3 Methods
3.1 Stepwise
Blastocyst Vitrification
Procedure
Vitrification of blastocysts needs to be undertaken utilizing a
“closed system” (HSV: High Security Vitrification Kit; Cryo Bio
System, L’Aigle, France; FDA 510(k) clearance for cleavage stage
embryos in blastocysts) (see Notes 1 and 2) after a two-step loading
with cryoprotectant agents at 24
C (see Table 1) (see Notes 3 and
4). If assisted collapsing is done prior to vitrification, then the
blastocyst needs to be put on an inverted microscope equipped
with a laser system (Zilos-tk or Lykos, Hamilton Thorne), the
junction of two trophectoderm cells in each blastocyst needs to
be located, and one shot (100% power, 500 μs pulse length) was
applied. Then the blastocysts can be moved back in the incubators
for 5–10 min. Briefly, blastocysts have to be placed in equilibration
solution, which is the base medium (M199 with 20% Serum Substitute Supplement (SSS) containing 7.5% (v/v) ethylene glycol
(EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO)). After 8 min,
the blastocysts need to be washed quickly in vitrification solution,
which is the base medium containing 15% (v/v) DMSO, 15% (v/v)
Table 8
A comparison of retrospective data from the cryopreservation program (Fertility Centers of Illinois,
Chicago) of vitrified day 5 and day 6 blastocysts without AC (group A) and with AC (group B) using a
closed carrier system in patients younger than 35 years old between 2007 and June 2019
Technique
Group A (no AC)
Group B (with AC)
Less than 35 years old
Less than 35 years old
Day 5
Day 6
Day 5
Day 6
Patient’s age (y)
31.4 Æ 2.3
31.6 Æ 0.6
31.4 Æ 2.2
31.7 Æ 2.2
Transfers
647
379
1913
634
Blastocysts warmed
1095
684
2614
930
Blastocysts survived
1075 (98.2)
669 (97.8)
2608 (99.8)
926 (99.6)
Blastocysts transferred
1049
652
2598
916
Blastocysts transferred (mean)
1.6
1.7
1.4
1.4
Implantations
449 (42.8)
215 (33.0)
1438 (55.4)
406 (44.3)
Positive pregnancy/VET
380 (58.7)
193 (50.9)
1377 (72.0)
423 (66.7)
Clinical pregnancy/VET
339 (52.4)
166 (43.8)
1208 (63.1)
339 (53.5)
Ongoing/delivered pregnancies
296 (45.7)
137 (36.1)
1079 (56.4)
285 (45.0)
**P < 0.001 VET vitrified embryo transfer
Values are numbers unless otherwise described, and percentages are indicated between brackets
Vitrification of Human Blastocysts
509
3.1 Stepwise
Blastocyst Vitrification
Procedure
Vitrification of blastocysts needs to be undertaken utilizing a
“closed system” (HSV: High Security Vitrification Kit; Cryo Bio
System, L’Aigle, France; FDA 510(k) clearance for cleavage stage
embryos in blastocysts) (see Notes 1 and 2) after a two-step loading
with cryoprotectant agents at 24
C (see Table 1) (see Notes 3 and
4). If assisted collapsing is done prior to vitrification, then the
blastocyst needs to be put on an inverted microscope equipped
with a laser system (Zilos-tk or Lykos, Hamilton Thorne), the
junction of two trophectoderm cells in each blastocyst needs to
be located, and one shot (100% power, 500 μs pulse length) was
applied. Then the blastocysts can be moved back in the incubators
for 5–10 min. Briefly, blastocysts have to be placed in equilibration
solution, which is the base medium (M199 with 20% Serum Substitute Supplement (SSS) containing 7.5% (v/v) ethylene glycol
(EG) and 7.5% (v/v) dimethyl sulfoxide (DMSO)). After 8 min,
the blastocysts need to be washed quickly in vitrification solution,
which is the base medium containing 15% (v/v) DMSO, 15% (v/v)
Table 8
A comparison of retrospective data from the cryopreservation program (Fertility Centers of Illinois,
Chicago) of vitrified day 5 and day 6 blastocysts without AC (group A) and with AC (group B) using a
closed carrier system in patients younger than 35 years old between 2007 and June 2019
Technique
Group A (no AC)
Group B (with AC)
Less than 35 years old
Less than 35 years old
Day 5
Day 6
Day 5
Day 6
Patient’s age (y)
31.4 Æ 2.3
31.6 Æ 0.6
31.4 Æ 2.2
31.7 Æ 2.2
Transfers
647
379
1913
634
Blastocysts warmed
1095
684
2614
930
Blastocysts survived
1075 (98.2)
669 (97.8)
2608 (99.8)
926 (99.6)
Blastocysts transferred
1049
652
2598
916
Blastocysts transferred (mean)
1.6
1.7
1.4
1.4
Implantations
449 (42.8)
215 (33.0)
1438 (55.4)
406 (44.3)
Positive pregnancy/VET
380 (58.7)
193 (50.9)
1377 (72.0)
423 (66.7)
Clinical pregnancy/VET
339 (52.4)
166 (43.8)
1208 (63.1)
339 (53.5)
Ongoing/delivered pregnancies
296 (45.7)
137 (36.1)
1079 (56.4)
285 (45.0)
**P < 0.001 VET vitrified embryo transfer
Values are numbers unless otherwise described, and percentages are indicated between brackets
Vitrification of Human Blastocysts
509