Embryo Culture Systems
The earliest stages of human development, until day five or six after fertilization, normally occur in the woman’s fallopian tube (or oviduct). However, after in vitro fertilization (IVF), much of this period of early development occurs in the laboratory. The conditions under which the embryos are “cultured” have been carefully formulated to provide an environment that mimics – as closely as possible – that of the fallopian tube.
Recently, commercially prepared culture media (generally termed sequential media) have become available. These media support embryo development in the laboratory for up to six days. By allowing the embryo to reach the blastocyst stage, we can make a more stringent selection of those to be transferred during an IVF cycle. As a result, these systems may be preferable for patients who would prefer or benefit from a one- or two-embryo transfer.
Embryo Development and Selection
Eggs retrieved from the ovaries are inseminated with sperm during therapeutic in vitro fertilization (IVF). Fertilization must be confirmed by the embryologist and embryo development carefully monitored thereafter. On the first, second and third days of development, embryo quality is evaluated based on key morphological markers, including the number of cells, cell size and symmetry, multinucleation (more than one nucleus in each cell) and the presence of cytoplasmic fragmentation. The thickness of the zona pellucida, the protective shell surrounding the developing embryo, is also a consideration for embryologists as they select the “best” embryos for replacement in the uterus.
Although some morphologically manifested abnormalities are known to reduce implantation and pregnancy rates – especially when all available embryos are affected – laboratory techniques may ultimately change the outcome. Selective assisted hatching may be used on embryos with thick or abnormal zonae and fragment removal, in conjunction with assisted hatching, may improve the chances of implantation when embryo development is hampered by cytoplasmic fragmentation.
Cleavage Rate, Cell Number and Symmetry
The rate of cleavage (cell division) is an important predictor of an embryo’s developmental potential. Evidence indicates that early cleavage, embryos with four cells on day 2, and embryos with seven to nine cells on day 3 result in higher implantation rates and establish more pregnancies than those with fewer or more cells at those time-points. Based on this, we preferentially replace seven to nine cell embryos on day 3 and consider others for cryopreservation if they meet additional quality standards.
Uneven cleavage is common among human embryos developing in-vitro and there is general agreement that replacement of embryos with this characteristic results in lowered pregnancy and implantation rates. This may be due to an unequal distribution of cellular components among uneven cells or the occurrence of more nuclear abnormalities among them. As a result, these embryos generally are not selected if others are available.
Embryos without fragmentation or minor fragmentation are selected for replacement and cryopreservation, provided that they are at an appropriate stage of development. If the only embryos available have extensive fragmentation, selection is based on the degree and the pattern of fragmentation. In these cases, application of assisted hatching and fragment removal may restore developmental potential and improve the overall pregnancy and implantation rate in patients with an otherwise poor chance for pregnancy.
A. A normally fertilized egg showing two pronuclei
B. An uneven 6-cell embryo with one multinucleated cell
C. An 8-cell embryo with minor cytoplasmic fragmentation
Following the first division, some blastomeres in human embryos show multiple nuclei rather than the normal single nucleus. Possible causes are the lack of appropriate oxygen levels during follicular development or a rapid response to hormones during ovarian stimulation. Regardless of the cause, implantation and pregnancy rates decrease with increasing proportion of embryos with multinucleated cells replaced in the uterus. These embryos also have a considerably reduced ability to reach the blastocyst stage in extended culture. The selection of such embryos for replacement is avoided if at all possible
Blastocyst Culture and Transfer
The first human pregnancy after in vitro fertilization (IVF), reported in 1976 by Robert Edwards and Patrick Steptoe, was achieved after replacement of a single blastocyst in the uterus. Although that pregnancy was ectopic (occurring outside the uterus) and was not delivered, it demonstrated the feasibility of extended embryo culture and replacement at the blastocyst stage.
In the years that followed, blastocyst transfer was virtually abandoned because it was very difficult to obtain blastocysts in culture using the available technology. It was also argued that reducing the time spent in culture to a minimum may be beneficial to the embryo and improve pregnancy results. However, in 1985, IRMS Scientific Director Dr. Jacques Cohen and his colleagues at Bourn Hall Clinic in England reported the first successful pregnancies after replacement of frozen-thawed blastocysts; this generated renewed interest in blastocyst culture and replacement. The recent re-emergence of this technology is a result of major advances in culture techniques and media preparations that more consistently support the development of early human embryos for five to six days.
At the blastocyst stage, an embryo contains approximately 100 cells. It has developed to the point at which, following hatching from the zona pellucida, its implantation is imminent. The cells on the outer layer of the blastocyst eventually form the placenta, the sac that protects and nurtures the developing fetus during pregnancy, while the fluid-filled inner core of the blastocyst contains cells that will form the fetus. Blastocysts are easily recognizable, allowing for relatively easy selection by the embryologist. By contrast, embryo selection during earlier cleavage stages is more difficult and requires more experience.
IRMS and many other IVF programs reserve blastocyst replacement for patients who are under the age of 40, produce 10 or more eggs and are at risk for high order multiple pregnancy. The eggs must be fertilized normally and exhibit good development during the first three days in culture. It is becoming increasingly evident that many patients and embryos do not benefit from this treatment modality. For instance, patients with poor embryo morphology are encouraged to have replacement of day 3 embryos with assisted hatching and fragment removal, since these embryos do not tolerate extended periods in culture. Those over 40 years of age may be more likely to achieve an ongoing pregnancy after preimplantation genetic diagnosis and replacement of embryos that appear free of genetic abnormalities that can affect implantation and miscarriage rates.
Studies at IRMS and elsewhere indicate that pregnancy rates for blastocyst transfer are similar to those resulting from day 3 embryos. However, implantation rates may be higher. Also, since replacement is limited to two blastocysts, high order multiple pregnancies with three or more fetuses are largely avoidable – as they are when day 3 replacements are limited to two embryos in patients with very good pregnancy prognosis.
Articles with additional information on Blastocyst Culture can be found in our research articles page.
Disadvantages of Blastocyst Replacement
While nearly 80% of fertilized eggs survive through the third day in culture, even under ideal circumstances, only about 30%-40% become well-structured blastocysts by the fifth day. Although embryos are carefully examined on day 3 and selected for extended culture, there are no guarantees that they will continue to develop. As a result, a couple with viable embryos on day three could end up with no blastocysts for replacement. Moreover, there may be fewer embryos available for cryopreservation and the freeze/thaw process is less successful for blastocysts than those frozen at earlier stages of development.
There is also concern regarding the sex ratio among children born after blastocyst replacement. At least one large study suggests that more males than females are born. Another possible risk of this treatment is the apparent increase in the incidence of monozygotic (identical) twinning, and the associated obstetric complications of such pregnancies.