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.

Until recently, the medium used for embryo culture has been a relatively simple solution of salts and nutrients. For most patients, this standard IVF culture medium supports the development of high quality embryos and results in high rates of implantation and pregnancy. However, a small number of patients have embryos that do not appear to develop well under standard culture conditions. For these patients, we consider the use of alternate culture systems.

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.

We also may use sequential media to benefit patients who experienced poor embryo quality in previous IVF cycles. Since they differ significantly in formulation from standard culture media, it is possible to provide embryos of repeat patients with a substantially different culture environment than that of previous IVF cycles.

There is considerable variation in embryo quality between patients. We know from our experience with co-culture that embryos from one patient may respond more favorably to one culture system than another. While the standard embryo culture medium has proven to be superior in the vast majority of our patients (we have used it to treat more than 6,000 patients), a small, selected group of patients may benefit from alternative culture systems.

 

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.

Fragmentation

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.

Multinucleation

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.

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