Specialised procedures for the embryo

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).