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The Embryo Lab


From Fertilization to the transfer…During the oocyte retrieval the oocytes are visualized by the embryologist, collected and placed in culture medium specially designed to give nutrients to the developing egg (oocyte) and embryo.
The oocyte, spermatozoa and embryo are placed in incubators to maintain the ideal conditions for their growth in vitro. The correct temperature and pH are maintained to mimic physiological conditions.

Several hours later the oocytes are ready to be placed with the spermatozoa.
If the method ICSI is necessary, then the cells surrounding the oocytes are removed in order to inject the single spermatozoa into center of the oocyte.
If ICSI is not needed then the oocytes are added to culture medium containing approximately 100,000 spermatozoa.

The next day, whether "classic" IVF or ICSI has been performed, fertilization is confirmed by observing 2 pro-nuclei (one containing half the number of chromosomes from the mother, the other containing the other half from the father).

The fertilized oocytes (zygotes) are selected and further cultured until the day of embryo transfer either a) on the 2nd day (2-4 cell stages) b) 3rd day (6-8 cell stage) or c) 5th/6th day (blastocyst stage).

In some cases a small incision will be made in the outer protein coat surrounding the embryo to assist implantation. (Assisted Hatching).
If many oocytes are fertilized and are of "good quality" the surplus of embryos may be frozen for a possible future attempt.
The embryo transfer is a painless procedure, performed without anesthetic-usually taking 5-10 minutes and can be likened to a simple PAP test. The embryos are placed in a catheter and introduced into the uterus vaginally.
The number of embryos replaced depends on their "quality", age of the patient and outcome of previous IVF attempts..



Conventional IVF involves the culturing of eggs and sperm overnight.
The sperm is prepared by centrifugation that separates the most motile of sperm from the rest of the sample.
Several hours after the egg collection a specific number of sperm e.g. 100,000 sperm are incubated with each egg.
It may take several hours for the sperm to penetrate the outer cells surrounding the oocyte before fertilization happens.
The eggs are then left until the following morning

When factors suggest that there might be a problem with the fertilization of the eggs.
In cases of low numbers of viable sperm or low motility, ICSI may be suggested as a method to increase chances of fertilization.
The sperm is prepared exactly as for conventional IVF.
A few hours after egg collection the eggs are stripped of the cells surrounding the outside the egg. The purposes of these cells in the fallopian tube is to stop sperm arriving at the egg at the same time, and are removed to visualize the egg for injection.
Using a powerful microscope and micromanipulators, the egg is held by a micropipette controlled by delicate joysticks.
A tiny needle picks up a single sperm. One sperm for each egg is injected into the cytoplasm (center) of the egg.
The eggs are then left until the following morning.
ΝΕW: IMSI technique (intracytoplasmic morphologically selected sperm injection) - a recently-developed innovative technique in the area of in-vitro fertilization (IVF) and complements the existing ICSI technique (intracytoplasmic sperm injection).

Culture of embryos


From this moment onwards whether the fertilization was by IVF or ICSI, the embryos are treated the same.16-20 hours after fertilization the pronuclei (pn) can be visualized. Eggs containing 2pn (DNA from the mother, and DNA from the father) are classified as fertilized and separated from the unfertilized.
The embryos developments are observed and a choice is made for which day of transfer.

 Assisted hatching


An Embryo undergoing Assisted Hatching.

A protein coat called a Zona Pellucida surrounds the embryo.
The developing embryo must escape from this outer layer to implant. It has been suggested that in some cases making a hole in the Zona Pellucida can facilitate the implantation of IVF embryos. (Assisted Hatching).
It is known that culturing embryos in vitro can increase the hardness of the zona and several studies have shown a beneficial effect on IVF results after Assisted Hatching.

Many recommend assisted hatching in patients >38 yrs or in patients with visibly thicker zonae than normal.

The hole in the zona pellucida is done by micromanipulation of the embryo with the hole being made either mechanically by needle, by chemical means or by laser.

The procedure is usually done just prior to embryo transfer and risk of damaging the embryo is very rare.

Blastocyst stage Transfer

A blastocyst is a stage of embryo development after 5 or 6 days of culture.

Shortly before implantation the embryo forms this structure consisting of an outer layer of cells (that will become the placenta) and an "inner cell mass" which will form the embryo itself.

The blastocyst increases in size and escapes from its protein coat (hatching).

Until recently it has not been possible to grow embryos to this stage with a great deal of success but owing to better understanding of the embryo's nutritional needs new culture media has been developed.

Normally embryo transfer occurs 2 or 3 days after egg collection, but transfer at the blastocyst stage can now be offered.

The advantages?
Blastocysts have a high implantation potential. Patients having blastocyst to transfer have a greater chance of pregnancy.

The disadvantages?
Not all embryos reach the blastocyst stage.
Not all patients have an embryo transfer because no embryos have developed to blastocysts.

When discussing blastocyst stage transfer you could consider
1) How many embryos are available
2) The possibility of an IVF attempt without embryo transfer.


Cryopreservation of embryos and sperm
For many years freezing semen samples have been a possibility.
Human semen was first successfully cryopreserved, with subsequent pregnancies and births in 1953.
Nowadays freezing semen and testicular tissue samples are a routine procedure in the embryological laboratory. The samples can be frozen before a treatment cycle to be thawed when needed.
Equally important is the cryopreservation of embryos. If a sufficient number embryos are available, then one option is to freeze the surplus (spare) embryos for possible use at a future date.
Usually embryos are frozen on the first day after fertilization, on the second day (4 cell stage) or at the stage of blastocyst formation (5/6 days after fertilization).
Cryopreservation gives us the possibility to store the embryos that will not be transferred in the present cycle for a later date without the cost and inconvenience of a stimulated IVF cycle. Also, in rare occasions when we wish to delay the transfer to avoid the risk of ovarian hyperstimulation syndrome or when we wish to wait for a time when endometrial receptivity will give us a better chance of pregnancy.
Storage is in liquid nitrogen at -196*C.
Although there is no theoretical time limit of storage you will be asked to sign a consent form according to the IVF center policy / IVF legislation ascertaining to the length of time allowed for Cryopreservation.
If the couple at a future date wishes to transfer their embryos/semen to another IVF center this is of course a possibility by transporting their samples in miniature liquid nitrogen containers can "Dry Shippers".



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