Embryo culture conditions: what embryos like best.

In London this May, there will be an unusual event: the celebration of two million children delivered after in vitro fertilization (IVF) procedures. The first “test tube” baby, Louise Brown, was born on 25 July 1978, demonstrating for humans what had already been shown for the mouse and the rabbit, namely that an oocyte could be fertilized by sperm in vitro, transferred to the reproductive tract, and establish a successful pregnancy. By the mid-1980s there were a few hundred IVF children, mostly conceived at Bourne Hall, near Cambridge, where Patrick Steptoe, who retrieved the eggs, and Robert Edwards, the embryologist and human IVF pioneer, established their clinic and research laboratory. What was originally labeled as “immoral, unethical and dehumanizing” (http:// news.bbc.co.uk./1/hi/health/3093429.stm; Ref. 1) has now become mainstream, transforming the treatment of infertility in both women and men, and bringing the gift of children to countless couples world wide. One birth in 50 in Western societies is now the outcome of IVF. Despite the triumph of IVF, there remain concerns about the procedures. Although the success of such artificial reproductive technologies (ART) has improved over the last 25 yr, a successful outcome for any couple is not ensured (2). Many embryos simply fail to implant, and there also remains the daunting outcome of multiple pregnancies, a problem that would be most easily overcome by reducing the number of embryos transferred to one. Finally, there remains the worry that IVF babies are at greater risk than natural conception babies for developmental abnormalities (3). For most species, it is the intrinsic quality of the oocyte before it is fertilized that primarily determines whether a zygote has the potential to progress through pregnancy (4). In general, human oocytes used for IVF are derived from a clutch of eggs collected by endoscopy after ovarian stimulation. Not all these eggs are of equivalent quality. Selection of embryos for transfer after they have divided only once or twice is usually based on their morphological appearance according to criteria that most embryologists recognize as arbitrary and imprecise (5). Accordingly, there has been an increased tendency to culture the embryos for several days before transfer (6). Because embryos that cleave fastest and reach the blastocysts stage earliest are likely to be the most developmentally competent (4), there is an obvious logic to this practice. Although oocyte quality is crucial for successful IVF, the culture conditions in which the zygote forms and subsequently divides plays a large part in determining whether that oocyte potential can be realized (4). Unfortunately, in vitro culture can lead to epigenetic changes in the embryonic genome (7) and also influence gene expression in a global manner (4, 8–10), which together might well result in adverse outcomes on development. The best known example in animal models is the “large offspring syndrome” frequently noted in ruminant species after embryos have been cultured in vitro under less than optimal conditions (3, 4). There are also indications that human IVF offspring might have a higher frequency of abnormalities at birth (11, 12). The number of low birth weight IVF babies raises another concern, namely whether these children will demonstrate subsequent overcompensatory adolescent growth, hypertension, and other aspects of FOAD syndrome (fetal origin of adult disease), which has been noted in naturally conceived babies of low birth weight (see Ref. 3). Culture media are now available that allow embryos to progress to blastocysts at rates comparable with those occurring within the uterus (12), raising the hope that such embryos will be free of the epigenetic marks introduced as a result of the stress of in vitro culture. Many of these media are based rather loosely on the concentrations of ions, amino acids, and sugars found in the reproductive tract of the female at the time of egg release, fertilization, and development (13). In the studies of Sjöblom et al. (14), for example, the focus of this commentary, a defined medium completely devoid of protein, allowed more than 95% of murine two-cell embryos flushed from the oviduct of naturally bred mice to advance to blastocysts. A key question is whether these blastocysts are of equivalent quality to those naturally conceived and developing in vivo. Some recent studies with mouse embryos have been reassuring in this regard in that they have shown that optimizing a culture medium in terms of its ability to promote embryo growth (15) or omitting a suspect ingredient such as fetal bovine serum (16) can avoid certain postnatal developmental and behavioral consequences attributed to the prior in vitro embryo culture. On the other hand, these studies also imply that minor deviations from optimal practice can lead to subtle, unintended consequences on the resulting pups. Oviductal and uterine fluids are certainly more complex than any of the culture media now commonly used for human, rodent, and bovine embryos and contain, among their mysterious ingredients, bioactive factors produced by the mother. Sjöblom et al. (14) examined the effects of one such factor, a cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), which is known to be present in the reproductive tract and to have a positive effect on embryo cell number, by including it during culture of their mouse embryos to the blastocyst stage. These cultured embryos were then transferred to recipient females and examined either late in gestation or at various times after birth. Prior culture in regular medium without the cytokine led to lower fetal growth, a more rapid compensatory growth after birth, increased body mass as adults and greater fat deposits in the abdomen compared with controls that had not been cultured as embryos. Males were particularly susceptible to these outcomes. Surprisingly, the embryonic exposure to GM-CSF greatly reduced all these side effects of culture except the adiposity. The lower fetal growth of previAbbreviations: ART, Artificial reproductive technologies; GM-CSF, granulocyte-macrophage colony-stimulating factor; IVF, in vitro fertilization.