Introduction and basic concept of genetics

The increasing knowledge of genetic background of various illnesses, including reproductive disorders are cause of closer cooperation among medical genetics and reproductive medicine with assisted reproduction and embryology. Thus a new branch of medical genetics has been created – reproductive genetics, which specializes on improving pre-conception, pre-implantation and classic pre-natal diagnosis of genetically caused pathological states, using method of classical and molecular cytogenetics and molecular genetics. Reproductive genetics together with specialized genetic counselling focuses on providing complex medical – preventive care of married / partner couples with reproduction disorders.

Methods of assisted reproduction enable to achieve pregnancies even in cases, where natural way of conception reproduction fails. Although sterility and loss of reproduction represent an important selective mechanisms, it is known that besides gynecologic, andrologic, hormonal or immunologic factors, etiology of infertility is also caused by genetic factors – monogenic (one defective gene), chromosomal and multifactor (common effect of more genes and other factors of environment). Assumption is that genetically caused barriers including chromosomal anomaly and gene mutation is present in 15% of men and 10% of women with fertility disorder. Some genetic factors – such as chromosomal aberration and mutation of some genes have been known for many years, in case of others a link to specific part of chromosome is suspected, but majority still remains undisclosed. Overcoming genetic barriers using modern methods of assisted reproduction could mean transferring genetically caused sterility (microdeletion of sexchromosome Y), some illnesses (e.g. cystic fibrosis, thrombosis tendencies, etc.) and syndromes (chromosome anomaly) onto offspring.

After several years of hard work, Sanatorium Pronatal has reached an agreement with insurance companies for medical genetic operations, including laboratory examinations, thus diagnostic treatment of unfertile couples has been optimised, making medical genetics more affordable. Our medical genetic department has started functioning 1.10.2002 as specialized workplace offering services in areas of genetic consultancy and laboratory treatments of our patients. Based on results of an initial genetic consultation with an infertile couple, including genealogical analysis, we undergo a cytogenetic examination, followed by DNA diagnosis of CFTR gene mutation for cystic fibrosis, Leiden mutation of F5 gene for plasmatic factor FV and mutation G20210A in gene coding prothrombin (thrombosis tendencies, spontaneous miscarriage and gynecological complications) and microdeletion (small losses) on Y chromosome (men’s reduced fertility and infertility). Upon results of cytogenetic and molecular genetic examination couple is introduced to all risks for offspring prior to IVF, an objective pre-implantation or prenatal genetic diagnosis is ensured. We have introduced pre-implantation diagnosis of chromosomal anomaly into our routine practice, using FISH method in an early stage of embryo prior to transfer into the uteri (see picture 1). Picture of risk-involving couple

(Pic. 1) Risk pair - Scheme showing genetic examination of couple with reproductive disorders and pre-implantation genetic diagnosis (PGD) in Sanatorium Pronatal

It is understood that genetic examination of donors of gametes is undertaken (genetic consultation, cytogenetic examination and examination of CFTR mutation of cystic fibrosis genes).

In parallel we work on improving methodical routine with goal to increase scope of examinations of defects of other genes causing sterility for specific group of patients, increasing pre-implantation genetic diagnosis accuracy by extending the karyotype examination from a single embryonic cell, examination of frequency of genetically pathological sperm using FISH method in cases of sever disorders of spermatogenesis and cytogenetic examination of sporadic miscarriages after infertility treatment of methods of assisted reproduction.

DNA and Human chromosomes

Human genetic information is carried in deoxyribonucleic acid (DNA). It is a specific kind of bio-macromolecule that is able to carry information about human trait and character. Parts of DNA represent specific genes, while every gene has got its exactly specified place in the DNA (locus).

DNA is especially present in the nucleus (genomic DNA). Only a small portion is preserved in small cellular organs – mitochondria (mitochondrial DNA), which occur in cytoplasm of the cell. Genomic DNA is present in all cells that have nucleus (cytoblast), with exception of grown red blood-corpuscles – erythrocytes, that have practical relevancy to sampling of biological material in genetic examination.

DNA combined with protein complex, RNA, ions and enzymes make up chromatin. Multiple shortening of chromatin strands create small structures – chromosomes. If chromatin comprises of active genes, we are speaking about euchromatin. However some chromosomes are created by so called heterochromatin, which harbors non-active genes. Heterochromatin differs from genetically active eurchromatin with its functional and colored characters.

Normal, healthy individual carries 46 chromosomes in somatic cells (i.e. in body cells). Out of that 22 pairs that is 44 chromosomes are so called autozomes and 2 chromosomes represent sex chromosomes – gonozomes. While women have 2 sex chromosomes XX, men carry sex chromosomes X and Y. Thus women‘s physiological karyotype is 46,XX (pic. 2) and men‘s physiological karyotype is 46,XY (pic. 3). Set of 46 chromosomes in somatic cell is labelled as diploid.

(Pic. 2) women’s karyotype 46, XX

(Pic. 3) men’s karyotype 45,XY

During cell splitting chromosomes, thus also genetic information it includes, accurately splits into newly created daughter cells. Mature gametes posses only half set that is 23 chromosomes, which are labelled as haploid. All normal, mature female eggs carry one venereal chromosome X, mature male sperm carry either X chromosome or Y chromosome. Because human somatic (body) cells carry chromosomes in pairs – males are exception – each gene is present in two forms, called alleles. Both alleles can be normal, in some cases mutated (damaged) or one normal and one mutated allele. There is only one chromosome from each pair present in gametes that carries only one allele – normal or mutated.

There are several parts of chromosomes that we can distinguish (pic. 4). Each chromosome has got a narrow part – primary constriction, where centromere is located. Centromere plays an important role during cell splitting of chromosomes into daughter cells. Centromere splits chromosome into 2 parts – short limb (labelled p) and long limb (labeled q). End of both chromosome limbs is called telomere. Acrocentric chromosomes no. 13, 14, 15, 21 and 22 harbour centromere and also section of secondary constriction, which splits from its short limb a small fragment – satellite. A thin bridge connects short limb and the satellite. It is where cytoblast (or seed) is organized.

(Pic. 4) Morphology of chromosome

Breakdown of chromosome into each part is important for accurate description of chromosome and changes it undergoes.

Chromosomes differ from each other in size, position of centromere and striping, which shapes in various colouring processes. These colouring processes enable us to recognize each chromosome, accurately state its number and to asses changes in its structure (numeric and structural anomaly of chromosomes). Conventional, homogenous colouring is applied on whole chromosome, G- and R- striping method create system of light and dark stripes, where R-stripes are seen as negative of G-stripe. C-striping is colouring of centromere areas of all chromosomes and heterochromatine parts of some chromosomes. Other, less used colouring methods enable us to resolve some specific situations.

Chromosome set of an individual or cell organized under international cytogenetic classification is called karyotype..

Cytogenetic examination

Cytogenic examination of karyotype is a demanding process, requiring team of specialized experts (pic. 5 – right side). Periphery blood drawing is followed by delivering biological material to cytogenetic laboratory. Cells are then cultivated for 3 days in special medium – liquid environment, containing all nutritious elements need for growth and splitting of cells. After 72 hours of cultivation an element is added to the cells, ceasing cell splitting in stage of metaphase, or pro-metaphase. Following hypotonic solution causes cell bursting and splitting off cytoplasm. After fixation cell suspension is dropped on a support glass and after drying it is colored using a particular method, G-stripe technique is most standard. The process finishes by microscopic evaluation of the chromosome and assessing type of karyotype of treated person.

Molecular-genetic treatment

Gene defect – mutation – represent small changes that are undetectable under a microscope. Its presence is examined using modern molecular genetic methods (pic. 5 left side). After periphery blood drawing, DNA is isolated from blood cells. Using a specialized procedure – polymerase chain reaction, a segment of taken DNA that needs to be examined is multiplied. Depending on type of diagnosed mutation a splitting product PCR reaction enzymes is carried out – using restrictive endonucleasis, which split specifically amplified fragments into 2 parts. Next step is separation of each fragment in electrical field on agarose or polyacrylamide gel, which is then documented. Subtracting size of fragment determines presence of normal or mutated allele.

(Pic. 5) Cytogenetic and molecular genetic treatment scheme