Aberrant methylation of placental development genes in chorionic villi of spontaneous abortions with trisomy 16.

In humans, aneuploidy is incompatible with the birth of healthy children and mainly leads to the death of embryos in the early stages of development in the first trimester of pregnancy. Trisomy 16 is the most common aneuploidy among spontaneous abortions of the first trimester of pregnancy. However, the mechanisms leading to the death of embryos with trisomy 16 remain insufficiently investigated. One of these potential mechanisms is abnormal placental development, including aberrant remodeling of spiral arteries. Spiral artery remodeling involves the migration of trophoblast cells into the maternal spiral arteries, replacing their endothelium and remodeling to ensure a stable embryonic nutrition and oxygen supply. This is a complex process which depends on many factors from both the embryo and the mother. We analyzed the methylation level of seven genes (ADORA2B, NPR3, PRDM1, PSG2, PHTLH, SV2C, and TICAM2) involved in placental development in the chorionic villi of spontaneous abortions with trisomy 16 (n = 14), compared with spontaneous abortions with a normal karyotype (n = 31) and the control group of induced abortions (n = 10). To obtain sequencing libraries, targeted amplification of individual gene regions using designed oligonucleotide primers for bisulfite-converted DNA was used. The analysis was carried out using targeted bisulfite massive parallel sequencing. In the group of spontaneous abortions with trisomy 16, the level of methylation of the PRDM1 and PSG2 genes was significantly increased compared to induced abortions (p = 0.0004 and p = 0.0015, respectively). In the group of spontaneous abortions, there was no increase in the level of methylation of the PRDM1 and PSG2 genes, but the level of methylation of the ADORA2B gene was significantly increased compared to the induced abortions (p = 0.032). The results obtained indicate the potential mechanisms of the pathogenetic effect of trisomy 16 on the placental development with the participation of the studied genes.

Expression of the NUP153 and YWHAB genes from their canonical promoters and alternative promoters of the LINE-1 retrotransposon in the placenta of the first trimester of pregnancy.

The placenta has a unique hypomethylated genome. Due to this feature of the placenta, there is a potential possibility of using regulatory elements derived from retroviruses and retrotransposons, which are suppressed by DNA methylation in the adult body. In addition, there is an abnormal increase in the level of methylation of the LINE-1 retrotransposon in the chorionic trophoblast in spontaneous abortions with both normal karyotype and aneuploidy on different chromosomes, which may be associated with impaired gene transcription using LINE-1 regulatory elements. To date, 988 genes that can be expressed from alternative LINE-1 promoters have been identified. Using the STRING tool, genes (NUP153 and YWHAB) were selected, the products of which have significant functional relationships with proteins highly expressed in the placenta and involved in trophoblast differentiation. This study aimed to analyze the expression of the NUP153 and YWHAB genes, highly active in the placenta, from canonical and alternative LINE-1 promoters in the germinal part of the placenta of spontaneous and induced abortions. Gene expression analysis was performed using real-time PCR in chorionic villi and extraembryonic mesoderm of induced abortions (n = 10), adult lymphocytes (n = 10), spontaneous abortions with normal karyotype (n = 10), and with the most frequent aneuploidies in the first trimester of pregnancy (trisomy 16 (n = 8) and monosomy X (n = 6)). The LINE-1 methylation index was assessed in the chorionic villi of spontaneous abortions using targeted bisulfite massive parallel sequencing. The level of expression of both genes from canonical promoters was higher in blood lymphocytes than in placental tissues (p < 0.05). However, the expression level of the NUP153 gene from the alternative LINE-1 promoter was 17 times higher in chorionic villi and 23 times higher in extraembryonic mesoderm than in lymphocytes (p < 0.05). The expression level of NUP153 and YWHAB from canonical promoters was higher in the group of spontaneous abortions with monosomy X compared to all other groups (p <0.05). The LINE-1 methylation index negatively correlated with the level of gene expression from both canonical (NUP153 - R = -0.59, YWHAB - R = -0.52, p < 0.05) and alternative LINE-1 promoters (NUP153 - R = -0.46, YWHAB - R = -0.66, p < 0.05). Thus, the observed increase in the LINE-1 methylation index in the placenta of spontaneous abortions is associated with the level of expression of the NUP153 and YWHAB genes not only from alternative but also from canonical promoters, which can subsequently lead to negative consequences for normal embryogenesis.

Comparative cytogenetics of anembryonic pregnancies and missed abortions in human.

Miscarriage is an important problem in human reproduction, affecting 10-15 % of clinically recognized pregnancies. The cases of embryonic death can be divided into missed abortion (MA), for which the ultrasound sign of the embryo death is the absence of cardiac activity, and anembryonic pregnancy (AP) without an embryo in the gestational sac. The aim of this study was to compare the frequency of chromosomal abnormalities in extraembryonic tissues detected by conventional cytogenetic analysis of spontaneous abortions depending on the presence or absence of an embryo. This is a retrospective study of 1551 spontaneous abortions analyzed using GTG-banding from 1990 to 2022 (266 cases of AP and 1285 cases of MA). A comparative analysis of the frequency of chromosomal abnormalities and the distribution of karyotype frequencies depending on the presence of an embryo in the gestational sac was carried out. Statistical analysis was performed using a chi-square test with a p <0.05 significance level. The total frequency of chromosomal abnormalities in the study was 53.6 % (832/1551). The proportion of abnormal karyotypes in the AP and MA groups did not differ significantly and amounted to 57.1 % (152/266) and 52.9 % (680/1285) for AP and MA, respectively (p = 0.209). Sex chromosome aneuploidies and triploidies were significantly less common in the AP group than in the MA group (2.3 % (6/266) vs 6.8 % (88/1285), p = 0.005 and 4.9 % (13/266) vs 8.9 % (114/1285), p = 0.031, respectively). Tetraploidies were registered more frequently in AP compared to MA (12.4 % (33/266) vs. 8.2 % (106/1285), p = 0.031). The sex ratio among abortions with a normal karyotype was 0.54 and 0.74 for AP and MA, respectively. Thus, although the frequencies of some types of chromosomal pathology differ between AP and MA, the total frequency of chromosomal abnormalities in AP is not increased compared to MA, which indicates the need to search for the causes of AP at other levels of the genome organization, including microstructural chromosomal rearrangements, monogenic mutations, imprinting disorders, and epigenetic abnormalities.

Karyotype evaluation of repeated abortions in primary and secondary recurrent pregnancy loss.

PURPOSE: To study the contribution of embryo chromosomal abnormalities in primary and secondary recurrent pregnancy loss (RPL) and to analyze the recurrence of chromosomal constitution in miscarriages from the same couple. METHODS: Retrospective study of abortion karyotypes in RPL families based on the mother's primary or secondary RPL status (563 embryo specimens, 335 samples from primary, and 228 samples from secondary RPL). RPL was defined as two or more consecutive miscarriages. One hundred eight cases of recurrent embryo/fetal loss in 51 families were analyzed to assess the probability of having the same karyotype pattern (recurrent normal or recurrent abnormal) in both previous and subsequent pregnancy loss. The karyotypes of abortions were established using standard cytogenetic analysis, as well as interphase fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH). RESULTS: The frequency of aberrations was 43.9% in abortions from primary RPL versus 52.6% in secondary RPL (p = 0.041). Women 35 years of age or older were the main contributors to this difference. The odds ratio of a subsequent abortion having the same karyotype pattern (normal or abnormal) as the previous one was 6.98 (p = 0.0013). CONCLUSION: The frequency of abnormalities is higher in abortions from the secondary RPL versus primary RPL group, and this difference is due to the relative deficiency of miscarriages with abnormal karyotypes in older women with primary RPL. The probability of having the same karyotype pattern (recurrent normal or recurrent abnormal) in the previous and subsequent abortion is increased significantly compared with chance.

[Methylation status of line-1 retrotransposon in chromosomal mosaicism during the early stages of human embryonic development].

Early stages of human embryonic development are characterized by spatio-temporal coincidence of events of total epigenetic genome reprogramming and elevated level of mosaic forms of numerical chromosome abnormalities. It is possible that the abnormal reprogramming of various regions of the genome can lead to violations of local epigenetic chromatin organization and gene expression, affecting the correct chromosome segregation during mitosis. In this study, a comparative analysis of the methylation index of LINE-1 retrotransposon, which is largely reflecting the methylation profile of the genome, is performed in placental tissues of spontaneous abortions with complete and mosaic forms of aneuploidy, and with a normal karyotype, as well as in the control group of induced abortions of the first trimester of pregnancy. It was shown that extraembryonic mesoderm and chorionic cytotrophoblast of spontaneous abortions with chromosomal mosaicism are characterized by the highest index of LINE-1 methylation among all groups studied. At the same time excessive hypomethylation of transposable genetic element recorded in spontaneous abortions with normal karyotype. It is suggested that violations of parental genomes demethylation during epigenetic reprogramming at preimplantation stages of development may be associated with an increased frequency of mitotic errors in chromosome segregation, leading to the formation of a mosaic karyotype.

[SKEWED X-CHROMOSOME INACTIVATION IN HUMAN MISCARRIAGES].

Sex ratio in first trimester of pregnancy is skewed due to preferential elimination of female embryos. It could be resulted from aberrant X-chromosome inactivation. X-chromosome inactivation was analyzed in extraembryonic tissues of miscarriages and induced abortions with 46, XX karyotype. In chorion cytotrophoblast of both miscarriages and induced abortions observed either random or skewed X-chromosome inactivation. In extraembryonic mesoderm of the control group, random inactivation was observed, whereas 15% of miscarriages had skewed X-chromosome inactivation. The highest frequency of skewed inactivation of one of the parental homologues was observed in the groups of blighted ovum pregnancy and embryos from women with recurrent pregnancy loss. It was suggested that in these cases compartmentalization of cells in the blastocyst probably leads to predominance of cell with mutant active X-chromosome among the cells of inner cell mass carrying the aberrations that are incompatible with normal embryonic development.

[Clinical and genetic analysis of idiopathic intellectual disability based on array comparative genomic hybridization].

In this study authors searched for chromosomal aberrations in 71 children with developmental delay or idiopathic mental retardation using Human Genome CGH Microarray Kits 4×44K and 8×60K (Agilent Technologies, USA). Microdeletions and microduplications, as well as CNV, which may be related to intellectual disability and associated with regions of known hereditary diseases or chromosomal syndromes were identified in 14 (20%) children (these patients are described in this article). During the analysis, candidate genes localized within the regions of aberrations and associated with development and functioning of nervous system were denoted.

[Epigenetic effects of trisomy 16 in the human placenta].

The methylation profiles of the placental tissues of human embryos with normal karyotype and trisomy 16 were compared using Infinium HumanMethylation27 BeadChip array (Illumina, United States). Numerous differences between the extraembryonic tissues with diploid and aneuploid karyotypes were observed. The extraembryonic mesoderm of embryos with trisomy 16 appeared to be less methylated compared to the diploid tissue, whereas the cytotrophoblast of aneuploid embryos was hypermethylated. The presence of the supernumerary chromosome was shown to influence the epigenetic profile of the genome changing the level of methylation of CpG sites of all chromosomes. However, the biggest number of differentially methylated loci was found on the chromosome 16. Besides, more often the epimutations were tissue-specific. The hypomethylated genes in both tissues belong to the groups of genes responsible for different metabolic processes, whereas the hypermethylated genes control the processes of development, cell adhesion, immune response, and response to stimulus.

[Dynamics of methylation changes within functional groups of genes during breast cancer progression].

For the first time in a comparative perspective the epigenetic status of the benign proliferative processes, breast cancer, and metastases to regional lymph nodes was studied using DNA methylation microarray "GoldenGate Cancer Panel I" ("Illumina", USA). The functional groups of differentially methylated genes were identified in each set of samples. The genes that regulate cell proliferation and mobility were methylated in samples with benign proliferative processes. An aberrant methylation of the genes responsible for cell differentiation and proliferation, as well as protein phosphorylation and cell mobility was observed in the samples with malignant phenotype. Differential methylation of the genes that regulate cell adhesion, the formation of anatomical structures, angiogenesis, immune response, signal transduction, and protein phosphorylation was found in the samples with metastases to regional lymph nodes in comparison with the morphologically unaltered breast epithelium. The tissues from the benign proliferative processes and metastases to regional lymph nodes were generally characterized by a relatively lower level of epigenetic variability in comparison with the tissues of the primary tumor.

[DNA methylation profile in human placental tissues].

For the first time using genome-wide Infinium HumanMethylation27 BeadChip Array (Illumina, USA) DNA methylation pattern was determined in the human cytotrophoblast and extraembryonic mesoderm. These tissues are the derivatives of trophectoderm and inner cell mass of blastocyst and have substantial differences in dynamics of epigenetic genome reprogramming during early stages of differentiation. The genome of the extraembryonic mesoderm cells has been shown to be more methylated compared to the cytotrophoblast similarly to other mammalian species. Differences in methylation pattern of single CpG-dinucleotides and dinucleotides localized within promoter CpG-islands have been found. It has been shown that the majority of single CpG-dinucleotides in both tissues were methylated whereas promoter CpG-island sites were not. Comparative analysis revealed 202 differentially methylated genes in extraembryonic mesoderm and 40 genes in cytotrophoblast. These genes are responsible for diverse biological processes. However, in the extraembryonic mesoderm the main functional groups included genes responsible for DNA binding and transcriptional factors' activity whereas in the cytotrophoblast--for transport and protein and cytokine secretion.

[Methylation profiling of the cell cycle regulating genes in placenta of human embryos with chromosomal mosaicism].

To date the investigation of epigenetic mechanisms of cell cycle regulation, ensuring genomic stability maintaining and accurate transfer of hereditary information to daughter cells, is of a considerable interest. Development of the up-to-date molecular technologies allows determining methylation pattern of the whole genome at a single round of analysis. In this work for the first time the epigenetic status of placental tissues of human embryos with mosaic karyotype was studied using the genome-wide Illumina Infinium HumanMethylation27 BeadChip Array (Illumina, USA). The groups of genes, related to the cell cycle and its regulation and containing differentially methylated CpG-sites in their promoter regions, are determined. The methylation level of oncogenes (ARHGEF1, RGF5), tumor-suppressors (APC2, BRACA2, DCC, GRLF1, RB1, TP73, TSPYL2, VHL) as well as genes, participating in chromosome segregation regulation (CNTROB, GMNN, PROCR, TACC1), was changed most frequently.

[Skewed X-chromosome inactivation in human embryos with mosaic trisomy 16].

The sex ratio and X-chromosome inactivation were analyzed in placental tissues of human spontaneous abortuses with pure and mosaic forms of chromosome 16 trisomy. The sex ratio value was found to decrease with an increase in the share of cells with the trisomic karyotype, which suggests differential survival of embryos belonging to different sexes. The pattern of X-chromosome inactivation in cells of extraembryonic mesoderm in the control group of embryos and in spontaneous abortuses with the level of trisomy 16 below 80% corresponded to random X-inactivation, whereas in most embryos with a frequency of trisomy 16 exceeding 80% skewed inactivation was observed. Our results support the hypothesis about the existence of an autosomal transfactor influencing the initiation of X-chromosome inactivation and suggest its possible localization on chromosome 16.

[Epigenetic aspects of early lymphogenic metastasis in breast cancer].

Metastasizing is one of the key stages in tumor development. Understanding this process is necessary for effective diagnosis, therapy and prediction of clinical outcome. Some recent data suggest the possibility of metastatic phenotype cells appearance at the early stages of tumor evolution in contravention with generally accepted hypothesis of linear metastatic process development. In this study we have performed a comparative analysis of the array-based DNA methylation profile in biopsy samples of patients with benign breast disorders, breast cancer and lymphogenous metastases. In some cases the biopsy samples dated back to different stages of the same tumor. For the analysis the GoldenGate Methylation Cancer Panel I was used. The DNA methylation level in 1,505 CpG-sites was similar in samples from patients with benign breast disorders and lymphogenous metastases. Our data support the hypothesis of the early appearance of cell clones responsible for the tumor limphogenous dissemination. Epigenetic component apparently plays an important role in this process.

[Estimation of the methylation status of the promoter region of the cell cycle gene P14ARF in placental tissues of spontaneous abortuses with chromosomal mosaicism].

The methylation status of the promoter region of the cell cycle gene P14ARF was studied in the extraembryonic mesoderm and in the chorion cytotrophoblast of 46 human spontaneous abortuses with chromosomal mosaicism. Aberrant methylation of alleles of this gene was revealed for the first time in placental tissues of 9% of embryos. The identified epimutations were found to be characteristic of embryos with aneuploid cell clones of postzygotic origin. It is suggested that epigenetic inactivation of loci responsible for the regulation of cell division and for segregation of chromosomes is associated with the occurrence of mosaic forms of the karyotype at early stages of human embryonic development.

[Epigenetic inactivation of the RB1 gene as a factor of genomic instability: a possible contribution to etiology of chromosomal mosaicism during human embryo development].

The methylation status of the cell cycle control gene RB1 has been studied in placental tissues of spontaneous abortions of the first trimester of pregnancy with mosaic variants of numerical chromosomal abnormalities verified by a molecular genetic examination. Aberrant methylation of the gene promoter region has been revealed for the first time in 20% of embryos with chromosomal mosaicism that died in utero. A maximum frequency of epimutations was recorded in a group of embryos with a low level of abnormal cells for which mitotic errors are most likely to determine the formation of mosaic aneuploidy in primary euploid zygotes. It has been suggested that aberrant epigenetic genomic modifications at early stages of human embryonic development can be one of the mechanisms promoting genomic instability realized in the form of mosaic abnormalities of the karyotype that are incompatible with the normal course of embryogenesis.

[Retrospective molecular-cytogenetic characteristics of tetraploidy in early human embryolethality].

The ploidy level of noncultivated extraembryonic tissues was studied by fluorescence in situ hybridization in 30 human I trimester spontaneous abortions with tetraploid or diploid-tetraploid karyotype after conventional cytogenetic analysis. Only thirteen embryos (43 %) were verified to be tetraploid that provides evidence for the hypothesis of placental cell polyploidization during long-term in vitro cultivation. It is shown that preferred compartmentalization of tetraploid cells in the inner cell mass derivatives is associated with blighted ovum - the most severe type of human embryo dysmorphogenesis.

[Polymorphism of trinucleotide repeats at loci FRAXA and FRAXE in the population of Tomsk]. / Polimorfizm trinukleotidnykh povtorov lokusov FRAXA i FRAXE u zhitelei g. Tomska.

Polymorphism of CGG and GCC trinucleotide repeats, whose expansions at the FRAXA and FRAXE loci have been identified as causative mutations in two forms of mental retardation, was studied in Slavic population of Tomsk. At the FRAXA locus a total of 31 allelic variants ranging from 8 to 56 copies of CGG repeat with two modal classes of 28-29 and 18-20 repeat units (with the frequencies of 24.6 and 11.5% respectively) were revealed. Compared to other populations, this locus was characterized by unusually high frequency of intermediate alleles with the sizes of more than 40 CGG repeat units (12.4%). Since intermediate repeats of the FRAXA locus were more prone to instability than normal alleles, it was suggested that Slavic population of Siberia had higher risk of the development of FMR1 dynamic mutations, giving rise to the Martin-Bell syndrome. The FRAXE allele frequency distribution was demonstrated to be normal with 18 allelic variants ranging from 9 to 27 GCC repeat units. In the population of Tomsk this locus had higher than in other populations frequency (26.7%) of short (less than 15 repeat units in size) alleles. In addition, in the Tomsk population both loci were characterized by high level of heterozygosity and low frequencies of modal allele classes. These results can be explained by the high level of outbreeding typical of the population of Siberia.