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Oocytes: The oocyte retrieval begins approximately 34-36 hours after the shot of hCG. The hCG initiates oocyte maturation (nuclear and cytoplasmic) and biochemical and structural changes to the follicle. In general, oocytes are collected from about 85-90% of the follicles greater than 15 mm at the time of hCG. The oocytes will be covered by a large mass of cumulus cells (pic left). Although these cumulus cells aid in the discovery of the oocytes from the follicle fluid, they also obscure the view of the oocyte making the assessment of oocyte maturity difficult. In some male factor patients these cumulus cells are removed. In generally, about 80% of the oocytes will be mature at the time of oocyte retrieval.

Fertilization: Fertilization begins when the initial sperm penetrate the oocyte and is incorporated into the cytoplasm. Subsequently both the female and male chromatin will form pronuclei. These structures are similar to the nuclei of somatic cells and can be observed approximately 12-18 hours after sperm insemination or injection. At the time of fertilization check (Day 1), the embryologist will check the presence of two pronuclei as evidence of normal fertilization (pic left). Normal fertilization rates are about 70-75% of the oocytes inseminated. In addition, approximately 3-5% of oocytes will show the presence of three or more pronuclei are observed at the time of fertilization check (pic right). In most cases this is due to more than one sperm penetrating the oocyte at the same time. Such embryos have the abnormal complement of chromosomes and are hence discarded. The penetration of the oocyte is usually restricted to one sperm due to a hardening of the oocyte shell initiated by the first sperm penetration. However, this hardening takes several minutes to form, an occasionally another sperm will penetrate the oocyte prior to hardening completion.

Cleavage: Approximately 20-24 hours after sperm insemination or injection, the embryo will undergo its first cleavage division. Subsequently every 12-20 hours the embryo will divide again until day 3 or development (pic left day 2; pic right day 3). These divisions are maternally controlled and have little to do the paternal genome. In fact, it not until some time late day 3 or early day 4 that the embryonic genome (maternal and paternal combined) is activated, sometime between the eight- or 16-cell stage of development.

Compaction: After activation of the embryonic genome there is a clear morphological change to the embryo call compaction which is observed on day 4 (pic left). During the compaction stage embryo cells start to adhere to each other very tightly, forming tiny holes between them call gap junctions for communication and form a morula. This interaction between cells is also induces the cells of the embryo to loose their totipotency (ability of each cell to form an embryo on its own).

Compaction: About 24 hours after the formation of a morula, a central fluid-filled cavity forms called a blastocoel (pic right). It is at this stage where the first differentiation of cell types occurs. The cells that line the periphery form the trophectoderm which gives rise to the placenta. A second group of cells in the shape of a ball forms call the inner cell mass (ICM) goes on to form the baby (7 o'clock position in pic). The ICM cells are the controversial embryonic stem cells, which can form any cell of the body. In general, about 25-65% of the fertilized oocytes will form blastocysts. This can vary depending on maternal age, stimulation as well as other maternal and paternal intrinsic factors.

When is the best day to do embryo transfer: Day 3 or Day 5?
Currently, a large percentage of clinics have the ability to culture embryos to day 5 or 6. However, mostly clinics do some patients on day 3 and others on day 5 or 6. In general, the RSC favors day 3 transfers in older patients (greater than 38), in cases of poor embryo development already by day 3, or when number of available embryos is low (less than 4). However, each patient is individually evaluated based on the former described and other factors. In general, the pros and cons to each are described below:

Day 3

Pros:

• Embryos are placed in the uterus sooner
• More likely to have a transfer
• Possible higher pregnancy rate when embryo quality low

Cons:

• Less embryo quality information
• Embryos are transferred into the uterus prior to normal physiological time
• Higher numbers of embryos transferred; higher multiple pregnancy rate

Day 5

Pros:

• Much more embryo quality information
• Embryos get transferred into uterus at their normal physiological time
• Lower number of embryos transferred leaded to lower multiple pregnancy rate

Cons:

• No embryos may reach the blastocyst stage and no embryo transfer will occur
• Culture media may not support embryo development whereas uterus may have