Time- lapse technology in clinical se2ng: outcome results
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1 10 th Croa5an Congress on Gynecological Endocrinology, Human Reproduc5on and Menopause Brijuni, September 10 13, 2015 Time- lapse technology in clinical se2ng: outcome results Nikica Zaninović, MS, PhD, HCLD, ELD Director, Embryology Lab PI, Stem Cell Deriva?on Lab Associate Professor CRM, Weill Cornell Medical College of Cornell University New York, USA N. Zaninovic has no conflict of interest
2 Time- lapse imaging systems EmbryoScope Incubator + microscope, 20X Users annota5ons and algorithms EEVA: in incubator, mathema5cal algorithms, no annota5ons, dark field, 10x Primovision: in incubator, LED light, 10x ESCO: incubator + microscope ASTEC: in incubator, red light, 10x
3 Implemen7ng EmbryoScope at CRM, Weill Cornell November 2011: 1 st E instrument (20% O 2 ) 2012 two E instruments (5% and 20% O 2 ) 2013: 6 E instruments (all 5% O 2 ) March 2013 EmbryoScope incubation for all IVF patients 2014: 8 E instruments 2015: 11 E instruments (server incorporated) Over 4,500 cycles in E Babies born (March 2015): 1, IVF 128 DER 124FET >500 ongoing preg. in 2014/15 pending deliveries
4 Human Embryo Development 2 nd PB ICSI 2 PN Syngamy 2 cell 4 cell 8 cell 16 cell I st 2 nd 3 rd 4 th Morula St. Cav Mor BL- 1 BL- 3
5 Op5mal embryo development: Time- lapse
6 Selec5on of human embryos for ET using Time- lapse (Cornell) Two - step process 1. Embryo de- selec5on Selec5on of embryos with abnormal karyokinesis, cytokinesis or other abnormali5es for exclusion
7 Selec5on of human embryos for ET using Time- lapse (Cornell) Two - step process 1. Embryo de- selec5on Selec5on of embryos with abnormal karyokinesis, cytokinesis or other abnormali5es for exclusion 2. Embryo selec5on Selec5on of embryo(s) with op5mal morphokinetcs
8 Embryo De- Selec5on
9 Abnormali5es of human embryo development iden5fied by Time - lapse: Embryo de- selec5on DUCS: Direct Unequal Cleavages Cell Fusion NUK: Nuclear division without cytoplasmic division I cell MNU (Mul5nucleated 1- cell) Premature compac5on Compac5on of few cells, par5al blastocyst Cell extrusion Blastocyst collapse
10 Direct Unequal Cleavages (DUCS) < 5 hr DUC DUC DUC 3 4-9
11 DUCS: frequency and outcome 5,597/21,368 (26.2%) embryos % DUC- 1 DUC- 2 DUC- 3 Non DUC DUCS Frequency Ducs /total ducs BL cryo D5/6 Impl. (FH) 20.2 No LB with DUC- 1
12 DUCS: Chromosomal Analysis 310 PGS cycles; 202/1,462 DUCS (13.8%) biopsied embryos (D3 or D5 bx) % EUP% ANU% CxA% TotalANU DUC- 1 all DUC- 2 all DUC- 3 all NonDUC
13 Chances of DUCS embryos to be euploid BL (PGS data) DUC- 1 (1-3) DUC- 2 (2-4) DUC- 3 (4-6) Non- DUC Good embryos D3 27% 58% 82% 56% BL D5/6 8.2% 19% 40% 61% Euploid BL 25% 28% 40% 38% Euploid % 1-3% 5% 16% 24%
14 Embryo selec5on using Time- Lapse kine5c parameters
15 Op5mal Embryo Kine5c? t6 t8 t0 t2 t3 t4 t5 t7 t9 tm tsb tb teb ICSI or PNf Hours Individual cleavage 7me points Insufficient for embryo selec5on
16 Op5mal Embryo Kine5c? t6 t8 t0 t2 t3 t4 t5 t7 t9 tm tsb tb teb ICSI or PNf t3- t2 CC2/P2 t5- t4 t8- t5 t4- t3 S2/P3 t5- t3 t8- t5/t4- t3 t3- t2/t5- t2 or other combina5ons t5- t2 Hours Hours Individual 7me points + Synchronized cleavages Enhanced embryo selec5on
17 Embryo Selec5on Cleavages 1 st 2 nd 3 rd 4 th t6 t8 t0 t2 t3 t4 t5 t7 t9 tm tsb tb teb ICSI or PNf Time interval between cleavages t6 t8 t0 t2 t3 t4 t5 t7 t9-16 tm tsb tb teb ICSI or PNf Cleavage stage dura5ons Embryo morphology: fragmenta5on, cell symmetry.. Can blastocyst development be predicted by embryo kine5c by day 3? Can embryo kine5c by day 3 predict embryo viability/implanta5on? Can we develop universal embryo selec5on model(s) based on embryo kine5c?
18 EmbryoScope vs. standard incubator Retrospec7ve analysis 2011 (S) vs (S) vs (E) Based on the live births Excluded: PGS, cryo U/F, CC, split cycles In Press
19 IVF cycles 2011 (S) (S) (E) Number of pa7ents Number of ET cycles Number of oocytes (avg. 12.9) (avg. 10.8) (avg. 13.1) (avg./cycles) Number of MII (Avg.) (Avg. 10.5) (Avg. 8.7) (Avg. 10.9) Number of 2PN (Avg.) 9646 (Avg. 7.8) 7401 (Avg. 6.2) (Avg. 8.3) Number of embryos ET (Avg.) 3907 (Avg. 2.7) 3664 (Avg. 2.6) 3846 (Avg. 2.6) Number of D3- ET Number of D5- ET (%) 136 (9.3%) 91 (6.4%) 290 (19.3%) (few D2, D4 or D6 ET) Number of SET (D5)(%) 20 (14.7%) 22 (24.2%) 70 (24.1%) Number of cycles with BL cryo (%/cycle) 401 (27.5%) 310 (21.2%) 633 (42.1%) Number of BL frozen (avg./cryo cycle) 1011 (Avg. 2.5) 718 (Avg. 2.3) 2113 (Avg. 3.3)
20 Overall clinical outcome data for CP (sac seen), OP (per LB), IMPL (LB per ET) and LB cycles (per ET) between 2011 S, S and E cycles. % /1459* 536/1421** 698/1505* ** 582/1505* ** S 2011 S E /1505* ** /1459* 421/1421** 473/1459* 416/1421** /3846* ** /3907* *** /3664** *** 0 CP/ET OP/ET Impl. LB/#emb.ET LB/ET * E vs. S 2011 P=0.01 ** E vs. S P=0.01 ***S vs. S 2011 P=0.05, X 2
21 Ongoing pregnancies per ET (LB/ET) by maternal age in S 2011, S and E groups. % /483* ** S 2011 S E /418* 142/290* ** /321* 115/ / /327** 54/ /274** 105/325 38/ /268 11/164 15/181 16/227 0 < >42 * E vs. S 2011 P=0.01 ** E vs. S P=0.01, X 2
22 Implanta7on rate (number of LB/ embryos ET) by maternal age in S 2011, S and E groups % /910* ** S 2011 S E /645** /848* 190/684* ** /779* / / /618** 60/766 43/ /882 17/601 51/796 12/565 19/723 < >42 * E vs. S 2011 P=0.01 ** E vs. S P=0.01, X 2
23 Ongoing pregnancies per ET (LB) in ICSI cycles by maternal age: S vs. E groups % / /361 90/189* S E /195* 75/244 72/ /1093* 403/1097* 20 32/216 35/ /195 12/150 0 < >42 total X 2 :, * p<0.01 Implanta5on rate (LB/embryos ET) in ICSI cycles by maternal age: S vs. E groups % /689* S E /485* 122/455* /435* 83/666 90/ /2845* 515/2811* /638 37/547 18/621 14/489 0 < >42 total X 2, * p<0.01
24 Ongoing pregnancies per ET (LB) in inseminated cycles by maternal age: S vs. E groups % /122 44/84 34/79 52/101 30/81 37/92 S /52 19/62 E / / /32 3/31 0 < >42 total All NS Implanta5on rate (LB/embryos ET) in inseminated cycles by maternal age: S vs. E groups % /221 61/160 68/229 40/183 49/254 32/216 S E /1035* 147/819* 23/219 13/158 3/112 1/102 < >42 total X 2, * p=0.02
25 Ongoing pregnancies per ET (LB) in day 3 ET cycles by maternal age: S vs. E groups % / / /237* S E /245* 98/308 89/276 44/262 46/ /1324* 417/1214* 10 16/225 15/177 0 < >42 total * p<0.05 Implanta5on rate (LB/embryos ET) in day 3 ET cycles by maternal age: S vs. E groups % / / /590* S E /567* 106/ / /3493* 536/3347* /785 51/733 19/717 17/588 0 < >42 total * p<0.01
26 Percent of day 5 ET in correla5on with maternal age % /483 S 2011 S E / / /418 43/320 35/ /336 18/ / /327 27/274 6/220 5/164 4/181 91/ /325 4/268 2/227 < >42 total
27 Miscarriage cycle rate by maternal age % 13 S 2011 S E /321 34/336 24/220 26/ /418 24/327 30/483 21/274 24/290 28/325 25/320 22/268 20/227 11/ / / / / < >42 Total All NS between groups
28 % BL cryo cycles E 2013 S 2012 BL cryo D5/6 E vs. S (excluded: CRYO U/F, CC, PGD/S, DER) Overall < >42 Avg. BL frozen E 2013 S Overall < >42
29 SET vs. TET (January to July 2014); fresh vs. frozen; IVF and DER % 100 % DER: 50% D5- ET IVF CP IVF FH DER CP DER FH 80 NS 70 NS NS NS 0 SET fresh SET frozen TET fresh TET frozen N SET TET
30 Implanta5on rate in PGS- FET (n=176) vs. FET (n= 902) cycles PGS FET < >42 < >42 < >42 GS Imp% FH Imp% LB Imp% PGS: day5/6 bx; 24 chr screening
31 Embryology Lab: Director: Supervisors: Embryologists: IVF Andrologists: Acknowledgements Zev Rosenwaks, MD, Medical director Nikica Zaninovic, MS PhD, HCLD, ELD Robert Clarke, PhD, HCLD, ELD Zhen Ye, BS Carol- Ann Cook, MS Rosemary Berrios, MS Richard Bodine, MS Hang Yin, PhD Jiying Hao, MD Sam Jones, BS Qiansheng Zhan, MD, PhD Christopher Norberg, BS Sandra Benedict, MS Pavle Josipovic, PhD June Hariprashad, BA Dina Weiss, MS Ameeta Bahia, BS Laboratory Assistants: Katherine Larkins, BS Rosa Robles, MA Administra7on: Naomi Lourie, BA (manager) Abigail O Connell, BA IVF physicians, nurses and administra7ve staff Special thanks: Joel Goldschlag Jose Tabora- Sierra, PhD (sta7s7c) Jonas Malmsten
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43 Day 0 2 nd PB ICSI 2 PN Syngamy 2 cell 3 cell 4 cell 5 cell 8 cell 9 cell 16 cell Morula St. Cav Mor BL- 1 BL- 2 BL- 3
44 2 nd PB ICSI 2 PN Syngamy Day 1 Day 1 2 cell 3 cell 4 cell 5 cell 8 cell 9 cell 16 cell Morula St. Cav Mor BL- 1 BL- 2 BL- 3
45 2 nd PB ICSI 2 PN Syngamy 2 cell 3 cell 4 cell 5 cell Day 2 8 cell 9 cell 16 cell Morula St. Cav Mor BL- 1 BL- 2 BL- 3
46 2 nd PB ICSI 2 PN Syngamy 2 cell 3 cell 4 cell 5 cell 8 cell 9 cell 16 cell Morula Day 3 St. Cav Mor BL- 1 BL- 2 BL- 3
47 2 nd PB ICSI 2 PN Syngamy 2 cell 3 cell 4 cell 5 cell 8 cell 9 cell 16 cell Morula Day 4 Day 4 St. Cav Mor BL- 1 BL- 2 BL- 3
48 2 nd PB ICSI 2 PN Syngamy 2 cell 3 cell 4 cell 5 cell 8 cell 9 cell 16 cell Morula St. Cav Mor BL- 1 BL- 2 BL- 3 Day 5
49 Time- line of human embryo development in vitro (from Veeck and Zaninovic, An atlas of human blastocyst, 2003) 42h 66h 72h 90h 100h 114h 24h - 26h 120h 144h 0h D0 D1 D2 D3 D4 D5 D6
50 Conven7onal annota7on vs. 7me- lapse Cell stages at fixed 5me Conven5onal D1 9AM D1 3PM D2 10AM D3 9AM D4 9AM D5 9AM 3D hr 2PN hr hr hr hr hr Cell stages when occur, exact 5ming Time- lapse D1 9AM T2 T3 T4 T5 T6 T7 T8 Comp. Cav. Blast. Kinetics 4D hr 2PN ??????
51 EmbryoScope vs. standard incubator Cornell valida5on
52 Study design 3E E S (standard) 5.8 % CO % O 2 37 º C 5.8 % CO % O 2 37 º C 5.8 % CO % O 2 37 º C 3E vs. E (Study 2) E vs. S (Study 1) 3E vs. E vs. S 3E vs. S (study 3) Culture medium: In house sequen5al medium; ph: , in incub ; Osm: mosm
53 Study 1. EmbryoScope (20% O 2 ) vs. S (20% O 2 ) on sibling oocytes; n=77 pa7ents E S P No. 2PN Day2 cell number (Avg. + SD) NS Day 2 fragmenta5on (Avg. + SD) NS Day 3 cell number (Avg. + SD) Day 3 fragmenta5on (Avg. + SD) NS > 4- cells day 2 (48 hours) 307/491 (62.5%) 271/466 (58.2%) NS > 8- cells day 3 (66 hours) 206/491 (42.0%) 166/466 (35.6%) No. embryos ET (%) n=163* 93/163 (57.1%) 70/163 (42.9%) < 0.05 D3- ET 67/93 (72.0%) 60/70 (85.7%) D5- ET 26/93 (28.0%) 10/70 (14.3%) No. embryos frozen (D5/6) (%) n=186* 104/186 (56.0%) 82/186 (44.0%) < 0.05 Embryos used (ET + frozen)* 197/491 (40.1%) 152/466 (32.6%) < 0.05 * 74 pt. with ET, 3 pt. all cryo (not included) Paitred t- test for avg. X 2 for propor5ons
54 Study 2. EmbryoScope (5% O 2, 3E) vs. EmbryoScope (20% O 2, E) on sibling oocytes; n=28 pa7ents 3E E P No. 2PN Day2 cell number (Avg. + SD) NS Day 2 fragmenta5on (Avg. + SD) Day 3 cell number (Avg. + SD) Day 3 fragmenta5on (Avg. + SD) NS > 4- cells day 2 (48 hours)* 75/154 (48.7%) 65/142 (45.8%) NS > 8- cells day 3 (66 hours)* 65/154 (42.2%) 54/142 (38.1%) NS No. embryos ET (%) n=78 44/78 (56.4%) 34/78 (43.6%) < 0.05 D3- ET 34/44 (77.3%) 30/34 (88.2%) D5- ET 10/44 (22.7%) 4/34 (11.8%) No. embryos frozen (D5/6) (%) n=60 37/60 (61.7%) 23/60 (38.3%) < 0.05 Embryos used (ET + frozen) 78/154 (50.6%) 57/142 (40.1%) < 0.05 DUCS 28/154 (18.2%) 23/142 (16.2%) NS * DUCS excluded Paitred t- test for avg. X 2 for propor5ons
55 Each dot represent the mean 3E E 3E E 3E E 3E E 3E E 3E E 3E E T2 T3 T4 T5 T6 T7 T8 7me Incub N Minimum Maximum Mean Std Dev Lower Quar7le Median Upper Quar7le p- value (from Wilcoxon to test Median) 2 3E E E E E E E E E <.0001 E E <.0001 E E <.0001 E
56 Study 3. EmbryoScope (5% O 2, 3E) vs. S (20% O 2 ) on sibling oocytes; n=35 pa7ents 3E S P No. 2PN Day2 cell number (Avg. + SD) NS Day 2 fragmenta5on (Avg. + SD) NS Day 3 cell number (Avg. + SD) Day 3 fragmenta5on (Avg. + SD) NS > 4- cells day 2 (48 hours) 165/242 (68.2%) 130/208 (62.5%) NS > 8- cells day 3 (66 hours) 137/242 (56.6%) 83/208 (40.0%) No. embryos ET (%) n=94 64/94 (68.0%) 30/94 (31.9%) < 0.05 D3- ET 47/64 (73.4%) 21/30 (70.0%) D5- ET 17/64 (26.6%) 9/30 (30.0%) No. embryos frozen (D5/6) (%) n=95 55/95 (57.9%) 40/95 (42.1%) < 0.05 Embryos used (ET + frozen) 119/242 (49.2%) 70/208 (33.7%) < 0.05 Paitred t- test for avg. X 2 for propor5ons
57 ICSI day 0 (DO) vs. ICSI day 1 (D1) on sibling oocytes; n=77 pa7ents DO D1 P No. 2PN Day2 cell number (Avg. + SD) Day 2 fragmenta5on (Avg. + SD) NS Day 3 cell number (Avg. + SD) Day 3 fragmenta5on (Avg. + SD) NS > 4- cells day 2 (48 hours)* 200/330 (60.6%) 145/281 (51.6) > 8- cells day 3 (66 hours)* 171/330 (51.8%) 108/281 (38.4%) < 0.05 No. embryos ET (%) n= /161 (62.1%) 61/161 (37.9%) < 0.05 D3- ET 70/100 (70%) 41/61 (67.2%) D5- ET 30/100 (30%) 20/61 (32.8%) No. embryos frozen (D5/6) (%) n= /162 (62.3%) 61/162 (37.7%) < 0.05 Embryos used (ET + frozen) 201/330 (60.1%) 122/281 (43.4%) < 0.05 DUCS 74/330 (22.4%) 72/281 (25.6%) NS * DUCS excluded Paitred t- test for avg. X 2 for propor5ons
58 1=DO; 2=D1 Wilcoxon Signed Ranked test ICSI D0 vs. D1: 77 pt. t- 5mes
59 % ICSI 2PN Fer5liza5on rate: 898 emb. in E (2013) vs. 900 emb. in S (2012) cycles Retrospec5ve analysis, avg. MII=NS E S * * * * * * 50 overall < >42 X 2 * p<0.05
60 1 emb ET KID (Known Implanta5on Data) KID + 2 emb ET KID -? KID?
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