специализирани процедури при ембриона в болница "Надежда"

reproductive medicine

Specialised procedures for the embryo

Specialised procedures for the embryo include Embryoscope time-lapse monitoring and culture, preimplantation genetic testing (PGT), and assisted hatching. These are only available on recommendation by your fertility consultant and the embryologists. Another specialised procedure is preparing embryos for cryopreservation at the Embryology lab.

биолог от Андрологична лаборатория на болница "Надежда" извършва подготовка на сперматозоиди за инвитро процедура

Embryo culture

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.

Embryo development assessment

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.

TIME LAPSЕ embryo culture in Embryoscope

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.

Blastocyst biopsy for PGT

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.  

Preimplantation genetic testing (PGT)

PGT is a method for very early genetic diagnosis to select healthy IVF embryos before transferring them into the uterus. It involves the biopsy of polar bodies, blastomeres or trophectoderm cells from the embryo and subsequent DNA isolation and genetic analysis. PGT is subdivided into three main groups of tests: PGT-А (preimplantation genetic testing for aneuploidies, or the wrong number of chromosomes); PGТ-SR (preimplantation genetic testing for structural rearrangements); and PGT-M (preimplantation genetic testing for monogenic disorders).

PGT-А is recommended in advanced maternal age, recurrent miscarriages, failed previous IVF cycles, severe male factor. PGT-SR is applied in families where one or both parents are carriers of structural chromosome rearrangements, such as balanced translocations, inversions, or duplications/deletions. PGT-M is indicated in families where one or both partners are carriers or have a monogenic disease, so they risk transferring it to the offspring. 

PGT is justifiable in cases when it is known that the disease or condition of one of the partners could result in a stillbirth or a demise in early childhood. PGT has its grounds too in patients with diseases that have a longer lifespan but with multiple incurable and severe, often progressing physical or mental disabilities. Typically those are rare genetic diseases that are life-threatening or chronic debilitating illnesses with no effective or accessible treatment. In such cases, using PGT,  only healthy embryos can be selected for transfer. In this way, the mother is spared the health risks and the psychological trauma that would ensue if a spontaneous pregnancy would need to be terminated following results from invasive prenatal testing (chorion biopsy, amniocentesis) confirming genetic abnormalities of the foetus.

Thanks to the significant technological advances in assisted reproduction and genome analysis, PGT has the potential to be an indispensable tool in combating genetic disease and a crucial approach to its prevention.


We are proud that the team of the Genetics lab of Nadezhda hospital were the first who introduced preimplantation genetic diagnosis in Bulgaria back in 2008.

PGT is routinely performed at Nadezhda hospital for couples in which one of the partners has a chromosomal abnormality (a balanced structural translocation, inversion or other chromosomal rearrangements; mosaicism in one of the partners). Our team has years of experience with such cases.

In about 2–4% of couples with recurrent miscarriages, one of the partners is a carrier of the so-called balanced chromosome mutations – reciprocal and Robertsonian translocations, inversions, etc. This type of mutation is called familial because it is usually inherited by a healthy parent and is seldomly new. The carrier is typically healthy, but a certain percentage of their sex cells have a genomic imbalance. Such gametes have a poor fertilising capacity, resulting in implantation failure, miscarriages or the birth of offspring with chromosomal disorders. With carriers of chromosomal rearrangements, the miscarriage risk is higher with every subsequent pregnancy, varying according to the mutation type.

Since all the embryo’s chromosomes are usually tested, not only those involving the chromosomal rearrangement, the technique provides an opportunity to discover additional mutations in the remaining chromosomes.

Indications for this type of preimplantation testing are families where one or both partners are carriers of monogenic defects for diseases with different inheritance types: autosomal recessive, autosomal dominant, X-linked (sex-linked), and hereditary cancer syndromes (germline mutations).


Autosomal recessive disorders

With two parents who are both carriers of an autosomal recessive mutation, the risk for transmission to the offspring is 25%. Diseases with this inheritance type are beta-thalassemia (Cooley’s anaemia), cystic fibrosis, epidermolysis bullosa, spinal muscular atrophy, various metabolic disorders, etc.


Autosomal dominant disorders

With this type of inheritance, the risk is 50% for every child. This group includes Charcot-Marie-Tooth disease, neurofibromatosis, myotonic dystrophy, Huntington’s disease, osteogenesis imperfecta, familial adenomatous polyposis, and achondroplasia, among others.


Х-linked disorders

The risk can vary between 25% to 50% depending on the inheritance type, recessive or dominant. Diseases in this group include Duchenne muscular dystrophy, fragile X syndrome, haemophilia, incontinentia pigmenti, focal dermal hypoplasia, or Goltz syndrome. These disorders only affect one of the sexes, so healthy embryos of the opposite sex could be selected for transfer. Another option for selecting healthy embryos is DNA analysis for the specific mutation if available technology would allow it.

At Nadezhda hospital, PGT for the following monogenic diseases is successfully performed:

  • Duchenne muscular dystrophy;
  • Haemophilia;
  • Myotonic dystrophy;
  • Huntington’s disease;
  • Fragile X syndrome;
  • Epidermolysis bullosa;
  • Beta-thalassemia (Cooley’s anaemia).

Nadezhda hospital’s Genetics lab is partnering with the National Genetics lab at Maichin dom University Hospital, Sofia, to offer the best and most affordable options for PGT-M to all couples who might need it.


Options for doing a PGT-M test are finalised after medical genetics counselling with our specialists in medical genetics.

Indications for PGT-А include advanced reproductive age of the female, recurrent miscarriages, previous failed IVF cycles, severe male infertility.

In PGT-А, embryos are analysed for the total number of chromosomes. Approximately 70% of embryos resulting from spontaneous conception or an IVF procedure are lost before birth. Most of them are lost during the first trimester, some even before implantation. The primary cause for this is chromosome abnormality known as aneuploidy, resulting from the loss or addition of chromosomes.

At least 85% of aneuploidies originate in the egg. Sperm have a significantly lower contribution, to only 7-8% of all cases. The remaining aberrations occur accidentally during cell division in the early embryo stages.

It is known that, on average, more than 50% of embryos have a chromosomal abnormality. Since this percentage increases with age, this is probably the main reason women have difficulties conceiving and have more miscarriages with advancing age. The high incidence of chromosomal mutations adds to the benefit of doing PGT-A of embryos during IVF.

Some chromosomal abnormalities allow the pregnancy to be carried to term and live birth to occur (Down syndrome, Edwards syndrome, Turner syndrome, etc.); others result in preterm birth, miscarriage or embryonic development arrest even before implantation.

PGT can be performed only with in vitro fertilisation (IVF). The IVF for PGT itself includes controlled ovarian stimulation, oocyte retrieval by follicular aspiration, collection of sperm from the male partner, egg fertilisation, embryo biopsy, PGT analysis and the transfer of healthy embryos into the uterus.

Different biopsy techniques are used, such as polar body biopsy, blastomere biopsy of early embryos, and trophectoderm biopsy of blastocyst embryos.

The biopsy is done using a laser to make a tiny opening into the egg’s outer layer, through which one or two polar bodies are aspirated. This approach is informative only about maternal mutations, as it does not account for the paternal genome contribution or for mutations arising after fertilisation. The procedure typically does not affect the further development of the embryos.

Typically only one or two cells are extracted by the biopsy on day three after fertilisation, when the embryo is at the 6-8 blastomeres stage. The main disadvantage of this approach is the relatively high incidence of natural mosaicism in 3-day embryos. It is possible that the analysed cell has defects, while all the rest are normal; as well as the opposite scenario – the analysed blastomere to be normal, while the remaining cells have defects. In PGT-M, the isolation and amplification of DNA from a single cell also bears risks of ADO (allelic dropout – a partial amplification failure affecting one allele). This would result in misdiagnosis and is the main reason why this approach has been losing popularity in clinical applications.

This is the most popular approach currently. It is performed on day five or six after fertilisation, at the blastocyst stage when the embryo consists of 100–150 cells. The blastocyst is like a balloon with a small ball inside, composed of two layers of cells. The cell group on the inside is the one from which the foetus develops. The outer surface cells surrounding the cavity are called the trophectoderm, and they give rise to the future placenta. In trophectoderm biopsy, 5–7 cells predecessors of the future placenta are taken, and no future foetal cells are affected. The advantage of trophectoderm biopsy is that the analysis uses a larger number of cells and has higher precision and a lower risk of misdiagnosis due to mosaicism and ADO.

Preimplantation genetic testing bears minimal risks for the further development of the eggs and embryos. The risk of biopsy-related damage is less than 0,2% in oocytes and less than 1% in embryos. Approximately 5% risk of misdiagnosis exists (a false positive or a false negative) due to the embryo’s natural mosaicism or technical limitations of the method used. The analysis results could indicate that there is not one normal embryo of all tested, so that no embryo transfer can be done. In some cases, results could be impossible to interpret.

PGT success rates directly correlate with the success rates of the IVF clinic; therefore, PGT should be attempted only ат the best IVF clinics, after in-depth medical genetics counselling. If pregnancy occurs, in all cases, non-invasive prenatal testing is recommended to confirm PGT results. There is no increased risk of congenital anomalies in children born from a PGT-IVF procedure.

Currently, PGT of polar bodies, blastomeres and trophectoderm cells is available at Nadezhda hospital. Depending on the genetic mutation that needs to be analysed, different methods can be used. The genetics lab has equipment available for all current technologies of genetics analysis:

  • Fluorescent in situ hibridisation (FISH);
  • DNA microchip analysis (Array comparative genomic hybridisation (aCGH));
  • DNA Real-time polymerase chain reaction (RT-PCR);
  • Next-generation sequencing (NGS).

Assisted hatching

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.

Egg/ embryo cryopreservation

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.

Contract for egg/ embryo cryopreservation/ destruction

If you are having your embryos or eggs frozen, you will sign a contract for cryopreservation and storage, which specifies all the conditions of freezing and the period of storage of part or all of the viable genetic material, the rights and obligations of the parties, the methods of payment and termination of the contract. After the expiry of the contract, if you so wish, it may be extended by signing an annex. If you require any assistance or further information, please contact tel. 0887 701 927.


In accordance with Regulation No. H-2 of 12 July 2023 on assisted reproduction activities of the Ministry of Health

Art. 71. Good reasons for the withdrawal and destruction of ova, spermatozoa and zygotes are:

  1. the written certified wish of the patients;
  1. the expiry of the maximum storage period requested by the patients when signing the declaration of informed consent prior to cryopreservation;
  1. the risk of cross-contamination or transmission of bacterial or mycotic infection;
  1. the risk of cross-contamination or transmission of a transmissible disease (HIV, hepatitis B and C, syphilis, etc.);
  1. the expiry of more than 6 months after the requested storage period for unclaimed sperm, ova or zygotes;
  1. the death of the spouse/partner, irrespective of the existence of a declared wish during his/her lifetime, in respect of spermatozoa or zygotes, except in the cases referred to in paragraph 5.9 of Section IV of Annex 1 to Art. 1, т. 1;
  1. the death of the spouse/partner, notwithstanding the existence of a declared wish during lifetime, in respect of oocytes or zygotes;
  1. the establishment of a disease in an anonymous donor which can be transmitted to the offspring or the recipient;
  1. the impossibility of securely identifying donors or tracing the path of genetic material due to incorrect or missing labelling or confusion during processing.

See also

Specialised procedures for the male partner
Specialised tests for the female partner
Embryology lab
Assisted reproduction technology