Specialised procedures - embryo

Embryos are cultured in Petri dishes in drops of culture medium overlayed with mineral oil that prevents evaporation. Petri dishes are placed in incubators at a tem

perature of 37°C, in a gas mix containing 6% CO₂ and 5% oxygen. Embryos are cultured for 2 to 5 days when the procedure is completed by embryo transfer or embryo cryopreservation.

The routine method for monitoring and assessing embryo development is by taking out the Petri dish containing the patient’s embryos from the incubator and placing it under the microscope once daily. Each embryo is evaluated individually, and various parameters are registered according to its development stage, such as the number of cells, symmetry, fragmentation, compaction, blastocyst size, etc.

• On day one after fertilisation, two pronuclei can be observed in normally fertilised eggs.
• On day two, the embryo should have reached the 2–4 cells stage.
• On day 3, the embryo should be at 6–8 cells;
• On day 4, cell division continues, the cells should be 12–16, and compaction should be starting;
• On day, blastocysts are formed.

Data is recorded in the electronic file and saved in the IVF cycle report. We monitor the development of each embryo individually, and thus we evaluate its quality.

The embryoscope combines a last generation incubator with an integrated microscope, camera and specialised software for monitoring and analysis of embryo development. It is a complete closed system where embryos can be cultured and observed without being taken out of the incubator. This reduces the stress for embryos and is a prerequisite for their better development. Embryo culture conditions are excellent and remain stable at all times. The embryoscope controls environmental parameters such as temperature and gas composition round the clock, with data being logged for each patient.

The camera documents embryo development every 10 minutes in seven different planes. In contrast to conventional microscopy, the embryoscope allows the observation of the dynamics of embryo development – the way embryo morphology is changed over time, providing data for development analysis, with continuous monitoring of embryo cleavage and transformations at every single moment. The data collected can be stored and used to build a mathematical model assisting the assessment so that the embryos with the highest implantation potential are selected. The possibility to choose the best embryo improves the chances of success.

Some embryos have an abnormally thick outer layer, or “shell”. To facilitate their “hatching” or breakthrough and subsequent implantation, careful thinning and opening of the outer layer, the zona pellucida, can be done by a laser before embryo transfer. Cryopreservation is thought to result in the thickening or hardening of the zona, so we apply assisted hatching to facilitate implantation.

PGT (preimplantation genetic testing) is a group of genetics techniques that allow us to analyse the genetic material of the embryo before its implantation. In this way, miscarriages and medical abortions can be avoided if an affected embryo is identified. The test is done at the blastocyst stage, by performing a biopsy of trophectoderm cells. The capacity of PGT includes looking for a particular gene, a check of the number of one or more chromosome pairs, as well as full chromosome analysis. The biopsy is performed using a laser integrated into the microscopes, through which a tiny portion of cells is cut out. Micropipettes are used to extract the material for genetic analysis, after which the embryo is frozen until the results come out.  

Embryo transfer typically takes place on days 2 to 5 after fertilisation. Your fertility consultant will decide on the time of embryo transfer, taking into account the specifics of each case. Before the procedure, the best embryos are selected based on their development and morphokinetic scores. The embryos chosen for transfer are placed in a Petri dish with a nourishing medium. An individual sterile catheter is prepared and attached to a syringe. When the doctor is ready to proceed, the embryos are loaded into the catheter in a small drop near its tip. The doctor introduces the loaded catheter through the cervix and into the uterus under ultrasound guidance and deposits the embryos at the best position for implantation.

Cryopreservation is used to maintain the viability of cells over long periods. It is achieved by freezing and liquid nitrogen storage at ultralow temperatures – 196°C. At Nadezhda hospital, we employ vitrification, the most modern and effective cryopreservation technique. Vitrification is a freezing method with ultra-fast cooling rates. Upon contact with liquid nitrogen, the intracellular water transitions into a glass-like (vitrified) state without forming ice crystals. Ice crystal formation is the leading cause for loss of cell viability if freezing is attempted with no special cryoprotectants or techniques. In vitrification, commercially developed media are used to replace a part of intracellular water with cryoprotectants. In this way, cells have additional protection from cryogenic damage. After incubation in a cryoprotectant solution, the oocytes or embryos are placed onto special devices (the so-called straws) and directly submerged into liquid nitrogen. The technique is highly reliable, yielding more than 95% preserved viability and development potential, both in embryos and oocytes.

Through the ‘Keep Hope Alive’ project, Nadezhda Hospital is actively engaged in fertility preservation for cancer patients and patients with autoimmune and rheumatoid diseases.