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Collection and assessment of eggs through follicle puncture

The embryologists collect the eggs from the aspirated follicular fluid at the time of the ovarian puncture. The eggs are counted, washed and transferred to petri dishes which provide a special nutrition medium. The petri dishes are placed in an incubator where egg maturation continues.

Fertilization with classic IVF

In this method, sperm cells and eggs are placed together in a petri dish and the process of fertilization occurs spontaneously. The sperm cells overcome on their own the layer of cumulus cells and reach the shell of the egg and only one of them goes through and merges with it. For this type of fertilization is necessary the semen analysis to be good enough or with slight deviations. The staff at the andrology laboratory has prepared in advance the sperm cells for the fertilization. Next, a procedure for their purification and capacitation is carried out and their count and motility is measured. In the embryology laboratory we add the received sperm cells to the eggs in optimal concentration and then they are returned to the incubator. The next morning, the eggs are washed from the sperm and cumulus cells and are placed one by one into drops of nutritional medium. The eggs are checked and those which have fertilized normally are returned back to the incubator for further culturing.

Different functional defects in eggs or sperm cells can prevent fertilization. There are still no tests developed to identify the problem. In case of no success with IVF fertilization, ICSI is preferable in next attempts.

ICSI

In this method embryologists inject selected sperm cell directly into the cytoplasm of the egg. For this purpose, before proceeding to fertilization, the eggs undergo a gentle enzymatic and mechanical treatment for complete removal of the accompanying cumulus cells. Before the ICSI, these are placed in small drops in petri dishes. There is a small drop with a dense medium in each of these petri dishes and the sperm cells are added to the drop. The drop slows down their movement, which allows to see their morphology and to select the best ones. The selected sperm cell is immobilized and aspirated into the injection needle. In this procedure a special micromanipulator with special sterile individual micropipettes is used in order to perform the injection.
ICSI fertilization applies in cases of poor semen analysis, severe male factor, after testicular sperm aspiration or extraction, when thawed eggs are used, but also when the number of eggs is too small, poor egg quality, previous failures with classic in vitro, etc.The method of fertilization is discussed with your physician before egg collection.

Classic IVF/ICSI

In some cases, the eggs can be divided and both methods to be applied.

IMSI

This is an ICSI procedure where ultra high magnification is used to select the sperm cells before the fertilization. We usually work with a 60x magnification and we use glass bottom petri dishes. In IMSI a special lens is used which allows 100x magnification; an immersion oil which improves the image is also used. The method is based on morphology.

Selection of sperm cells with hyaluronic acid. This method is based on the ability of the sperm cell to adhere to the molecules of the hyaluronic acid, which is the main component of the extracellular environment of the cumulus that surrounds the egg. It is deemed that these sperm cells are mature and well prepared. The method is mainly functional.

Visualization of the spindle of the eggs (Spindle view). A polarized light which visualizes the spindle and the inner layer of the shell of the egg is used. A good egg has only one spindle. The location of the spindle is usually near the polar body, but in many cases it is shifted, which changes the angle of injection in an ICSI procedure. By the morphology of the spindle we can predict the further development of the egg. This technique also allows assessing whether the maturation is complete. If the egg is in a telophase of meiosis /the chromosomes have not yet separated /, it is needed to wait before the injection.

Activation of eggs through calcium ionophore

In certain patients a very low rate or lack of fertilization in a previous ICSI procedure is observed. In such cases, the eggs are incubated in a special medium containing calcium ionophore. Deficiency of calcium in the egg is one of the reasons for egg activation failure after injection of the sperm cell inside the cytoplasm.

Embryo culturing

The embryos are cultured in petri dish with nutritional medium drops covered with liquid paraffin, which prevents them from evaporating. The petri dish is placed in an incubator at a temperature of 370 C, 6% carbon dioxide and 5% oxygen. The cultivation lasts for 2-5 days by the time of embryo transfer or cryopreservation of the embryos received.

Estimate of developmental stage

The standard way of monitoring and evaluating embryo development is by taking the vessel containing the embryos out of the incubator once daily, in the morning, and placing it under a microscope. The embryos are monitored one by one and different parameters are recorded depending on the stage of development, number of cells, evenness of the cells, fragmentation, compactization, size of the blastocysts, etc.
• On day 1 following insemination, normally fertilized eggs have 2 pronuclei.

• On day 2 the embryo should divide at 2 to 4 cells.

• On day 3 the embryo is at 6 to 8 cells.

• On day 4 the division continues (12-16 cells) and normally the cells start to compact and cannot be counted. At this stage, the embryo is called morula.

• On day 5 blastocysts are formed. As a blastocyst, the embryo has an average of about 60 cells. At this stage a cavity in the embryo is formed and the cells differentiate to trophoblast, which participates in the formation of the placenta and inner cell mass of which the fetus develops.

The data is entered in the electronic file and stored in the protocol for the specific procedure. We track the individual development of each embryo and thus evaluate its quality.

Time-lapse embryo culturing in EmbryoScope

EmbryoScope combines the latest generation embryo incubator with a built-in microscope, a camera and a specialized software for monitoring and analysis of embryo development. It is a comprehensive system where embryos can develop and be monitored without taking them out. This reduces stress on the embryos and provides conditions for better development. The conditions for embryo culturing are very good and remain stable throughout the process. The EmbryoScope monitors round-the-clock all parameters such as temperature and gases and data are stored for each patient.

The camera captures the development of the embryo every 10 minutes, in seven focal planes. Unlike conventional monitoring under a microscope at a certain time, with EmbryoScope we can monitor the development in its dynamics, that is, how morphology changes over time. We are able to analyze the development and to track the division and changes in morphology in detail at any time. The analysis data are stored and can be used in future studies. We аrе building а mathematical model which will facilitate the selection of embryos with the highest implantation potential, which is different for each hospital. By choosing the most suitable embryo we increase the chances of success and the overall success rate.

Embryo transfer

The procedure for returning the embryos into the uterus occurs 2 to 5 days of fertilization. Your treating physician decides which day is most suitable for your embryo transfer. For embryo transfer we select the best embryos comparing their division and other morphological indicators. The selected embryos are placed in a petri dish with nutritional medium. An individual sterile catheter with syringe is prepared. When the doctor is ready to perform the manipulation, the embryos are drawn to the tip of the catheter in the form of a small drop. The catheter penetrates the cervix and under ultrasound guidance the embryos are placed in a suitable area in the uterus.

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