Human embryo culture media
Culture media for embryo development must meet the metabolic needs of preimplantation embryos by addressing amino acid and energy requirements based on the specific developmental stage of the embryo.
Culture media for embryo growth was first described in 1912 for the growth of an embryo of a rabbit. Later on in 1949 mouse embryos were grown in culture media from the 8-cells stage to blastocysts. These culture media, like Earle, Ham's F10, Tyrode's T6 and Whitten's WM1 were based on different salts and were constructed to support the development of somatic cells and cell lines in culture. These culture media, known as physiological salt solutions were used by Robert Edwards for his first successful IVF attempt. These media were formulated for use with or without serum supplementation, depending on the cell type being cultured. The Ham's Nutrient Mixtures were originally developed to support growth of several clones of Chinese hamster ovary (CHO) cells, as well as clones of HeLa and mouse L-cells.
In 1984-1985 special media were developed for human IVF. Menezo and his colleagues published a paper, in 1984, describing a new concept in Human Embryo culture. They suggested adding serum albumin as a source for amino acids. The serum protein ensures that oocytes and embryos do not adhere to the glass surface of the pipette used to manipulate them. The medium entitled B2, is still in use today. In 1985 Quinn et al. published in the journal Fertility and Sterility a formula entitled Human Tubal Fluid (HTF), which mimics the in vivo environment to which the embryo is exposed. The formulation of HTF was based on the known chemical composition of the fluids in human fallopian tubes as known at that time. This medium is based on a simple balanced salt solution without amino acids; however, the concentration of potassium was adjusted to that measured in the human female reproductive tract. This medium was found to be better compared with earlier media developed.
The supplementation of the HTF medium with either whole serum or with serum albumin became a gold standard for the production of culture medium for human embryos transferred on day 2 or day 3 of culture.
Over the years, further basic research on the metabolism of preimplantation embryos revealed that there are specific needs depending on the developmental stage of the embryo. Energy source requirements evolve from a pyruvate-lactate preference while the embryos, up to the 8-cell stage, are under maternal genetic control, to a glucose based metabolism after activation of the embryonic genome that supports their development from 8-cells to blastocysts.
The above observation leads to the development of the first commercial media. The culture media developed was based on HTF: both media were free of inorganic phosphate, glucose and amino acids. Pool and his colleagues formulated HTF which was free of glucose and phosphate.
Later experiments performed by Gardner and his colleagues supported these findings, further demonstrating the changing metabolic needs of the embryo between its cleavage stages up to 8-cells and later stages up to blastocysts. Cleaving embryos use pyrovate and lactate as energy sources and non-essential amino acids (NEAA) for protein metabolism. From the 8-cell stage the major energy source is glucose and for protein metabolism the embryos use essential amino acids (EAA). Gardner et.al. also showed significant differences in the concentrations of various metabolites between the fallopian tube and the uterus.
These findings lead Gardner and his colleagues to formulate the composition of two culture media G1 and G2 that are to be used in sequence. G1 supports the in-vitro development of the fertilized oocyte, the zygote, to the 8-cell stage, and G2 from 8-cells to blastocyst. Several modifications to these media were formulated also by other groups. Sequential media are now being used successfully in IVF treatment all over the world.
The composition of the embryo culture medium.
Culture media containing a phosphate buffer or Hepes organic buffer are used for procedures that involve handling of gametes outside of the incubator, flushing of follicles and micromanipulation.
PH and Osmolality: Most culture media utilize a bicarbonate/CO2 buffer system to keep PH in the range of 7.2-7.4. The osmolarity of the culture medium must be in the range of 275-290 mosmol/kg.
Culture conditions: Embryos should be cultured under paraffin oil, which prevents evaporation of the medium preserving a constant osmolarity. The oil also minimizes fluctuations of pH and temperature when embryos are taken out of the incubator for microscopic assessment. Paraffin oil can be toxic to gametes and embryos; therefore, batches of oil must be screened and tested on mouse embryos before use in culture of human embryos.
Temperature: The human oocyte is temperature-sensitive and a humidified incubator with a temperature setting of 37.0-37.5°C must be used for oocyte fertilization and embryo culture.
Water: The medium is composed of 99% water. Purity of the water is crucial, and is achieved by ultrafiltration.
Protein Source: Albumin or synthetic serum are added in concentrations of 5 to 20% (w/v or v/v, respectively). Today the commercial media includes synthetic serum in which the composition is well known.
Sources for protein supplements (serves today for research only)
Human cord serum (HCS) (difficult to obtain)
Human serum albumin (HSA)
Fetal calf serum (FCS)
Bovine serum albumin (BSA)
Commercial IVF media
Salt solution in MTF
Nacl, Kcl, Kh2PO4, Cacl22H2o, Mgso47H2O, NaHCO3
Carbohydrates are present in the female reproductive tract. Their concentrations vary throughout the length of the oviduct and in the uterus, and are also dependent on the time of the cycle.
Together with the amino acids they are the main energy source for the embryo. Culture media that support the development of zygotes up to 8-cells contain pyruvate and lactate. Some commercial media are glucose free, while others add a very low concentration of glucose to supply the needs of the sperm during conventional insemination.
Media that support the development of 8-cell embryos up to the blastocyst stage contain pyruvate and lactate in low concentrations and a higher concentration of glucose.
Supplement of the culture medium with amino acids is necessary for embryo development.
Media that support the development of zygotes up to 8-cells are supplemented with non essential amino acids. Proline, serine, alanine, aspargine, aspartate, glycine, glutamate.
Media that support the development of 8-cell embryos up to the blastocyst stage are supplemented with essential amino acids. : Cystine, histadine, isolucine, leucine, lysine, methionine, valine, argentine, glutamine, phenylalanine, therionine, tryptophane.
Their role in the culture medium is unclear.
The majority of ART laboratories use culture media containing antibiotics to minimize the risks of microbial growth. The most commonly used antibiotics being Penicillin (β-lactam Gram-positive bacteria disturbs cell wall integrity) and Streptomycin (Aminoglycoside Gram-negative bacteria disturbs protein synthesis). The anti-bacterial effect of penicillin is attributed to its ability to inhibit the synthesis of peptidoglycan, unique glycoproteins of the bacterial cell wall. Streptomycin and gentamycin belong to the aminoglycoside group of antibiotics which exert their antibacterial effect by inhibiting bacterial protein synthesis. The use of genthamicine is still controversial and it is not being used by every laboratory.
EDTA is used as a chelator in medium that supports the embryo from the zygote stage to 8-cells and prevents abnormal glycolysis.