Fresh Embryo at Blastocyst Stage: The cells are elongated and pressed against one another. The inner and outer cells are clearly visible, as is the cavity.

Two Vitrified Embryos at Blastocyst Stage After Warming: Though their appearances differ, both embryos implanted and created viable pregnancies.

This embryo looks perfect, as if it was never frozen. The outer and inner cells are clearly visible, as is the cavity.

This embryo has rounded, more dissociated cells resulting from shrinkage during incubation in cryoprotectant, (as cells shrink they pull away from each other). The cavity is small, but visible.

Here are some images from the different techniques that can be used for gender selection

To read more about this process, see our Ask The Experts post

The sequence shown below shows an 8-cell embryo on day 3 (#1) in which the cells are beginning to press up against one another (see 2 cells flattening against each other). After about 16 hours, the cells have divided again to produce a 16-cell embryo (#2) and they have compacted together even more. By day 4 of life (#3) the embryo is completely compacted to the point that individual cells can no longer be seen. At this stage the embryo is called a morula from the Latin for mulberry. The compaction process results in a fluid tight seal between cells and the embryo can now pump fluid into the center of the ball of cells. The cavitating morula (#4) also allocates cells to the inside and outside (#5) of the growing cavity or cyst. The outer cells will form the placenta of the implanting embryo and the inner cells will form the embryo proper or fetus, as well as some of the extraembryonic membranes such as the yolk sac. When the cyst is formed and cells have been allocated, the embryo is called a blastocyst (#6). The quality of a blastocyst is determined by the number of cells in the outer and inner populations as well as the degree of expansion of the cyst.

– Joe Conaghan, PhD, HCLD

Related Post: Day 3 vs. Day 5 Transfer

The morning after egg retrieval and the introduction of the egg to sperm, an embryologist observes the eggs and records their fertilization status. The following photographs reveal what the embryologist may see and the factors they examine when evaluating fertilization.

This is a mature, unfertilized egg. The presence of a polar body (circled in red) indicates that the egg was mature and ready to be fertilized. The absence of pronuclei and a 2nd polar body indicates that the egg failed to fertilize. Note the single sperm (arrow) attached to the outside of the eggshell.

This is a normally fertilized egg with two pronuclei (circled in blue) and two polar bodies (circled in red).

This is an abnormally fertilized egg with three pronuclei (circled in blue). In this case, because there is only one polar body, (circled in red), we would suspect that there are 2 maternal pronuclei in addition to the paternal pronucleus.

This is an abnormally fertilized egg with only one pronucleus, (circled in blue) and one polar body (circled in red).

This is an abnormally fertilized egg with four pronuclei (circled in blue).

Photo 2 Pro-nuclei:
This is a normally fertilized egg with 2 pronuclei. One nucleus contains half of the woman’s DNA and the other nucleus contains half of man’s DNA.

Photo 3 Pro-nuclei:
This is an abnormally fertilized egg with 3 pronuclei. One nucleus is from the woman’s DNA, but the other two pronuclei are from two different 2 sperm (each sperm contributing 50% of their DNA) that penetrated this egg. The resulting embryo will typically die within 48 hours because it has 50% more DNA than normal.

Photo Grade 1:
This embryo has even, symmetrical cell sizes. There are no cellular fragments. This is a beautiful grade 1, 8-cell embryo.

Photo Grade 2:
The embryo is developing slowly. It has 5 cells, however it should have 8 cells at this stage. Each of the 4 cells visible in this view are different sizes. This is a grade 2 embryo.

Photo Grade 3:
Although this embryo has 8 cells, many cellular fragments are present. These are scattered throughout the embryo. Cell sizes are uneven. This embryo is a grade 3. One positive characteristic is the flattening of cells against each other. This shows that the embryo is alive and attempting to develop.

In graph A (pregnant) DNA fragmentation index is nice and low at 7.5%. You can see clearly that there are very few sperm (7.5%) with moderate or high fragmentation and that most of the sperm are bunched tightly together with very little fragmentation. These healthy sperm were able to establish and maintain a pregnancy.

In graph B (not pregnant), the sperm DNA is much more unstable and there is a fairly even spread of low, moderate and high fragmentation. The DNA fragmentation index is 65% and these sperm were unable to establish a viable pregnancy.

Below we outline the five stages of embryo development: Fertilized Oocyte, Four Cell Embryo, Eight Cell Embryo, Morula, and Early Blastocyst.

1. Embryos are stepped through increasing (freeze) or decreasing (thaw) antifreeze concentrations in a 4-well plate.	2. Once saturated with antifreeze, the embryos are loaded into small straws.	3. The straws are placed in a controlled- rate freezer.
4. Once frozen, the embryos are placed in state-of-the-art, computer controlled storage tanks.	5. The straws snap into labeled metal canes. They are barely visible through the mist of the cooling nitrogen gas.	6. After thawing, it is not unusual to see one or more dead cells in an embryo. Arrow is to 1dead cell in 4-cell embryo.

To successfully freeze, or cryopreserve any cell, its water must first be removed. Otherwise, the water expands into ice crystals as it freezes, and this process bursts or kills the cell. To maintain embryo viability during cryopreservation, the embryo is bathed in antifreeze, which draws the water out of the cells while the antifreeze enters. (1) Made with propylene glycol supplemented with sucrose, the antifreeze is not harmful in any way, provided the embryos are kept cool.

After this first step, the embryos are loaded into small straws (2) and placed in a controlled-rate freezer, which cools the embryos at a rate of -0.3°C/minute until they reach a temperature of -38°C. (3) Then they are placed in, and stored in liquid nitrogen at -196°C. (4,5)

To thaw the embryos, the straw(s) are first warmed to room temperature and the embryos unloaded into a Petri dish. Then they are stepped through decreasing concentrations of antifreeze until eventually all the antifreeze has been removed and the embryos have been rehydrated. (1)

There is tremendous variability in how well embryos tolerate the freeze/thaw procedure, although surprisingly, it has little to do with the quality of the embryos at the time of freezing. A poor quality embryo will endure the process just as well as a good quality one. However, since poor quality embryos have a very low possibility of implanting, they are often not worth freezing.

While every care is taken to protect the embryos during the process, some embryos will have one or more burst cells (6) after they have been thawed. This cell loss results either from puncture by tiny ice shards around the embryo or from rupture as water rapidly enters the cell during thawing.

In 2003, over 80% of embryos survived freezing and thawing with one or more cells intact. On average, 2 out of every 3 cells in an embryo tolerated the process, and we consider an embryo with 50% or more of their cells surviving as having a normal chance of implanting after transfer. If an embryo thaws with fewer than 50% of its cells alive, we usually recommend thawing another embryo if one is available. At PFC 66% of embryos thaw with 50% or more of their cells intact.

Sperm morphology refers to the size and shape of the sperm.
The amount of abnormal sperm is high in humans compared to other mammals, but most of the defects are subtle. Obvious flaws, such as 2 tails or 2 heads, account for only a small percentage of the defects. Most inadequate sperm appear as too small, or with kinks in the tail or abnormally shaped heads. We are satisfied if a semen sample has 14% sperm with perfect morphology. Individuals with overall low sperm count may not have enough normal sperm in the sample to achieve fertilization. Even though sperm morphology issues are almost impossible to correct, they can be overcome by using ICSI to insert the sperm into the egg.