Protein Macromolecules

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Historically, the most commonly used protein source in human IVF and embryo culture was patient's serum, added to the culture medium at a concentration of 5% to 20%. In some programs, fetal cord serum was used in preference. The use of serum in embryo culture media has several inherent drawbacks: the considerable expense and time required for its collection and processing (and screening of the fetal cord serum), the risk of infection to the laboratory staff, as well as the added stress to the patient. Serum contains many components which are poorly characterized. Furthermore, proteins in serum have macromolecules attached, such as hormones, vitamins and fatty acids, as well as chelated metal ions and pyrogens (184,185). As the concentration of such macromolecules and other serum components varies between patients and even within the menstrual cycle, it makes any comparison between batches of medium which contain serum almost impossible. Furthermore, serum from several groups of patients such as those with endometriosis, PCO or unexplained infertility appears to be embryo-toxic (186-190). There are several reasons for the elimination of serum from mammalian embryo culture systems. From a physiological perspective the mammalian embryo is never exposed to serum in vivo. The fluids of the female reproductive tract are not simple serum transudates (191), but rather specialized environments for the development of the embryo (30). Serum can best be considered a pathological fluid formed by the action of platelets. More disturbing however, is the growing evidence that serum is detrimental to the developing mammalian preimplanta-tion embryo in culture. Studies on the embryos of mice, sheep and cattle have demonstrated that serum in the culture medium induces morphological, metabolic, genetic and ultrastructural changes in blastocysts cultured from the zygote stage. The trophectoderm of such blastocysts develops a vesicular appearance due to the sequestering of lipid in the blastomeres (37,102,128,192,193).

Furthermore, when ruminant pronucleate embryos were cultured to the blastocyst stage in the presence of serum they possessed mitochondria with abnormal folding of the cristae, possibly associated with reduced oxidative capacity (128,192). Such blastocysts exhibited elevated levels of lac-tate production, plausibly associated with mitochondrial damage and impaired oxidative capacity (102). Finally, the inclusion of serum in the culture medium is associated with the birth of abnormally large lambs after the transfer of blastocysts to recipient ewes (128,194). Such data is of great concern and the mechanism(s) by which serum imparts such detrimental effects is the focus of much research. The over expression of certain growth factor genes in this phenomenon is a plausible mechanism (195-197). For example, fetal overgrowth in the sheep following embryo culture in the presence of serum has been associated with a decreased expression of M6P/IGF-IIR through loss of methylation (196). M6P/IGF-IIR has a role in fetal organogenesis. Interestingly, this locus although imprinted in mice, sheep and cows, is not imprinted in the human (198). Subsequently, this specific absence of imprinting may mean that the human embryo is less susceptible to epigenetic disturbances. This may therefore explain why Menezo and colleagues (199) have not reported any adverse effects in children following blastocyst co-culture in the presence of serum, but that studies on mice, sheep and cattle have all revealed long term effects on embryos cultured in the presence of serum.

So why was serum included in human embryo culture media? Undoubtedly the main reason for the inclusion of serum in media used in human IVF is the limited ability of simple salt solutions and tissue culture media to support embryo development in the absence of serum. In a suboptimal medium serum can act as a chelator and a buffer to minimize pH fluctuations when medium is outside of a CO2 environment. It may serve to supplement simplistic media with known regulators of embryo development such as amino acids, whereas when added to more complex tissue culture media such as Ham's F-10, it may help by binding the embryo toxic transitional metals present. However, with the development of more physiological embryo culture media designed to fulfill the changing requirements of the embryo, and the inclusion of appropriate chelators, the requirement for serum in embryo culture has been eliminated (37,38,40,46,200). Serum should now reside only in the annals of embryo culture, and certainly not in the media.

Protein can be added to culture media in the form of serum albumin. The addition of a macromolecule such as serum albumin prevents gamete and embryos from becoming "sticky" whereby their surface charges make them stick to both glass and plastic. Macromolecules, therefore, facilitate gamete and embryo manipulation. Furthermore, albumin can negate the effects of toxins (201). Both human serum albumin (HSA) (46,202204) and bovine serum albumin (BSA) (205) have been used successfully in the culture of human embryos. The use of HSA requires adequate screening for HIV, hepatitis etc., while the use of animal products in human ART is no longer acceptable. More recently, several commercially available serum products have been used to great success in replacing serum in human embryo culture systems. These range from therapeutic albumin solutions (202,204,206) to globulin enriched albumin solutions such as Plasmanate (207), Plasmatein (208) and Synthetic Serum Substitute (SSS) (209-211). Of the latter products, SSS appears to be the most effective containing 84% HSA and 16% a- and p-globulins with less than 1% y-globulin. It has been proposed that the glycoprotein components of serum (a- and p-globulins) have a role in supporting embryo development in culture. Glycoproteins, which possess numerous hydroxyl groups, may confer benefit to the embryo by altering the solvent properties of the medium, making it more akin to the tubal environment (208,212). However, there have been no prospective randomized trials using such supplements.

Although serum albumin is a relatively pure fraction, it is still contaminated with fatty acids and other small molecules (213). The latter includes an embryotrophic factor, which stimulates cleavage and growth in rabbit morulae and blastocysts. This factor has been determined to be citrate (214). Not only are there significant differences between sources of serum albumin (215,216), but also between batches from the same source (215,217). Furthermore, some HSA preparations contain the preservative sodium caprylate, which binds to the hydrophobic domains of the proteins and therefore cannot be removed by dialysis (Pool TB. Personal communication, 1998). The effects of such a preservative on embryo development have yet to be determined. Therefore when using serum albumin or any albumin preparation, it is essential for each batch to be screened for its ability to adequately support embryo development in the mouse prior to clinical use. Importantly, recombinant human serum albumin has recently become available, which eliminates the problems inherent with using blood derived products, and certainly eliminates variability between lots. The use of recombinant human albumin has been validated in a prospective randomized trial and has been found to be equally as effective as human serum albumin in supporting IVF, embryo development in vitro, and subsequent pregnancies (218,219). Furthermore, the inclusion of recombinant human albumin appears to confer increased cryotolerance to those embryos cultured in its presence (11).

Finally, there is much interest in developing alternative macromole-cules to serum albumin, thereby facilitating the formulation of more defined culture media. The synthetic polymer polyvinyl alcohol (PVA) has been used extensively by Bavister's laboratory (220), although its effects on postimplantation development have yet to be fully elucidated in prospective trials. A major component of oviduct and uterine fluids are glycosaminogly-cans and proteoglycans. Similar to glycoproteins, glycosaminoglycans and proteoglycans have the capacity to attract cations such as sodium due to their high density of negative charges. Therefore these molecules will also alter the solvent properties of the medium. The glycosaminoglycan hyalur-onate can substitute for albumin when added to the culture medium and increase the cryotolerance of embryos (219,221,222). Interestingly, albumin and hyaluronate act in synergy to further increase mouse blastocyst development in culture. The addition of hyaluronate to the culture medium has also been shown to increase blastocyst development in porcine embryos (223). Of greatest significance however is the finding that the addition of hyaluronate to embryo culture medium significantly increases mouse blastocyst implantation and fetal development after transfer (221). Interestingly, this increase in viability was found to be due to the presence of hyaluronate in the medium used for transfer (Fig. 4) (221). As the human endometrium and embryo expresses the receptor for hyaluronate (224), it is plausible that hya-luronate is involved in the initial phases of blastocyst attachment to the endometrium. Therefore studies on the role of such glycosaminoglycans in human embryo development and transfer are warranted.

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100 Pregnancy Tips

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