The First World Congress On: Controversies in Obstetrics, Gynecology & InfertilityPrague, Czech Republic - 1999
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It has been claimed for along time that the longer the culture time is, the lowest the viability (Massip et al. 1984), even if most of the morphological aspect seems respected. Obviously, in vitro grown embryos have to be transferred to avoid any misleading observations based only on morphology. The ongoing concept was first that one culture medium could fit from fertilization to blastocyst (the ultimate embryonic phase in IVF): this concept is now obsolete. Blastocyst formation allows to understand most of the generic problems of embryogenesis (Paternal and maternal effects, cytogenetics...). Coculture was designed (Heyman et al. 1987). It was based in a first attempt to mimic either autocrine and paracrine interactions observed in vivo; The use of Vero cells (Mnzo et al. 1990) leads to a new approach based on the activity of the efficacy of transport epithelium and not on a specificity of genital tract cells. In this case, their is a continuous modification of the basic culture medium. However, already in this case, their is a strict dependency of the culture medium in coculture (Carney et al. 1990, Xu et al. 1992), also in relation with serum addition (Van Langendonckt et al. 1997).
In a first approach, a very simple medium can fit for fertilization. The commercial media are based on a Earle formula, with serumalbumin addition. A short fertilization time (Quinn et al., 1998) may lead to better embryo quality, without impairing fertilization rates.
Then during the first phase: 2 or 3 days post insemination, the medium has to have low glucose and phosphate concentrations. As the embryo cleaves under maternal store, it has to be protected against free radical injuries: Transcription for the protecting enzymes does not happen before genomic activation (El Mouatassim et al. 1999). EDTA and Hypotaurine are useful for this type of protection. Aminoacids are useful if not fully necessary. The notion of essential aminoacids has been evoked: it is yet impossible to define what is an essential aminoacid for the embryo; some metabolic pathways are present only in the embryo and not in the adult. Methionine is transported, used for methylations (through S-Adenosyl Met formation) and initiation of protein synthesis in the embryo (Mnzo et al 1989). Methylations are required for a normal mitochondrial function. Moreover, in the in vivo environment (the female genital tract), all the aminoacids are present.
Culture with the full set of amino acids in the first phase either using M3 medium (see Chouteau et al.1998) or Elliostep 2 gives excellent results (Table 1).
These data compete favorably with the ones published by Jones et al (1998) using S1/G1 in the first phase, where “essential aminoacids” are missing.
During the second phase, the culture medium has to be rich, containing reasonably high levels of glucose and free aminoacids. One important point is the presence of growth factors. It is not yet clear which one has to be added. However, recently we were obliged to stop our blastocyst program as blastocyst formation dropped: the negative effect observed for the second phase of culture was related to the absence of insulin during the second part of culture
One of the most important problem, in comparing culture media, is the patient indications: they are not similar from one group to the others.A good blastocyst program should have: Blastocyst formation around 45%, Ongoing mplantation rate of 25% per blastocyst. It is obvious that composition of culture media is important but more than one sequence of media can fit, if the basicr equirements are respected: this is mainly due to the rather high plasticity of embryo.
Carney E.W., Tobback C., Foote R.H. In vitro Cell. Dev. BIol.,26,629-635,1990 Chouteau J., Girard A.M., Sage J.C.etal. Human Reproduction, 13, 279-280, 1998.
El Mouatassim S., GuÃ©rin P., MÃ©nÃ©zo Y. Mol. Human Reprod., in press, 1999.
Heyman Y., MÃ©nÃ©zo Y., 1987. Iin “The Mammalian preimplantation embryo Regulation of growth and differentiation in vitro” B.D. Bavister ed., Plenum 175-191., 1987
Jones G.M., Trouson A.O., Gardner D.K. et al. Human Reprod.,13, 169-177,1998
Massip A., Van der Zwalmen P., Puissant F. et al. J. Reprod. Fertil., 71,199-204, 1984
MÃ©nÃ©zo Y., Khatchadourian Ch., Gharrib A.et al., Life Sciences, 44,1601-1609,1989
MÃ©nÃ©zo Y., Guerin J.F., Czyba J.C. Biol. Reprod. 42, 301-306, 1990
Quinn P., Lydic M.L., Ho M. et al. Fertil. Steril.,69,399-403,1998
Van Langendonckt A., Donnay I., Schuurbiers N. et al. J. Reprod. Fertil.,109, 87-93, 1997
Xu K.P., Yadav B.R., Rorie R.W. et al. J. Reprod. Fertil., 94, 33-43, 1992