Materials Used In Embryo Culture

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1. Various size pipette tips.

2. Appropriate embryo tested culture dishes.

3. Fertilization, cleavage, and blastocyst media.

Table 1

Comparison of sequential media and single-step medium culture protocols

Factors Sequential media Single-step medium

Table 1

Comparison of sequential media and single-step medium culture protocols

Change of medium

Require change of medium around D3 of culture

Can be used for complete preimplanta-tion period with no medium change

Energy substrates

Change on D3

No change

Presence of EDTA

Blastocyst medium does not contain EDTA that may reduce glycolysis

Contains EDTA that may compromise blastocyst development by slowing glycolysis

Effect of essential amino acids

Cleavage medium does not contain essential amino acids that may impair development from the zygote stage

Contains essential amino acids that may compromise early development

pH

Cleavage medium has a pH of around 7.20-7.25 which optimizes development from the zygote to 8-cell stage Blastocyst medium has a pH between 7.30 and 7.35 which is optimal for blastocyst development

pH is similar during the whole culture period unless the atmospheric CO2 concentration is changed on D3. Studies comparing sequential media to single-step medium culture have not optimized pH

4. Handling media (i.e., HEPES or MOPS buffered).

5. Appropriate protein source.

6. Embryo tested mineral oil.

7. Laboratory incubator.

8. Microscope.

3. Methods

1. The day before ovum pickup (OPU), label culture dishes. When making drops of medium, use a sterile pipette tip, rinsing the tip twice with culture medium before making the drops.

2. For in vitro fertilization (IVF). Prepare fertilization media in a sterile manner with appropriate protein and concentration. Place drops of fertilization medium into the dish according to laboratory protocol (see Notes 1 and 2).

3. Immediately cover the drops with oil and place the dish in the CO2 incubator (see Notes 3-5).

4. For intracytoplasmic sperm injection (ICSI). Prepare and place dishes of cleavage media in the CO2 incubator as above (see Notes 3-5).

5. When placing the dishes in the incubator, gently remove the lid of the dish and set it at an angle on the side of the dish to allow for complete gas exchange. Dishes must gas for a minimum of 4 h before use (or overnight).

6. On the day of OPU (D0) for IVF cases, prepare culture dishes with cleavage medium as in step 4.

7. For both IVF and ICSI on D0 or D1 before fertilization check. Prepare handling media according to laboratory protocol. Media can be left at room temperature overnight, or prepared early on the morning of D1 and warmed in air to 37 °C on a heating plate. In either case, warm the dishes to 37 °C on the morning of D1 before use.

8. For IVF late on D0 or early on D1. Prepare a wash dish with handling media.

9. On D1, or day of fertilization check, gently remove the cumulus cells by stripping oocytes according to laboratory protocol (see Note 6). Gently wash the stripped oocytes well in handling media. Then place fertilized oocytes in dishes of cleavage media. For ICSI'ed oocytes, successfully fertilized eggs can remain in cleavage medium. Zygotes can remain in cleavage media until D3 (see Note 7).

10. D2 zygotes can be examined according to laboratory protocol. Dishes of blastocyst media should be prepared as above and placed into the incubator at least 4 h prior to use on D3.

11. On D3 after blastocyst medium dishes have equilibrated for at least 4 h. For embryos that are to be cultured to D5/6, remove the embryos from the cleavage medium culture dishes and place into dishes of blastocyst medium (see Note 8).

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  • awet
    What are the thevmaterials used in embryo culture?
    4 years ago
  • Kenneth
    What are the materials used in embryo culture?
    4 years ago

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