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The Genetic Laboratory offers a large number of tests, mainly related to the diagnosis of reproductive problems - karyotyping (postnatal cytogenetic analysis, chromosome analysis); postnatal microarray analysis of microdeletion syndromes; prenatal genetic diagnosis via amniocentesis, chorionic villus sampling, non-invasive prenatal test; preimplantation genetic diagnosis/screening; microarray analysis of abortion material; DNA analysis for Y chromosome microdeletion; DNA analysis of congenital thrombophilia; HPV-genotyping.

We are proud that the lab is the first in Bulgaria to introduce pre-implantation genetic diagnosis of embryos.

Postnatal (postpartum) chromosome analysis (karyotyping, cytogenetic analysis)

The cytogenetic analysis is used to search for numerical and structural chromosome abnormalities. The chromosomes are obtained through leukocytes of peripheral venous blood taken after meal. The analysis is done under a light microscope at 1000-fold magnification.

Karyotyping is the first method of choice to search for genetic causes of male and female infertility. In these patients, cytogenetic analysis is mandatory, especially prior to an assisted reproduction, including insemination.

The indications for chromosome analysis include:

Partners with reproductive disorders: two or more miscarriages; lack of pregnancy up to 1 year; infertility of unknown etiology; birth of a child with malformation syndrome; stillbirth; azoospermia, severe oligoasthenoteratozoospermia, amenorrhea, early menopause; chromosome aberrations observed in prenatal diagnosis; proven family chromosomal re-arrangement in relatives.

Patients with a developmental disorder: multiple congenital abnormalities; suspected chromosomal disease based on clinical symptoms; abnormalities in growth, sexual differentiation and development; mental retardation and behavioral abnormalities.

DNA analysis for Y chromosome microdeletion

Patient DNA is isolated from venous blood after meals. Microdeletion (microscopic losses of genetic material) in the long arm of the Y chromosome (male sex chromosome) are a common cause of infertility in men. These are small chromosomal deletions (losses) which cover several adjacent genes, but are not large enough to be detected with conventional chromosome analysis.They are found much more frequently in men with azoospermia or severe oligozoospermia. Microdeletions are detected in 10-15% of men with azoospermia and respectively in 5-10% of men with severe oligozoospermia.

Attention should be paid that in assisted reproduction these microdeletions are always passed from father to son. Microdeletions are specifically related to the disruption of spermatogenesis. A matter of particularinterest is the AZF region (azoospermia factor region), which contains genes of key importance for sperm cells development and maturation.

DNA analysis of congenital thrombophilia

Patient DNA is isolated from venous blood after meals. About 40% of venous or arterial thrombosis are of hereditary character. Pregnancies of patients with confirmed thrombophilia should be closely monitored as they are at increased risk of complications such as preeclampsia, intrauterine growth retardation, placental abruption, premature birth, recurrent miscarriages and fetal distress. The DNA analysis panel of congenital thrombophilia includes:

Factor V Leiden

5% of Europeans are heterozygous carriers. The mutation results in substitution of the amino acid arginine with the amino acid glutamine at 506-th position in the protein. The amino acid substitution impairs the deactivation of factor V. In carriers of this mutation are detected increased levels of factor V and higher tendency to form blood clots. This results in a 5- to 8-fold increase in the risk of deep vein thrombosis and complications thereof. In the presence of additional factors, the risk can increase up to 35 times. There are evidences that this scenario predisposes to arterial thrombosis and obstetric complications. The homozygous form occurs in 0.06 to 0.25% of European population. There is approximately 60 times higher risk of deep vein thrombosis, arterial thrombosis and obstetric complications in people with the homozygous form. The presence of factor V Leiden in homozygous form is considered a high-risk thrombophilia.

FII G20210A

Between 1 and 2 % of Europeans are heterozygous carriers. Increased levels of factor II are associated with increased susceptibility to blood clotting and three-fold higher risk of deep vein thrombosis and complications thereof. The risk can increase up to 16 times if additional factors (for instance, oral contraceptives) are in place. There is no conclusive evidence that heterozygous carriers are more predisposed to arterial thrombosis and obstetric complications. А 0.00025 percent of the Europeans are homozygous carriers. Increased levels of this factor are associated with risk of deep vein thrombosis and complications thereof. The presence of factor FII G20210A in homozygous form is considered a high-risk thrombophilia.

MTHFR

The methylene tetrahydrofolate reductase (MTHFR) is a key enzyme in folate and homocysteine metabolism. C677T mutation in exon 4 of the gene leads to replacement of the amino acid alanine with valine and thus to a reduced enzyme activity. The homozygous form of the 677T allele in the MTHFR gene is associated with increased risk of cardiovascular complications and birth of a child with a neural tube defect.

Prenatal diagnosis

Prenatal diagnosis (PND) includes a variety of techniques and methods to clarify the health of the fetus before birth.

Prenatal diagnosis (PND) includes a variety of techniques and methods to clarify the health of the fetus before birth.

Amniocentesis with cytogenetic/microarray analysis

Amniocentesis is performed at 15-18 gestation weeks.

Under ultrasound control usually about 20 ml of amniotic fluid is aspirated. The sample is transported to the laboratory at room temperature. In routine cytogenetic analysis for about 14 days, there are enough propagated cells for chromosome analysis.

The early amniocentesis is performed at 14-16 gestation weeks, where less liquid is taken and the culture time is extended by 2-3 days.

The risk of interruption of pregnancy as a result of the manipulation is about 0.5%. An advantage of this method is that it allows almost all chromosome diseases to be diagnosed because all chromosomes are analysed. In the DNA microarray analysis, the amniocytes are not cultured, which significantly shortens the time to get the results. Besides almost all chromosomal disorders, the method is also informative for microdeletion syndromes such as Pradet-Willi, Angelman, Williams-Beuren, Di George and others.

Chorionic villus sampling with chromosomal microarray analysis

The chorionic villus biopsy is usually performed between gestation weeks 10 and 12. In ultrasound-guided transcervical or transabdominal procedure, 5 to 10 mg of villi of trophoblastic outer layer are aspired without alteration the integrity of the amniotic cavity. The diagnosis is relatively fast. A disadvantage of the method is a higher risk of miscarriage (1%).

Non-invasive prenatal test

The diagnostic method is based on analysis of free fetal DNA circulating in the peripheral blood of the pregnant woman. The free fetal DNA is derived from dead cells of the placenta and is continuously released into the bloodstream of the pregnant woman.

The required material is venous blood from the patient.

The advantages of the method are high detection ratio at less than 1% false positive results; early diagnosis (I trimester); promptness in obtaining the results (within 10 days); no risk to the pregnancy.

Among the limitations of the method are: informative only for trisomia 13, 18 and 21; not informative for partial trisomia in the studied chromosomes, mosaicism and fetoplacental discrepancies. A positive test result must be validated by invasive prenatal diagnosis via chorionic villus sampling or amniocentesis. When the results are normal, ultrasound monitoring of the pregnancy is recommended.

Microarray analysis of abortion material

The microarray analysis provides information on all chromosomal abnormalities of the aborted fetus. The chromosome analysis of abortion material affects the choice of therapy in couples with recurrent miscarriages.

Around 15-20% of pregnancies end up in abortion. Miscarriages occur in 50% of women over 40 years. Many studies have established a variety of reasons - genetic, age, antiphospholipid syndrome, uterine abnomalities, hormonal or metabolic diseases, infections, autoimmune diseases, quality of spermatozoa, harmful agents, etc. Undoubtedly, the largest share of early miscarriages (by gestation week 8) is due to chromosomal abnormalities. In 60% of the studied abortion materials a variety of chromosomal abnormalities that are non-hereditary are detected, and are usually associated with the advanced maternal age of the pregnant woman. The largest proportion is that of autosomal trisomies (three instead of two chromosomes); trisomies 16, 22 and 21 being the most frequent among them, followed by trisomies on 15, 18, 13, 9 and 14 chromosomes. The most common monosomy (one chromosome instead of two) is the single X chromosome, which is responsible for 10% of the miscarriages. One frequently detected abnormality is triploid where the fetus has 69 chromosomes instead of 46.

An advantage of the microarray analysis in comparison with a DNA test is that it provides an analysis of all chromosomes of the fetus. Karyotype testing is highly recommended in couples with two or more miscarriages.

Preimplantation genetic diagnosis / screening

IVF procedures are becoming increasingly successful thanks to the introduction of pre-implantation genetic diagnosis (PGD). PGD is a method for early detection of a number of genetic abnormalities in the embryo before it is transferred to the uterus; thus avoiding abortion later in case the fetus turns out to be affected. PGD is performed by examining I-st or II-nd polar body, or blastomere after biopsy on day-3 embryos (having 6 to 10 blastomeres) or trophectoderm biopsy on day 5. The pre-implantation selection of embryos can be divided into two groups: 1) PGS (pre-implantation genetic screening), i.e. screening for chromosomal aneuploidies, and 2) PGD (pre-implantation genetic diagnosis) - when one of the parents is a carrier of gene or chromosomal mutations. PGS indications could be advanced age of the mother (over 36 years), multiple miscarriages or a child born with abnormalities, previous unsuccessful IVF procedures as well as severe male infertility. The indications for PGD could be balanced structural translocation, inversion or another chromosomal re-arrangement; mosaicism in one of the partners; monogenic diseases (recessive or dominant); HLA typing - in parents whose child has a disease requiring stem cell transplantation from HLA-matched donor, sex selection (in X-linked diseases). Currently, ‘Nadezhda’ MHAT offers pre-implantation genetic analysis of chromosomal mutations via:

PGD/PGS of polar bodies (microarray analysis)

PGD/PGS of blastomere/trophectoderm (microarray analysis)

PGS by FISH analysis for embryo selection

PGD by FISH (translocations, inversions, mosaicism)

Why do chromosomes relate to fertility?

Approximately 70% of embryos derived from natural fertilization or IVF treatment are lost before birth. The majority of embryos are lost before the first trimester, and many of them even before implantation. The main reason for the loss of embryos, including miscarriages, are chromosomal abnormalities known as aneuploidies, resulting from loss or addition of chromosomes. It has been proven that more than 50% of the embryos have chromosomal abnormality. As the percentage increases with age, this is probably the main reason elder women to encounter difficulties in achieving pregnancy and to experience miscarriages more frequently. In connection with the high frequency of chromosomal mutations, there is a huge benefit in applying embryo pre-implantation genetic screening in the course of an IVF procedure.

Some chromosome abnormalities allow full-term pregnancy and birth of live children (Down syndrome, Edwards syndrome, Turner syndrome, etc.); others lead to premature birth, spontaneous miscarriage or suspension of embryonic development even before implantation.

The aneuploidy incidence in eggs increases as women age, trisomy 21 (Down syndrome) being the most common chromosomal abnormality in live births. The incidence of Down syndrome increases from 1 out of 900 births in women over 30 years of age, to 1 out of 230 in 37-year-old women and to 1 out of 20 in 46-year-old women.

At least 85% of the aneuploidies in the embryos occur in the egg. Sperm aneuploidies have a much smaller share of approximately 7-8%. The rest of aberrations occur randomly during cell division in the early embryonic stage.

Genetic techniques of pre-implantation diagnosis

FISH

This approach is considered more historically, as nowadays it is hardly ever used because of a number of disadvantages. Following a biopsy, one or two blastomeres are fixed on a slide which is used for the purposes of FISH analysis. The disadvantage here is that a limited number of chromosomes are explored, and there may be an overlap or interference of fluorescent signals or poor hybridization.

DNA microarray analysis (aCGH)

The implementation of the DNA microarray analysis for PGD is very promising. This technique allows the assessment of the genome of a single cell and opens opportunities for aneuploidy screening and detection of unbalanced translocations during the pre-implantation genetic diagnosis in polar cells, single blastomeres and trophectoderm cells.

Risks associated with biopsy and analytical procedures

The manipulation of embryos for the purpose of pre-implantation diagnosis also pose some minimal risks to their normal development. The risk of damaging the egg due to the biopsy is less than 0.2%, and that of the embryo – less than 1%. It is established that the risk of misdiagnosis (false positive or negative) is less than 5%. It is very important to select an appropriate stage to examine the embryo. For example, when testing a 3-day embryo for chromosomal mutations, one cell is taken at day 3 after fertilization when the embryo consists of 6-8 blastomeres. The problem with this approach is that this cell might be defective, but the rest to be normal. There is also another possibility: the tested blastomere to be normal, and the remaining cells to be defective (the so called mosaicism). This would lead to a misdiagnosis that the embryo has no chromosomal abnormalities, while in fact it has no chances of normal development. The risk of mosaicism reduces if trophectoderm cells from embryos at blastocyst stage are tested. For this reason, at present the trophectoderm biopsy is the choice of the best IVF clinics. Of course, for each particular case an individual approach is applied, as not all embryos can reach blastocyst stage outside the natural environment of the uterus. It should be kept in mind that there is a likelihood all embryos to have a genetic defect and therefore no normal embryos for transfer to be available. Although rarely, it is possible to get no reliable results from the microarray analysis. In any case, even after a PGD, when pregnancy occurs, it is recommended prenatal diagnosis in order to validate the result.

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