- Email: [email protected]
Oocyte dysmorphism is not a limiting factor for post-thaw survival following vitrification by cryotop method
TABLE 1. DNA integrity indices in day 5 and day 6 blastocysts in the two protocols Day 5 Blastocysts (n ¼ 32) Fresh (n¼14)
Short equilibration (n¼17)
Prolonged equilibration (n¼15)
84.36 8.76
95.59 4.12
DNA Integrity 93.94 6.64 Index %
Day 6 Blastocysts (n ¼ 35) Fresh (n¼13)
Short equilibration (n¼22)
DNA Integrity 95.47 4.25 77.61 16.65 Index %
Prolonged equilibration (n¼13) 72.22 11.44
CONCLUSIONS: Prolonged equilibration-vitrification protocol improves only day 5 blastocyst vitrification. Supported by: None.
P-522 APOPTOSIS IS NOT INDUCED BY SLOW CRYOPRESERVATION OR VITRIFICATION IN BIOPSED CLEAVAGE STAGE EMBRYOS. A. Kader, G. Mansour, A. Agarwal, R. Sharma, T. Falcone. Cleveland Clinic, Cleveland, OH. OBJECTIVE: Cleavage stage embryo biopsy makes embryos more vulnerable to development failure. Cryopreserved biopsied embryos are at a higher risk for developmental or implantation failure. The aim of our study was to 1) examine if apoptosis is induced in cleavage stage embryos after biopsy and 2) evaluate the extent of apoptosis following vitrification or slow cryopreservation of biopsied cleavage stage embryos. DESIGN: In Vitro, prospective. MATERIALS AND METHODS: 44 cleavage stage I embryos were biopsied and 1-2 blastomeres were removed by the aid of micromanipulator. After biopsy, 13 embryos were vitrified, 16 embryos were slowly cryopreserved and 15 embryos were immediately fixed in 4% formalin. Another group of 44 intact cleavage stage embryos were used. Of these 13 were used as fresh controls, 14 were vitrified and 17 were subjected to slow cryopreservation. Slow cryopreservation was done using Irvine propanediol based media. Vitrification was carried out using the Irvine vitrification media and cryotip loading devices. After thawing, all embryos were incubated in 10% albumin enriched HTF media for 4 h at 37 C with 5% CO2 and fixed. Embryos were stained for DNA damage using TUNEL assay. Blastomeres were mounted in Vectashield containing DAPI for counterstaining and examined by confocal microscopy. DNA integrity index in each embryo was calculated as percentage of TUNEL–ve to total number of blastomeres. RESULTS: DNA integrity index was comparable in fresh and biopsied groups. No further increase in apoptosis was observed following slow cryopreservation or vitrification. TABLE 1. Mean SD of DNA integrity index in intact and biopsied embryos
Intact Embryos (n ¼ 44) Parameter
Fresh (n ¼ 13)
Slow cryopreservation (n ¼ 17)
DNA Integrity Index (%)
100.00 0.00
96.17 5.55
Vitrification (n ¼ 14)
CONCLUSIONS: Embryo biopsy does not induce apoptosis in cleavage stage embryos. Both slow cryopreservation and vitrification do not induce apoptosis in biopsied cleavage stage embryos. Supported by: None.
P-523 OOCYTE DYSMORPHISM IS NOT A LIMITING FACTOR FOR POST-THAW SURVIVAL FOLLOWING VITRIFICATION BY CRYOTOP METHOD. I. Maldonaldo, A. Bermudez, A. Varghese, J. Gaytan, R. Sharma, A. Agarwal. Inmater S. C., Col; Lomas de Chapultepec, Mexico City, Mexico; Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH. OBJECTIVE: Oocyte and embryo morphology significantly affect the survival rates after slow freezing. Oocyte dysmorphism is a major concern for a majority of the embryologists although vitrification methods with oocyte survival rates >90% have been reported. The aim of our study aim was to examine if oocyte dysmorphism after denudation significantly affects survival rates following vitrification by cryotop method. DESIGN: Prospective study. MATERIALS AND METHODS: 336 oocytes obtained from 139 patients (<36 y) were individually vitrified and thawed using cryotop method. All of these oocytes had failed to fertilize after IVF-ICSI. Oocytes were divided in six groups based on the type of dysmorphisms: Group A ¼ vacuolated cytoplasm (n ¼ 60); Group B ¼ smooth endoplasmic reticulum cluster (n ¼ 52); Group C ¼ large perivitelline space (n ¼ 50), Group D ¼ granulated cytoplasm (n ¼ 60); Group E ¼ amorphous cytoplasm (n ¼ 51) and Group F ¼ normal morphology (n ¼ 60; controls). All the oocytes were equilibrated in an equilibration solution (ethylene glycol, 7.5% and 1, 2 propanediol; 7.5%) for 15 min and transferred into vitrification solution (VS; 15% ethylene glycol and 15% 1, 2 propanediol) for 1 min. They were immediately placed on the cryotop with a minimum volume of VS solution and submerged in LN2. The vitrified oocytes were thawed at 37 C for 1 minute using sucrose solution (1 Molar) followed by dilution solution (DS; 0.5 M of sucrose) for 3 minutes and washed X2 in wash solution (M-199 with 20% synthetic serum substitute) for 5 min each. They were examined for post thaw survival based on the dysmorphism. RESULTS: The post-thaw survival rate was comparable in all the groups. TABLE 1. Comparison of survival rates in different groups following minimal volume vitrification
Group A B C D E F
A
B
C
D
E
F
0.98 0.91 0.94 0.98 0.94
0.93 0.96 0.99 0.96
0.96 0.93 0.96
-
0.96
-
0.96 1.0
No statistical differences were observed among different groups by chisquare analysis. CONCLUSIONS: Vitrification by cryotop technology using minimal volume approach increases both cooling and warming rates. The rapid transfer of heat helps maintain the oocyte cytoarchitecture irrespective of the morphology unlike slow freezing. Oocyte morphology may not be a limiting factor in predicting the survival following minimal volume vitrification by cryotop method. Supported by: None.
100.00 0.00 P-524
Biopsied Embryos (n ¼ 44) Parameter
Fresh (n ¼ 15)
Slow cryopreservation (n ¼ 16)
Vitrification (n ¼ 13)
DNA Integrity Index (%)
100.00 0.00
99.11 3.57
98.29 6.16
FERTILITY & STERILITYÒ
SUCROSE IN THE FINAL EQUILIBRATION SOLUTION INTERFERES WITH THE EMBRYONIC DEVELOPMENTAL POTENTIAL OF VITRIFIED MOUSE METAPHASE II OOCYTES. H. Liu, L. C. Krey, J. A. Grifo. NYU Fertility Center, New York University School of Medicine, New York; New York University School of Medicine, New York. OBJECTIVE: Cryoprotectant concentrations, temperature and equilibration times are important factors that determine a successful cryopreservation
S283
Short equilibration (n¼17)
Prolonged equilibration (n¼15)
84.36 8.76
95.59 4.12
DNA Integrity 93.94 6.64 Index %
Day 6 Blastocysts (n ¼ 35) Fresh (n¼13)
Short equilibration (n¼22)
DNA Integrity 95.47 4.25 77.61 16.65 Index %
Prolonged equilibration (n¼13) 72.22 11.44
CONCLUSIONS: Prolonged equilibration-vitrification protocol improves only day 5 blastocyst vitrification. Supported by: None.
P-522 APOPTOSIS IS NOT INDUCED BY SLOW CRYOPRESERVATION OR VITRIFICATION IN BIOPSED CLEAVAGE STAGE EMBRYOS. A. Kader, G. Mansour, A. Agarwal, R. Sharma, T. Falcone. Cleveland Clinic, Cleveland, OH. OBJECTIVE: Cleavage stage embryo biopsy makes embryos more vulnerable to development failure. Cryopreserved biopsied embryos are at a higher risk for developmental or implantation failure. The aim of our study was to 1) examine if apoptosis is induced in cleavage stage embryos after biopsy and 2) evaluate the extent of apoptosis following vitrification or slow cryopreservation of biopsied cleavage stage embryos. DESIGN: In Vitro, prospective. MATERIALS AND METHODS: 44 cleavage stage I embryos were biopsied and 1-2 blastomeres were removed by the aid of micromanipulator. After biopsy, 13 embryos were vitrified, 16 embryos were slowly cryopreserved and 15 embryos were immediately fixed in 4% formalin. Another group of 44 intact cleavage stage embryos were used. Of these 13 were used as fresh controls, 14 were vitrified and 17 were subjected to slow cryopreservation. Slow cryopreservation was done using Irvine propanediol based media. Vitrification was carried out using the Irvine vitrification media and cryotip loading devices. After thawing, all embryos were incubated in 10% albumin enriched HTF media for 4 h at 37 C with 5% CO2 and fixed. Embryos were stained for DNA damage using TUNEL assay. Blastomeres were mounted in Vectashield containing DAPI for counterstaining and examined by confocal microscopy. DNA integrity index in each embryo was calculated as percentage of TUNEL–ve to total number of blastomeres. RESULTS: DNA integrity index was comparable in fresh and biopsied groups. No further increase in apoptosis was observed following slow cryopreservation or vitrification. TABLE 1. Mean SD of DNA integrity index in intact and biopsied embryos
Intact Embryos (n ¼ 44) Parameter
Fresh (n ¼ 13)
Slow cryopreservation (n ¼ 17)
DNA Integrity Index (%)
100.00 0.00
96.17 5.55
Vitrification (n ¼ 14)
CONCLUSIONS: Embryo biopsy does not induce apoptosis in cleavage stage embryos. Both slow cryopreservation and vitrification do not induce apoptosis in biopsied cleavage stage embryos. Supported by: None.
P-523 OOCYTE DYSMORPHISM IS NOT A LIMITING FACTOR FOR POST-THAW SURVIVAL FOLLOWING VITRIFICATION BY CRYOTOP METHOD. I. Maldonaldo, A. Bermudez, A. Varghese, J. Gaytan, R. Sharma, A. Agarwal. Inmater S. C., Col; Lomas de Chapultepec, Mexico City, Mexico; Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH. OBJECTIVE: Oocyte and embryo morphology significantly affect the survival rates after slow freezing. Oocyte dysmorphism is a major concern for a majority of the embryologists although vitrification methods with oocyte survival rates >90% have been reported. The aim of our study aim was to examine if oocyte dysmorphism after denudation significantly affects survival rates following vitrification by cryotop method. DESIGN: Prospective study. MATERIALS AND METHODS: 336 oocytes obtained from 139 patients (<36 y) were individually vitrified and thawed using cryotop method. All of these oocytes had failed to fertilize after IVF-ICSI. Oocytes were divided in six groups based on the type of dysmorphisms: Group A ¼ vacuolated cytoplasm (n ¼ 60); Group B ¼ smooth endoplasmic reticulum cluster (n ¼ 52); Group C ¼ large perivitelline space (n ¼ 50), Group D ¼ granulated cytoplasm (n ¼ 60); Group E ¼ amorphous cytoplasm (n ¼ 51) and Group F ¼ normal morphology (n ¼ 60; controls). All the oocytes were equilibrated in an equilibration solution (ethylene glycol, 7.5% and 1, 2 propanediol; 7.5%) for 15 min and transferred into vitrification solution (VS; 15% ethylene glycol and 15% 1, 2 propanediol) for 1 min. They were immediately placed on the cryotop with a minimum volume of VS solution and submerged in LN2. The vitrified oocytes were thawed at 37 C for 1 minute using sucrose solution (1 Molar) followed by dilution solution (DS; 0.5 M of sucrose) for 3 minutes and washed X2 in wash solution (M-199 with 20% synthetic serum substitute) for 5 min each. They were examined for post thaw survival based on the dysmorphism. RESULTS: The post-thaw survival rate was comparable in all the groups. TABLE 1. Comparison of survival rates in different groups following minimal volume vitrification
Group A B C D E F
A
B
C
D
E
F
0.98 0.91 0.94 0.98 0.94
0.93 0.96 0.99 0.96
0.96 0.93 0.96
-
0.96
-
0.96 1.0
No statistical differences were observed among different groups by chisquare analysis. CONCLUSIONS: Vitrification by cryotop technology using minimal volume approach increases both cooling and warming rates. The rapid transfer of heat helps maintain the oocyte cytoarchitecture irrespective of the morphology unlike slow freezing. Oocyte morphology may not be a limiting factor in predicting the survival following minimal volume vitrification by cryotop method. Supported by: None.
100.00 0.00 P-524
Biopsied Embryos (n ¼ 44) Parameter
Fresh (n ¼ 15)
Slow cryopreservation (n ¼ 16)
Vitrification (n ¼ 13)
DNA Integrity Index (%)
100.00 0.00
99.11 3.57
98.29 6.16
FERTILITY & STERILITYÒ
SUCROSE IN THE FINAL EQUILIBRATION SOLUTION INTERFERES WITH THE EMBRYONIC DEVELOPMENTAL POTENTIAL OF VITRIFIED MOUSE METAPHASE II OOCYTES. H. Liu, L. C. Krey, J. A. Grifo. NYU Fertility Center, New York University School of Medicine, New York; New York University School of Medicine, New York. OBJECTIVE: Cryoprotectant concentrations, temperature and equilibration times are important factors that determine a successful cryopreservation
S283