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Table 1 Description of slow-freezing/thawing protocols used in the study period, including formulations and methods

From: A monocentric analysis of the efficacy of extracellular cryoprotectants in unfrozen solutions for cleavage stage embryos

  Kit 1 provided by Cook Medical (USA) Kit 2 provided by Vitrolife (Sweden) Kit 3 provided by Vitrolife (Sweden)
Freezing solutions FS1: Cryo-PBS without CPAs
FS2: Cryo-PBS with 1.5 M 1.2-propanediol
FS3: Cryo-PBS with 1.5 M 1.2-propanediol and 0.1 M sucrose
FS1: Cryo-PBS without CPAs
FS2: Cryo-PBS with 1.5 M 1.2-propanediol FS3: Cryo-PBS with 1.5 M 1.2-propanediol and 0.1 M sucrose
FS1: Cryo-PBS without CPAs
FS2: Cryo-PBS with 1 M 1.2-propanediol and 0.5 M sucrose
Freezing method Step 1:
Equilibrate S1 for 10′ at 37° in air. and of S2 and S3 for 10′ at RT
Step 2:
Embryo incubation in S1 for 10′ at RT
Step 3:
Embryo incubation in S2 for 10′ at RT
Step 4:
Embryo incubation in S3 up to load in straws
Step 5:
Embryo loading and straws sealing
Step 6:
Loading of straws into the automatic freezer
Step 1:
Equilibration of S1. S2 and S3 for 15′ at RT
Step 2:
Embryo incubation in S1 for 5′ at RT
Step 3:
Embryo incubation in S2 for 5′ at RT
Step 4:
Embryo incubation in S3 up to load in straws
Step 5:
Embryo loading and straws sealing
Step 6:
Loading of straws into the automatic freezer
Step 1:
Equilibration of S1 and S2 for 15′ at RT
Step 2:
Embryo incubation in S1 for 10′ at RT
Step 3/4:
Embryo incubation in S2 and within 10′ embryo loading and straws sealing
Step 5:
Loading of straws into the automatic freezer
Automatic freezing program Start temperature at + 20 °C;
Lower to −7 °C at 3 °C per minute;
Soak for 10′;
Manual seeding of straws;
Hold for 10′;
− 7 °C to − 30 °C at 0.3 °C per minute;
− 30 °C to − 150 °C at 50 °C per minute;
Plunge into liquid nitrogen and storage.
Start temperature at + 20 °C;
Lower to − 7 °C at 0.2 °C per minute;
Manual seeding of straws;
Hold for 7′;
− 7 °C to − 30 °C at 0.3 °C per minute;
− 30 °C to − 150 °C at 50 °C per minute;
Plunge into liquid nitrogen and storage.
Start temperature at + 20 °C;
Lower to − 6 °C at 0.2 °C per minute;
Manual seeding of straws;
Hold for 10′;
− 6 °C to − 30 °C at 0.3 °C per minute;
− 30 °C to − 150 °C at 50 °C per minute.
Thawing solutions TS1: Cryo-PBS with 1.0 M 1.2-propanediol and 0.2 M sucrose
TS2: Cryo-PBS with 0.5 M 1.2-propanediol and 0.2 M sucrose
TS3: Cryo-PBS with 0.2 M sucrose
TS4: Cryo-PBS without CPAs
TS1: Cryo-PBS with 1 M 1.2-propanediol and 0.2 M sucrose
TS2: Cryo-PBS with 0.5 M 1.2-propanediol and 0.2 M sucrose
TS3: Cyo-PBS with 0.2 M sucrose
TS4: Cryo-PBS without CPAs
TS1: Cryo-PBS with 1 M sucrose
TS2: Cryo-PBS with 0.5 M sucrose
TS3: Cryo-PBS without CPAs
Thawing method Step 1:
Equilibrate TS1. TS2. TS3 and TS4 for 10′ at 20 °C
Step 2:
Thaw straws in air for 40″ and then in a water bath at 30 °C for 30″
Step 3:
Cut the end of a straw. Fit syringe. Cut the other end of the straw and expel contents into a petri dish
Step 4:
Embryos incubation in TS1 for 5′
Step 5:
Embryos incubation in TS2 for 5′
Step 6:
Embryos incubation in TS3 for 5′
Step 7:
Embryos incubation in TS4 for 5′ at RT and then for 5′ at 37 °C
Step 8:
Transfer embryos to equilibrated Cleavage Medium and incubate until transfer
Step 1:
Equilibrate TS1. TS2. TS3 and TS4 for 10′ at 20 °C
Step 2:
Thaw straws in air for 30″ and then in a water bath at 30 °C for 30″
Step 3:
Cut the end of a straw. Fit syringe. Cut the other end of the straw and expel contents into a petri dish
Step 4:
Embryos incubation in TS1 for 5′
Step 5:
Embryos incubation in TS2 for 5′
Step 6:
Embryos incubation in TS3 for 10′
Step 7:
Embryos incubation in TS4 for 6′ at RT and then for 4′ at 37 °C
Step 8:
Transfer embryos to equilibrated Culture medium and incubate until transfer
Step 1:
Equilibrate TS1. TS and TS3 for 10′ at RT
Step 2:
Thaw straws in air for 30″ and then in a water bath at 30 °C for 45″
Step 3:
Cut the end of a straw. Fit syringe. Cut the other end of the straw and expel contents into a petri dish
Step 4:
Embryos incubation in TS1 for 5′
Step 5:
Embryos incubation in TS2 for 5′
Step 6:
Embryos incubation in TS3 for 5′
Step 7:
Transfer embryos to equilibrated Culture medium and incubate until transfer
  1. FS1 Freezing solution number 1, FS2 Freezing solution number 2, FS3 Freezing solution number 3, PBS Phosphate-buffered saline, CPAs Cryoprotectants agents, RT Room temperature, TS1 Thawing solution number 1, TS2 Thawing solution number 2, TS3 Thawing solution number 3, TS4 Thawing solution number 4