The laboratory part of in vitro fertilization is initiated by transferring follicular fluid containing oocytes obtained by transvaginal follicle aspiration and ends by embryo transfer into the patient’s uterus. All accompanying processes take place in embryological laboratory. After transferring the follicular fluid to the embryological laboratory, all oocytes retrieved during the puncture are detected. Eggs are cleaned using pipettes and fine needles and placed in a medium inside a cultivation plate, which is labelled with the patient’s name.
The plate is then kept in a cultivation box, with optimal conditions or oocyte fertilization and embryo development. Partner’s sperms are added after 2 – 6 hours interval after egg retrieval. Contact of eggs and sperm can then take place and result in fertilization.
Whether the eggs have really been fertilized and whether fertilization was successful, can be verified 18 hours after the fertilization.
The result is registered, nutrient solution is replaced by a new one and cultivation continues. Two days after fertilization embryos consist of 2 – 4 cells and can be transferred into the uterus.
No more than 2 ( exceptionally 3 ) embryos are prepared for transfer. If there are more embryos obtained as a result of IVF treatment they are frozen and stored for a later embryotransfer.
Good quality supernumerary embryos with normal development are used for freezing (cryo-conservation). Prior to freezing, embryos are exposed to a cryoprotectant, which prevents damage during cryo-conservation. Grouped embryos (usually 2) are stored in special straws. Using programmable equipment, the temperature is slowly reduced to –140°C, when the embryos are stored in containers filled with liquid nitrogen. During thawing the straw with embryos will warm up to indoor temperature, cryo-conservation solution is removed and embryos are placed back to normal mediim in cultivation box. The cleavage of embryos is verified prior to transfer. Apart from embryos, it is possible to freeze male gametes (sperm) and recently also oocytes (non-fertilized eggs).
Vitrification is a modern technology for cryopreservation (freezing) of embryos and eggs, with better results, comparing to the classsical slow cooling method. Vitrification is the very rapid cooling method, avoiding the formation of intracellular ice crystals, the main reason of damage the cells of the embryo. Vitrification is optimal method of cryopreservation particulary for blastocysts and/or not-fertilized eggs, giving more than 90% o viability after warming
During extended cultivation, embryos are cultivated in a special media enabling their development to a higher embryonic phase. During this culture embryos, which do not cleave normally ( and are not able to implant), are identified. By this technique it is possible to choose the top quality embryos (mainly blastocysts) for transfer, thereby increasing the chance of successful treatment. The blastocyst is the most advanced embryonic stage achievable in conditions outside the patient’s body. Blastocysts have the highest implantation potential.
Not all embryos are, however, able to achieve this stage and it is possible that not a single embryo will be frozen even if there was a large number of embryos created and it is even possible that the transfer will not occur. This is the case when no embryo reaches the desired developmental stage.
If a couple is treated for male factor infertility (low count of sperm present in ejaculate, abnormal morphology or motility) or other immunological or so-called unclear (idiopathic) reasons or if in previous cycle fertilization failed, the technique of ICSI (intracytoplasmic sperm injection ) is used to fertilize the eggs.
For the purpose of oocyte fertilization, one sperm is aspirated inside a tiny glass pipette and then injected inside the ovum. After standard culture embryos are either ready for transfer or cryo-conservation.
PICSI (assisted fertilization) is a modified method of ICSI, making it possible to select a functionally competent sperm. Via this method a sperm of optimal maturity can be selected to fertilize an egg. The principle of this method lies in a binding of a mature sperm to a specially treated surface of the PICSI dish (Petri dish) coated with a layer of gel containing hyaluronan. This substance is an important natural part of egg shell. The head of a sperm bears a specific receptor that allows its binding to hyaluronan. At PICSI method, an embryologist selects those sperm that show any positive binding to hyaluronan.
The other micromanipulation technique used in embryological laboratory is assisted hatching. With a very delicate sharp needle (or special laser) the zona pellucida surrounding the embryo is opened to help the embryo to hatch. This technique is helpful especially in repeated IVF cycles, in elder women and in women with elevated FSH levels. On the contrary, according to our data it is not suitable for frozen-thawed embryos.
Primo Vision - Embryo evaluation with less stress!
Using Primo Vision Time-Lapse Embryo Monitoring System inside the incubator gives us the opportunity to observe all embryos without disturbing them or changing our daily routine. The system records what happens inside the incubator, giving us the chance to objectively assess embryo development, in order to transfer the best quality embryo.
Pre-implantation genetic diagnosis- PGD
The routine use of pre-implantation genetic diagnosis (PGD) was possible due to the advances in embryology and genetics. PGD enables to detect genetic abnormities (inherited or acquired) in embryos before they are transferred into the uterus. Using this technique only healthy embryos are transferred without a risk of induced abortion (in case of affected foetus) or the risk of a delivery of genetically handicapped child. For genetic analysis from each embryo in the eight-cell stage one to two blastomeres (cells) are biopsied for examination in the genetic laboratory. Only embryos with normal genetic information are suitable for transfer into the uterus. The embryo biopsy itself does not affect the embryo’s development.
Using this technique it is possible to diagnose also single gene abnormities. All presented techniques can be combined with the purpose to obtain the top quality embryos for transfer or cryo-conservation.