Prevention of chromosomal diseases through preimplantation genetic testing is an edge issue of current reproductive medicine and genetics. Embryo testing during in vitro fertilization cycles is designed to eliminate the transfer of aneuploid blastocysts, providing an increased probability of the take home babies, as well as reducing the risks of reproductive losses due to chromosomal abnormalities. The current state of the preimplantation genetic testing, its trends and perspectives are discussed.

Preimplantation genetic diagnosis/testing (PGD/PGT) for monogenic disorders is directed on prevention of the birth of the child with a hereditary disorders by means of testing embryos before implantation in IVF (in vitro fertilization). The high risk of severe form of a single gene disease is a strict medical indication for PGD for monogenic disorders at condition of contraindications and restrictions lack. Extension of PGD indications and genetic testing opportunities raises questions on standard regulation and ethical responsibility. Ethical questions are happening if a genetic risk is lower than the «high» or the disease cannot be classified as serious and if abnormal embryo transfer is proposed. Ethical aspects of PGD are considered in terms of basic ethical principles: beneficence, non-maleficence, autonomy and justice. In comparison with prenatal diagnostics, realization of these principles at PGD faces a number of additional difficult questions. The value of the human embryos and probability to have no children has to correspond to the valid risk and severity of a possible disease.

Genomic variability is the basis of genetic diversity and evolution and includes sequence and structural variability. Structural variability refers to variations in the number of copies of DNA (copy number variations - CNVs), ranging from 1000 bp up to several megabases (Mb) in size. Among them, some submicroscopic CNVs up to 3 Mb, can lead to clinical signs such as developmental delay, intellectual disability, congenital malformations and/or dysmorphic features, as well as autism spectrum disorders. A wide range of methods with different resolution is used for CNVs analysis. To date, chromosomal microarray analysis (CMA) is a universal method for CNVs detection. However, with the advent methods of next-generation sequencing, their applicability for CNV analysis is increasingly being estimated. Therefore, with the development of genome-wide technologies and bioinformatic tools for CNV analysis, there is an increasing need to confirm the obtained data in order to establish the true values of their sensitivity and specificity. In addition, information only about localization and gene content of CNVs is not enough for genetic counseling for the family. It is necessary to define structure and origin of the detected CNV to assess accurate recurrence risk of chromosome imbalance. For this purpose, molecular genetics and molecular cytogenetic methods are used. There are some methods of molecular genetics based on PCR with sufficient resolution to confirm submicroscopic CNV longer than 1000 bp. Analysis of submicroscopic CNVs by various modifications of FISH-method is limited by the length and specificity of DNA fragments in probes used in conventional FISH-protocols. Therefore, application of DNA probes of the order of several kb in length becomes relevant. If both group of methods allow to confirm CNVs detected by wide-genome technologies, than the latter are used to estimate the structure of chromosomal imbalance. Possibilities, advantages and disadvantages of different methods for CNVs verification are discussed.

Non-invasive prenatal testing (NIPT) of trisomy has recently become widespread in clinical practice in many countries, including Russia. Despite the fact that NIPT does not allow to estimate the number and structure of all chromosomes of the fetus (this is possible only when using karyotyping), this analysis has good sensitivity and specificity indicators, and also allows eliminating the risks accompanying invasive diagnostic procedures (amniocentesis, cordocentesis, chorionic villus biopsy, etc.). The sequences of cell-free fetal DNA (cffDNA), which are present in low concentrations in the blood of a pregnant woman and disappear after pregnancy, are mainly used for NIPT. Currently NIPT uses Next Generation Sequencing methods (NGS). The review presents main approaches and methods used for non-invasive prenatal testing of trisomy: massive parallel sequencing by the shotgun method (MPSS), targeted parallel sequencing (t-MPS) and approaches based on the study of single nucleotide polymorphisms (SNP). In review we also compare efficacy of their use for the detection of trisomy of chromosomes 13, 18 and 21 (Patau syndrome, Edwards syndrome and Down syndrome, respectively). When using MPSS, sequences of the whole genome are analyzed, and with t-MPS, only specific sequences of interest are analyzed. The article also discusses methods for analyzing mRNA, fetal epigenetic markers, and additional signal amplification methods (for example, DANSR, Digital Analysis of Selected Regions). The review presents the main reasons for the appearance of false (false positive and false negative) results in NIPT (mosaicism, the phenomenon of “twin resorption”, low levels of fetal DNA, etc.). A brief description of the current place of NIPT in clinical practice and prospects for the inclusion of NIPT in the prenatal screening scheme, as well as the main difficulties that limit the wider use of NIPT technology in the clinic, are given. Primarily, these difficulties are associated with the specificity of cell-free fetal DNA (concentration, individual differences, etc.), as well as ethical issues (genetic determinism, informed patient consent, etc.).

Introduction: Limited reproductive potential in humans and the progressive decline of the reproductive health of the population have led to the development of assisted reproductive technologies in recent decades. In order to improve pregnancy rates in couples with reproduction problems, preimplantation genetic screening was introduced into clinical practice. Cultivation of human embryos in vitro in in vitro fertilization cycles (IVF), as well as the possibility of obtaining genetic material during preimplantation genetic screening and diagnosis (PGS / PGD), allow us to estimate the frequency and spectrum of chromosomal abnormalities in human blastocysts. Aim: Analysis of the rate and spectrum of aneuploidies in human blastocysts obtained in the IVF-PGD cycles. Material and methods: A retrospective analysis of the molecular karyotypes of 113 blastocysts obtained in the cycles of assisted reproductive technology IVF-PGD from 47 women was carried out. The whole genomic amplification of DNA from trophectoderm cells was performed using the PicoPlex reagent kit (Rubicon Genomics, USA). Analysis of DNA samples obtained after whole genome amplification was carried out by array comparative genomic hybridization (aCGH) using a GenetiSure Pre-Screen microchip, 8×60K (Agilent Technologies, USA). Results: The efficiency of whole genome amplification was 97.3% (110/113). A balanced karyotype was established in 31% (34/110) blastocysts. The rate of a blastocyst with chromosomal imbalance in the group of women under 35 years old was lower (46.9%) compared to the rate of blastocyst with chromosomal imbalance in the group of women over 35 years old (81.0%) (p < 0.001). 74% of the identified chromosomal abnormalities were aneuploidy, 26% - structural chromosomal abberations. The distribution of aneuploidies had the following structure: autosomal trisomies (41%), autosomal monosomies (48%), aneuploidies of sex chromosomes (7%), autosomal tetrasomies (3%), autosomal nullisomies (1%). Aneuploidies of chromosomes 5, 15, 16, 17, 19, 21 and 22 were noted with the greatest frequency. Conclusions: Analysis of chromosomal aberrations in human embryos at the blastocyst stage showed a high frequency of chromosomal imbalance (69%) and a wide range of both numerical and structural abnormalities of chromosomes. PGS with aCGH allows the selection of blastocysts with a balanced karyotype. According to the results of blastocyst transfers in IVF-PGD cycles, clinical pregnancy occurred in 32 % of cases.

Recently, there has been an active introduction of high-resolution diagnostic methods for detecting chromosomal abnormalities in humans. Thus, the use of microarray diagnostics allows detecting 6% more chromosomal abnormalities in fetuses with a normal karyotype based on the results of standard cytogenetic methods. At the same time, as part of the prenatal diagnosis, aCGH is used in much smaller amounts. This is primarily due to the receipt of a large amount of information and difficulties with its interpretation. This paper presents the results of using the aCGH method in prenatal diagnosis at the Research Institute of Medical Genetics of the Tomsk National Research Medical Center. In the framework of prenatal diagnosis, 44 samples of fetal material were examined using the aCGH method. Prenatal diagnosis using microarray in 20 cases (45%) allowed to identify chromosomal abnormalities, a balanced karyotype was detected in 21 samples (48%). In three cases (7%) the result was not obtained due to insufficient quantity or low quality of the biological material. Among the 20 cases with chromosomal abnormalities, 4 samples with aneuploidy and 16 samples with CNV. In 16 samples of fetal material without aneuploidy, 13 deletions and 10 duplications were identified. All the adjustments included in this list are not represented or represented by isolated cases in the Database of Genomic Variants. When analyzing the clinical significance of the CNV, were identified 11 pathogenic, 5 probably pathogenic and 5 CNV with uncertain significance. Pathogenic rearrangements were represented by 7 deletions and 4 duplications identified in 10 samples. In addition to the “Primary Finding” of pathogenic CNVs, so-called “Incidental Finding” of pathogenetically significant rearrangements are also detected. The identification of “Incidental Finding” is characteristic of all genome-wide studies and leads to the emergence of ethical issues related to informing patients and the possible termination of pregnancy. In the present study, five “Incidental Finding” pathogenic CNVs and five rearrangements with uncertain significance were identified. The results of this work indicate the need to use modern molecular cytogenetic methods for the prenatal diagnosis of chromosomal abnormalities. It is necessary to take into account the emergence of new methodological and ethical issues that are most acute when using these methods in prenatal diagnosis.

Prenatal diagnosis of chromosomal diseases in Tomsk and the Tomsk region is an integral part of the medical care. In the Research Institute of Medical Genetics of Tomsk National Research Medical Center effective methods for chromosomal analysis at almost any gestational age are widely used. In the past five years, 3503 prenatal cytogenetic studies have been carried out and 300 cases (8,5%) of chromosomal abnormalities were detected. The highest percentage of chromosomal pathology (11,5%) was found in pregnant women within first trimester biochemical screening. A standard cytogenetic studying allows eliminates aneuploidy and polyploidy, structural anomalies, especially if they disrupt the morphology of chromosomes, but submicroscopic chromosomal abnormalities often remains not identified. Using of complex molecular cytogenetic technologies allows to exclude chromosomal pathology with the maximum probability.