The Embryo Culture SystemYasmin Magdi Abd-Elkreem

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In vivo is not in vitro in vitro environment is a very stressful place to

be: temperature and pH fluctuate; light exposure and high levels to oxygen. In addition, nutrients and energy sources may not be correct during all developmental stages.

A prerequisite for implantation and successful pregnancy, in vitro induced stress factors must be reduced to a minimum.

One way to reduce embryo stress is to use optimum culture system.

The Embryo Culture System Consists of :

1. The Media 2. Gas Phase3. Type of Medium overlay .4. The Culture Vessel .5. The Incubation Chamber .6. Ambient Air Quality .7. The Embryologists

themselves .

The Media Definition: is in vitro culture either of gametes , zygote, cleavage stage embryo,

post-comapction embryo and embryo transfer.

A culture medium is a foreign environment for the human embryo. Hence, the design of media is complicated, because the components must be selected, and their concentrations determined in order to minimize stress for the cultured embryo.

Upon first sight, embryo culture appears a rather simple procedures. In reality, it is far from it, requiring proactive quality control and quality assurance programs, together with a high level of training for embryologists.

The Media

The questions Albert Brachet tried to answer by culturing

preimplantation mammalian embryos

Is an egg or a young blastocyst capable of development outside the maternal organism?

If so, can it adapt itself to an artificial medium by creating special organs, or will it form its normal fixation and nutritional structures?

Brachet A (1913) Recherches sur le déterminisme héréditaire de l’œf des mammifères. Développement in vitro des jeunes vésicules blastodermiques de Lapin. Arch Biol (Lièges) 32, 205-248.

Brachet A (1912) Développement in vitro de blastodermes et de jeunes embryons de mammifères. C R Hebd Seances Acad Sci 155, 1191-1193.

First attempt to culture a mammalian embryo

The Media

“Simple media are used, including Earle’s solution with pyruvate, a medium designed to support mouse homologous human serum (7.5 – 8.6%). We prefer Earle’s solution containing pyruvate , with serum from each patient.”

Selection was based on commercially available media.

Edwards RG (1981) Test tube babies. Nature 293, 253-256.

Complex medium (B2/B3): Menezo Y, Testart J, Perrone D (1984) Serum

is not necessary in human in vitro fertilization, early embryo culture, and transfer.

Fertil Steril 42, 750-755. Simple medium (HTF): Quinn P, Kerin JF, Warnes GM (1985)

Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid.

Fertil Steril 44, 493-498.

First media to be tested in human First specifically designed chemically defined culture media for human embryos

1. The Media1.1 Composition Of Embryo Culture Media 1.1.1 Water

1.1.2 Ions1.1.3 Carbohydrates1.1.4 Amino Acids1.1.5 Vitamins1.1.6 Nucleic Acid Precursors1.1.7 Chelators 1.1.8 Antioxidants 1.1.9 Antibiotics1.1.10 Protein/Macromolecules 1.1.11 Hormones and growth factors1.1.12 Buffer System

1.2 Types Of Media For Embryo Culture 1.2.1 Simple Salt Soln with added

Energy Substrates1.2.2 Complex Tissue Culture Media1.1.3 Monoculture Media1.1.4 Sequential Media

1.3 Media Storage

1.1.1.Water Is the major component Making up around 99% of the contents The ability of embryos to develop is positively correlated to water qulaity Wittingham demonstrated that the development of 2-cell mouse embryos

to the blastocyst in culture was enhanced when the media was prepared using triple distilled water as opposed to double or single distilled water.

Endotoxin levels < 0.1 IU/mLWhittingham DG, J. Reprod Fertil Suppl 1971; 14:17

1.1.2.Ions Ions in any medium make the largest single contribution to osmotic

pressure. High K levels is benefit for sperm capacitation and in vitro embryo

development. High concentration of NaCl (125 mM)are detrimental to mouse embryo

development to blastocyst in vitro. Mg is not essential for embryo development to blastocyst stage. Ca is essential for embryos to undergo compaction in vitro. Phosphate ion has an inhibitory effect in case of presence of glucose,

however, it does not have inhibitory effect in the presence of amino acids.

1.1.3.Carbohydrates Glucose metabolism is also used to generate energy, early embryo is

unique in that it lacks the ability to metabolize glucose. high level of glucose in culture is responsible for retardation or

developmental arrest of cleavage-stage embryos. toxicity of glucose appears to be manifest in medium containing

phosphate and typically lacking amino acids. several studies determined that this inhibitory effect of glucose in the

presence of phosphate could be alleviated by the presence of amino acids, EDTA or vitamins.

lack of glucose in the media for the postcompaction-stage embryo resulted in impaired development to the blastocyst stage and a loss of viability

Martin KL & Leese HJ. Mol Reprod Dev 1995; 40: 436e443.

1.1.4.Amino Acids Among the most important components of all handling and culture media for the mammalian

oocyte and pre-implantation embryo is amino acids. amino acids begin to break down spontaneously forming by-products such as

ammonium. Prior the embryonic genome : 1. the embryo utilizes carboxylic acids and Amino Acids as energy sources.2. certain Amino Acids are known to function as biosynthetic precursor molecules.3. Certain Amino Acids acts as osmolytes, buffers of internal pH and antioxidants.4. Some of them acts as chelators especially for heavy metals. 5. Seven essential amino acids stimulate cleavage stage , Up to 8-cell stage non-essential amino acids and glutamine stimulate the cleavage stage. Post the embryonic genome:1. Non-essential amino acids and glutamine increase blastocoel formation and hatching.2.The essential amino acids stimulate cleavage rates and increase development of inner cell mass in the blastocyst.

1.1.5.Vitamins Acts as antioxidant to prevent perturbation in metabolism and loss of viability induced by

sub-optimal culture conditions. B-group vitamins are an integral part of carbohydrates and amino acids metabolism. Effect on embryo development remain largely unknown. Human zygotes can form blastocysts in culture in the absence of vitamins. B12, present in Ham`s F-10 causes a decrese in blastocyst expansion. Nicotinamide reduce embryo viability after transfer. The addition of vitamins as antioxidants to the culture media containing glucose and

phosphate helped to prevent a loss in respiration and metabolic control. The following possible vitamins are components of different ART culture media: ascorbic

acid, cyanocobalamin, folic acid and tocopherol. Moderate dosages of vitamins C and E were seen to reduce oxidative damage in mouse

embryo culture and improve their blastocyst development rate.

Lane M and Gardner DK. Best Practice & Reserch Clin Obstet and Gynaecology 1997; 21: 83-100.

Wang X et al. Fertil Steril 2002; 78: 1272-7.

1.1.6. Nucleic Acid Precursors Does not required for embryo development. Should be omitted from culture media. Hypoxanthine (present in Ham`s F-10) induced a block in a mouse

embryos at 2-cell stage in vitro. Adenosine and inosine are detrimental to embryo development at early

cleavage stage.

1.1.7.Chelators: EDTA Its usefulness is based on its role as a ligand and chelating agent, i.e. its

ability to “sequester” metal ions. many studies have demonstrated the beneficial effect of EDTA for

development of the embryo from the zygote stage. When postcompaction-stage embryos cultured with 100 micro-M EDTA,

have reduced ICM development and fetal development after transfer. biphasic effect of EDTA could be due to its effects in inhibiting glycolysis.

Nasr-Esfahani et al. J Reprod Fertil 1992; 96: 219e231.

Gardner DK et al. Mol Reprod Dev 2000; 57: 256e261.

1.1.8.Antioxidant UV-light and exposure to environmental levels of oxygen cause formation

of highly reactive radical oxygen species, ROS. Oxidative stress is one cause of embryo development retardation.

Using culture media containing a potent antioxidant is a simple way to achieve lower levels of ROS.

Supplementation of medium with superoxide dismutase (SOD) increased the development of mouse zygots beyond the 2-cell block to blastocyst.

There are a conflicting reports about adding antioxidants to embryo culture media.

Noda Y. et al. Mol. Reprod Dev. 1991; 31:28

1.1.9.Antibiotics Penicillin, streptomycin, gentamicin have routinely included in embryo

culture media. Improved cleavage rates of human embryos in medium free of antibiotics,

questioning the practice of routinely adding antibiotics to the culture medium.

Washing of embryos in a medium supplemented with antibiotics can remove any bacterial contamination.

Inclusion of antibiotcs in medium for sperm preparation is essential.

1.1.10.Macromolecules Common sources for macromolecules are proteins for culture media such

as human serum albumin or synthetic serum. Both are added at concentrations of 5 to 20%. Protein in the form of albumin is thought to maintain the stability of cell

membranes and chelate trace amounts of toxic components presented in culture water, media components and culture dishes.

Capillary membrane permeability and osmoregulation. serves to facilitate manipulation of gametes and embryos

1.1.11.Hormones and Growth factors Limited data on the direct action of hormones on the early embryo. Estrogen has negative impact, prolactin improve the rate of blastocyst

formation. Growth factors as: Insulin, IGF-I, IGF-II, EGF, TGF-α, TGF-β, PDGF-A, FGF-4, LIF

stimulates:

1- Cleavage 2- amino acid transport 3- protein synthesis 4- blastocoel formation 5- ICM development

pH refers to hydrogen ion concentration and is only meaningful when applied to aqueous (water-based) solutions.

pH is dynamic. pH range is 7.2 and 7.4. stable media pH is important to reduce environmental

stress in the culture environment and optimize assisted reproductive outcomes.

Intracellular pH (pHi) regulates protein conformation, glycolysis and a number of other critical metabolic and transport processes in the human oocyte/embryo. Maintaining pHi is imperative to esp. the early embryo.

Extracellular pH (pHe) in the culture medium is known to be a critical factor in establishing pHi.

1.1.12.Buffering system

The most popular pH regime thus describes ahigh-low-high standard, which seems to be the mostbeneficial pH standard regime in IVF.• Fertilization Higher pH (7.3-7.4)• Cleavage stage Lower pH (7.2-7.3)• Blastocyst Higher pH (7.3-7.4)

1.1.12.Buffering system The Regulation of pH is controlled by buffer and so we can classify media according to type of

used buffer to :1. CO2 /bicarbonate-buffered medium:• Using sodium bicarbonate-buffered media, the same physiological buffering system surrounding mammalian

cells.• Use CO2 incubators 5-7%.2. Phosphate-buffered medium:• Not requires CO2 environment.• maintain pH in air.• detrimental to embryo development in vitro. 3. HEPES-buffered medium:• used as a buffered medium for human oocyte collection and embryo handling.4. MOPS-buffered medium:• like HEPES , a potential advantage is that buffering capacity is less temperature dependent than HEPES.

1.2. Media types consisting of balanced salt solutions

with added carbohydrates glucose, pyruvate and lactate, and commonly supplemented with the patient’s serum.

These media lacked many components that are now known to be important for the maintenance of embryo physiology and health, such as amino acids.

Earle’s, T6 and HTF medium

complex tissue culture media, embryos used to be grown in tissue culture media.

designed to support immortal cell lines in culture and were far more complex, being supplemented with carbohydrates, amino acids, vitamins, nucleotides and metal ions.

Examples of such media are Ham’s F-10, MEM and TCM-199.

1-Simple media 2-Complex tissue culture media

None of these media were designed to support embryo development, they contain many components shown to be detrimental to embryo development in vitro, such as high levels of glucose, divalent metal ions, nucleotides and certain hormones, they are not able to support acceptable levels of blastocyst development.

Gardner DK & Lane M. New York: CRC Press, 1999, pp. 123e150.

a media should approximate the concentration to which the embryo is naturally exposed .

The embryo is capable of actively controlling ionic gradients etc, and is able to regulate its internal environment.

Consider the preimplantation period in at least two phases: pre- and post-compaction . Such a breakdown of the preimplantation period is of importance when one considers changes to medium formulations. Other considerations include the time at which the embryonic genome is activated

involves the simultaneous use of all the concentrations in a mixture because the effects of each component in the medium may depend on the concentrations of the other components .

concentrations are within ‘tolerable ranges’, the embryo itself will adapt and utilize whatever it requires .This philosophy led to a family of media in which all of the substances necessary to early embryological development are provided, and there is no need for a media change.

3-The sequential culture-“back-to-nature” principle

4-The monoculture “let-the-embryo-choose” principle

Many protocols can be used for the culture from fertilization to the blastocyst stage in an ART laboratory. In our lab: sequential media protocol, with an interrupted culture where two media of different compositions are used sequentially, change of medium occurs on day 2 of embryo culture.

1.2. Media types

3.Gas Phase Co2 (5-7%) required for:1- maintain pH of bicarbonate buffered media2- incorporate into protein and nucleic acids by the embryos. In vivo, oocytes and embryos are exposed to a maximum of ~5-8% O2 in

the reproductive system. Atmospheric O2 may lead to supraphysiological ROS levels, potentially causing oxidative stress (damage to cell organelles, lipids, membranes, DNA, gene expression), and ultimately poor embryo development.

Reduced O2 concentration results in enhanced development in vitro.

3.Type of Medium overlay maintain stable temperature, osmolality and pH. Changes in osmolality depend on volume, temperature and presence or

absence of oil overlay. The oil types commonly used are:1- mineral oil,2- paraffin oil 3-a mixture of both. Paraffin oil and mineral oil are chemically slightly different. The polycarbon

lipid tail of mineral oil contains more unsaturated bonds than the more saturated paraffin oil.

4.The Culture Vessel Use only non-toxic sterile disposables. Dishes for ICSI and embryo culture, test tubes for preparations and

aliquoting of media as well as pipettes are all made from different types of plastics. As all types of plastics are potentially toxic to embryos.

it is very important to use plastic labware that have been tested and developed for IVF purposes (Mouse tested vessels).

Tightly or loosely caped vessels is chosen according to type of used media.

5.The Incubation Chamber Important to maintain pH and temperature. The bicarbonate-buffered medium determines the equilibrium pH and

thus the required CO2 inflow in the incubator. Optimal pH control involves CO2 flow (5-7%) optimized for each

medium / embryo stage. Strict temperature monitoring in incubator at 37 ͦ C , Maintaining

constant temperature must be a top priority during all stages of culture. Deviations may harm metabolism and mitotic apparatus.

Door opening reduces incubator temperature. Repeated openings will cause permanent lowering of incubator (and medium) temperature.

6.Ambient Air Quality Embryos, esp. cleavage-stage embryos, are sensitive to environmental

toxicants, esp. Volatile Organic Compounds (VOC). The enclosed incubator environment may have concentrated VOC levels

several times higher than the ambient air. Careful attention should be dedicated to general air supply and purification

measures should be taken to effectively remove VOCs – esp. in metropolitan areas, where concentrations can be greatly elevated.

Osmolality human embryos require an osmolality range of 255-295 mOsm/kg for optimal

development. First, droplets (droplets were placed in the dish and covered with oil). Second, a

wash droplet (droplets were placed in a dish and covered with oil then, the original droplets were then removed and replaced by fresh medium droplets)

Data presented are representative of sampling after 5 min. Lower drop volume, higher temperature and the standard method all significantly increase media osmolality. No differences were apparent after 24 h.

Thank You For contact: E-mail: Yas.magdi@hotmail.com