Method and medium for in vitro culture of human embryos

Details Method and medium for in vitro culture of human embryos Method and medium for in vitro culture of human embryos

2001-02-20

2003-08-12

Crouch, Deborah (Department: 1632)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of culturing cells in suspension

C435S366000, C435S363000, C435S383000, C435S384000, C600S033000

Reexamination Certificate

active

06605468

ABSTRACT:

RELATED APPLICATIONS
This application is a §371 national phase filing of PCT/AU99/00499, having an international filing date of Jun. 18, 1999.
Infertility is a great concern to many couples who wish to conceive. The proportion of couples that are unable to conceive naturally is remarkably high. In the USA it is said that some 10-15% of couples of reproductive age are unable to have children, whereas in the United Kingdom the proportion has been estimated at 14%.
In the last 20 years or so some hope has been held out to infertile couples with the development of in vitro fertilisation (IVF) techniques. These IVF techniques generally take the form of stimulating the female to ovulate, contacting collected ova with sperm in vitro and introducing fertilised ova into the uterus. Multiple variations of this general process also exist. Despite considerable research and technical advances in the IVF field the rate of successful pregnancy following IVF treatment is still quite low and is in the order of 15 to 25% per cycle.
Undertaking an IVF program often causes great anguish, especially where there is no resultant successful pregnancy. It is presently believed that the poor success rate for IVF treatment is due to an extraordinarily high rate of early embryonic loss or implantation failure (Weinberg et al., 1988; Lenton et al., 1988).
The low efficacy of IVF, together with its high cost and the associated psychological trauma from repeated treatment failures make it desirable that improvements are made to the procedure. Current methods of increasing pregnancy rates during IVF treatment include placing multiple embryos (2-5) into the uterine cavity. This is not always successful and also carries with it a higher risk of multiple pregnancy.
In most in vitro fertilisation units embryos are transferred to the uterus 2 days after fertilisation (4-8 cells). One view is that the use of embryos at this early stage may contribute significantly to the low pregnancy outcome of IVF programs and that it is more desirable to use embryos at the blastocyst stage reached at day 5-7 of culture. The advantages suggested include improved synchronisation between embryo and uterus and the ability to select better quality embryos over the longer culture period. Blascocyst transfer may also help reduce the number of multiple births resulting from IVF, through allowing the selection of fewer numbers of highly competent embryos per transfer.
Unfortunately in standard culture media the majority of embryos (about 75%) fail to develop beyond the 4-8 stage. Nevertheless with certain clinical indications implantation of human embryos is performed at the blastocyst stage despite the low proportions of embryos that develop to blastocyst. Some recent studies have used co-culture techniques whereby embryos are co-cultured with feeder cells, for example Vero cells, which technique can more than double blastocyst formation rates (Ménézo et al., 1990; Plachot et al., 1995). There have been a number of studies using these co-culture techniques which have shown increased implantation rates after blastocyst transfer (Ménézo et al., 1992), particularly in women with repeated previous implantation failures (Oliveness et al., 1955; Plachot et al., 1955).
Co-culture is time consuming and expensive and concerns have been expressed about possible transfer of disease from contaminated cultures (Oliveness et al., 1955), in particular there is a concern relating to viral contamination which contamination is considered to be virtually impossible to fully eliminate. A safer and more practical approach is to attempt to produce a culture medium able to sustain embryo development through to the blastocyst stage that is independent of co-culture.
One approach to enhance in vitro embryo development without using co-culture techniques is to attempt to define factors that might be used to enhance embryo development in in vitro culture. A number of attempts have been already made to identify factors that might assist and amongst the promising factors are various stimulatory factors known as cytokines. One such factor, leukemia inhibitory factor (LIF) has already been indicated as being positive in this regard for humans (Dunglison et al 1996) and livestock species, U.S. Pat. No. 5,418,159.
One of the many factors also currently under investigation in both animals and humans relative to conception and embryo development is granulocyte-macrophage colony-stimulating factor (GM-CSF). However to date there has been no definite indication that a medium supplemented with GM-CSF would be sufficient to enhance the in vitro development of embryos to the blastocyst stage in a defined culture medium.
GM-CSF is a 23-29 kD glycoprotein which although secreted in a soluble form in vitro, is one of many cytokines known to be sequestered and immobilised in the ECM (extracellular matrix) in vivo through association with heparan sulphate. GM-CSF was originally characterised as a hemopoietic regulator and determinant of the maturation and behaviour of myeloid leukocytes in peripheral tissues. It is now known that GM-CSF is produced by a diversity of cell types including T-lymphocytes, monocytes, macrophages, fibroblasts, endothelial cells and epithelial cells.
The uterine epithelium has been identified by in situ hybridisation and in in vitro cell isolation studies as a major source of GM-CSF in the mouse uterus (Robertson el al 1992, Robertson et al 1994) and human oviduct and uterus (Zhao and Chegini 1994, Giacomini et al 1995). A role for GM-CSF in reproductive processes was supported by studies perturbing the cytokine environment during early pregnancy in vivo (Tartakovsky and Ben Yair, 1991) and experiments showing impaired fertility in genetically GM-CSF deficient mice (Robertson et al 1999).
Studies of radio-labelled ligand binding show clearly that murine blastocysts bind
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I-GM-CSF specifically, indicating that they express at least the low affinity form of the GM-CSF receptor. This conclusion was supported by RT-PCR analysis, which showed that blastocysts express mRNA for the &agr;-subunit of the GM-CSF receptor complex. A similar situation was found to exist in human embryos. GM-CSF-R was expressed at similar levels through the first four days of murine and human embryo development, from fertilisation to blastocyst stage. However mRNA for the &bgr;-subunit of the GM-CSF receptor complex was not detected in embryos of either species by the RT-PCR technique. Together, these data suggest that embryos express GM-CSF receptor from at least as early as fertilisation, but that it may be of the low affinity form. The embryo therefore falls into the same category as endothelial cells and other non-hemopoietic cells which exhibit a biological response to GM-CSF despite expressing only low affinity receptors. Although it seems clear in hemopoietic cells that the &agr;-subunit of the GM-CSF receptor cannot on its own transduce proliferative signal, it is not known whether the &agr;-subunit can in some circumstances initiate responses in cells in the absence of the &bgr;-subunit. The recent discovery of unconventional forms of the GM-CSF receptor in the human suggests that this may be possible.
It has also been shown that binding of cognate ligands to the GM-CSF receptor a subunit in isolation may mediate increased glucose transport via a phosphorylation-independent pathway (Ding et al., 1994). Recent experiments by the inventor show that culture with recombinant mouse GM-CSF (mGM-CSF) stimulates increased glucose uptake in murine blastocysts, to an extent achievable with known glucose transport stimulants such as insulin-like growth factor-1, suggesting that this cytokine may stimulate metabolism in murine embryos.
There is some evidence to indicate that GM-CSF also participates in regulation of embryonic growth. Conditioned media rich in mGM-CSF have been found to be effective particularly in promoting blastocyst development, particularly in the attachment of hatched blastocysts to serum attachment factors in plastic culture dishes (Robertson et al., 1991). T

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