Types of Media for Embryo Culture

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Culture media employed for clinical IVF vary greatly in their composition, yet there appears to be little difference between media in their ability to support development of the human embryo in vitro for up to 48 hours or in subsequent pregnancy rates after transfer (12). This has led to a great deal of confusion concerning the formulation of embryo culture media and the role of individual components in embryo development. An understanding of the role of culture media and their components has been hampered by the routine inclusion of serum in human embryo culture media. Serum has the ability to both mask potential embryo toxins and suppress the beneficial effects of other medium components. In light of this, there has been considerable research into the development of serum-free embryo culture media. Such studies have been invaluable in our understanding of the embryo's requirements during the preimplantation period.

Media used to culture the mammalian preimplantation embryo generally fall into one of four types.

Simple Salt Solutions with Added Energy Substrates

These media were originally formulated to support the development of zygotes from certain inbred strains of mice and their F1 hybrids (13). Examples of this type of media used in clinical IVF are M16 (14), T6 (14), Earle's (15), CZB (16), and KSOM (17). Derived from such types of media were human tubal fluid (HTF) medium (18,19), and P1 (20). As shown in Table 1 (21-25), there has been little change in the formulation of these media over the past 30 years. Such "simple" media are usually supplemented with either whole serum or serum albumin, and are used for the cleavage stage embryo only, i.e., pronucleate oocyte to the 8-cell stage.

Complex Tissue Culture Media

These media are commercially available and are designed to support the growth of somatic cells in culture, e.g., Ham's F-10 (Table 2) (26). Such media are far more complex, containing amino acids, vitamins, nucleic acid precursors, and transitional metals, and are usually supplemented with 5-20% serum. Importantly, such media were not formulated with the specific needs of the human embryo in mind, and they contain components which are now known to be detrimental to the developing embryo.

Simplex Optimized Media

This approach to formulate culture media depended on a computer program to generate successive media formulations based on the response of mouse embryos in culture (24,25). Once a specific medium was formulated, tested, and blastocyst development analyzed, the computer program would then generate several more media formulations for use in the next series of

Table 1 Composition (mM) of Simple Salt Solution with Added Energy Substrates used in Embryo Culture

Whitten

and

M16

KSOM

Basal XI

Pla

Whitten

Brinster

Biggers

(1971)

Earle'sa

HTFa

CZB

MTFd

(1993)

HTFa

(1998)

Component

(1957) (21)

(1965) (22)

(1968) (13)

(14)

(1971) (15)

(1981) (18)

(1985) (16)

(1989)(23)

(24,25)

(1995) (19)

(20)

NaCl

118.46

119.23

68.49

94.66

116.30

101.60

81.62

114.19

95.00

97.6

101.6

KC1

4.74

4.78

4.78

4.78

5.36

4.69

4.83

4.78

2.50

4.69

4.69

KH2P04

1.18

1.19

1.19

1.19

0.37

1.18

1.19

0.35

NaH2P04

1.02

CaCl2.2H20

1.71

1.71

1.80

2.04

1.70

1.71

1.71

2.04

2.04

MgS04.7H20

1.18

1.19

1.19

1.19

0.81

0.20

1.18

1.19

0.20

0.20

0.20

NaHC03

24.88

25.00

25.07

25.00

26.18

25.00

25.12

25.00

25.00

25.00

25.00

Ca Lactate

2.54

1.71

Na Lactate (D/L)

25.00

21.58

23.28

21.40

31.30

4.79

10.00e

21.4

21.4

Na Pyruvate

0.25

0.33

0.33

0.10

0.33

0.27

0.37

0.20

0.33

0.33

Glucose

5.55

5.56

5.56

5.55

2.78

3.40

0.20

BSA (mg/mL)

1.00

1.00

4.00

4.00

b

5.00

5.00

4.00

1.00

b

c

Ratios

Na/K

24.21

28.39

19.34

24.00

26.79

29.26

23.01

24.18

45.68

30.71

30.71

Ca/Mg

2.15

1.44

1.44

1.44

2.22

10.02

1.44

1.44

8.55

10.2

10.2

L/P

100

70.58

70.55

64.85

115.93

12.95

50.00

64.85

64.85

Note: CZB contains llO^iM EDTA, 1.0 mM glutamine, and 5.5 mM glucose after 48 hours of culture from the zygote stage. KSOM contains 10 EDTA and 1.0mM glutamine. Basal XI HTF contains 100EDTA and l.OmM glutamine. PI contains 50|xM taurine and 0.5^M citrate. Penicillin (100U/ mL) and streptomycin present (50^g/mL). Gentamycin present at lO^g/mL. aUsed in clinical IVF.

bMedium supplemented with human serum albumin. °Medium supplemented with synthetic serum substitute.

dModifications to these media have included the addition of specific groups of amino acids resulting in significant improvements to mouse zygote development in culture. epresent as L-Lactate.

Abbreviations'. HTF, human tubal fluid; CZB, Chatot, Ziomek and Bavister; MTF, mouse tubal fluid; KSOM, potassium simplex optimized medium; EDTA, ethylenediaminetetraceticacid; IVF, in vitro fertilization.

Table 2 Composition of Ham's F-10 Medium

Component Concentration (mM)

NaCl 126.60

KCl 3.82

MgSO47H2O 0.62

Na2HPO4 1.31

KH2PO4 0.61

NaHCO3 14.28

CaCl22H2O 0.30

CuSO45H2O 0.00001

FeSO47H2O 0.0030

ZnSO47H2O 0.0001

Phenol Red 0.034

Sodium Pyruvate 1.00

Calcium Lactate 1.00

Glucose 6.11

Alanine 0.10

Arginine 1.21

Asparagine 0.11

Aspartic acid 0.10

Cysteine 0.26

Glutamate 0.1

Glutamine 1.0

Glycine 0.1

Histidine 0.14

Isoleucine 0.02

Leucine 0.10

Lysine 0.20

Methionine 0.03

Phenylalanine 0.03

Proline 0.10

Serine 0.10

Threonine 0.03

Tryptophan 0.003

Tyrosine 0.12

Valine 0.03

Biotin 0.0001

Ca pantothenate 0.0015

Choline chloride 0.005

Cyanocobalamine 0.001

Folic acid 0.003

Inositol 0.003

Nicotinamide 0.005

Pyridoxine 0.001

(Continued )

Table 2 Composition of Ham's F-10 Medium (Continued)

Component

Concentration (mM)

Riboflavin Thiamine

0.001 0.003

Hypoxanthine Lipoic acid Thymidine

0.03

0.001

3.00

Note: Penicillin present at 100 U/mL. Streptomycin present at 50 mg/mL. Modifications as per the Center for Reproductive Medicine.

cultures. This procedure was performed several times to generate media that supported high rates of blastocyst development of embryos derived from the oocytes of outbred mice (CF1) crossed with the sperm of an F1 hybrid male, and were termed SOM and KSOM. Such media were subsequently modified by another laboratory to include amino acids (KSOMAA) (27). This last phase of medium development was based on previous studies on the mouse embryo (28) and did not involve the simplex procedure. This single medium formulation, KSOMAA, has been used to produce human blastocysts in culture (29). In such types of media, the embryo therefore has to adapt to its surroundings as it develops and differentiates.

Sequential Media

The approach taken in our laboratory has not only been to learn from the environment to which embryos are exposed in vivo (23,30), but also to study the physiology and metabolism of the embryo in culture, in order to determine what causes intracellular stress to the embryo (7,9,31-36). By being able to identify and monitor such stress, we have been able to develop stage specific culture media that substantially reduce culture-induced trauma. The development and characterization of such sequential media has been published in detail elsewhere (37-39).

Examples of sequential media include G1/G2 (Table 3) (37,40,41), universal IVF medium and M3 (42), and P1 together with blastocyst medium (43). Interestingly, medium M3 is a modification of Ham's F-10 and F-12, while blastocyst medium is a mosdification of Ham's F-10.

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