining Expected no. patients to achieve a 2nd child from their remaining frozen embryos Total Retrievals expected for Live Birth Total Retrievals expected for R 2 Live Births
R 15 Oocytes n (%)
10-14 Oocytes n (%)
398
292
345 (87%) 261 (66%) 262 (66%) 77 (19%) 116 (29%) 62 (53%)
230 (79%) 145 (27%) 138 (47%) 33 (11%) 37 (13%) 14 (38%)
0.007 0.001 0.001 0.004 0.001 0.001
284 (71%) 139 (35%)
151 (52%) 47 (16%)
0.001 0.001
(29%) and 37/292 (13%) still have R4 frozen embryos at the time of this study. Applying our frozen embryo transfer LBR from our cohort, we would expect at least 62/116 (53%) and 14/37 (38%) patients would have R1 further live birth. Thus, overall, we would expect 139 patients (35%) in whom R15 oocytes were retrieved to achieve R2 live births (thereby completing their family), compared with 47 patients (16%) in the 10-14 oocyte group (p¼0.001). CONCLUSION: There is no clear evidence that oocyte quality decreases with high embryo yield. Ultimately, more oocytes retrieved result in more embryos frozen and a higher cumulative LBR. With GnRH agonist trigger, coasting and/or freeze all cycles the risk of OHSS is almost eliminated. We believe the overall risks to the patient to be lower by reducing the need for fewer stimulated cycles. FINANCIAL SUPPORT: No direct financial support for this study. Other disclosures listed below. Dr. Alan Penzias: OvaScience Advisory Board, Reprosource Board of Directors, Recipient of Ferring Honoraria Dr. Michael Alper: Consultant for Ferring, EMD Serono, Reprosource and Good Start Genetics Dr. Denny Sakkas: Origio Board Advisor, Recipient of a grant from Ferring References: 1. Delvigne A, Vandromme J, Barlow P, Lejeune B, Leroy F. Are there predictive criteria of complicated ovarian hyperstimulation in IVF? Hum Reprod 1991 6:959–62.
e46
PCRS Abstracts
p (Fishers exact)
2. Simon C, Cano F, Valbuena D, Remohı J, Pellicer A. Clinical evidence for a detrimental effect on uterine receptivity of high serum estradiol levels in high and normal responder patients. Hum Reprod 1995;10: 2432–4.
Figure 1. Change in proportion of cycles with Day 3 versus Day 5/6 transfer between 2009 and 2014 (p<0.0001, Cochran-Armitage Trend test).
Vol. 105, No. 2, Supplement, February 2016