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.

A case report of Pallister-Killian syndrome with an unusual mosaic supernumerary marker chromosome 12 with interstitial 12p13.1-p12.1 duplication.

Pallister-Killian syndrome (PKS) is a rare inherited disease with multiple congenital anomalies, profound intellectual disability, and the presence in the karyotype of sSMC - i(12)(p10). The frequency of PKS may be underestimated due to problems with cytogenetic diagnosis caused by tissue-specific mosaicism and usually a low percentage of peripheral blood cells containing sSMC. Such tissue-specific mosaicism also complicates a detailed analysis of the sSMC, which, along with the assessment of mosaicism in different tissues, is an important part of cytogenetic diagnosis in PKS. Unfortunately, a full-fledged diagnosis in PKS is either practically impossible or complicated. On the one hand, this is due to problems with the biopsy of various tissues (skin biopsy with fibroblast culture is most often used in practice); on the other - a low percentage of dividing peripheral blood cells containing sSMC, which often significantly complicates the analysis of its composition and organization. In the present study, a detailed analysis of sSMC was carried out in a patient with a characteristic clinical picture of PKS. A relatively high percentage of peripheral blood cells with sSMC (50%) made it possible to perform a detailed molecular cytogenetic analysis of de novo sSMC using chromosomal in situ suppression hybridization (CISS-hybridization), multicolor FISH (mFISH), multicolor chromosome banding (MCB), array CGH (aCGH), and quantitative real-time PCR (qPCR), and short tandem repeat (STR) - analysis. As a result, it was found that the sSMC is not a typical PKS derivative of chromosome 12. In contrast to the classical i(12)(p10) for PKS, the patient's cells contained an acrocentric chromosome consisting of 12p material. Clusters of telomeric repeats were found at the both ends of the sSMC. Furthemore, the results of aCGH and qPCR indicate the presence of interstitial 8.9 Mb duplication at 12p13.1-p12.1 within the sSMC, which leads to different representations of DNA from different segments of 12p within cells containing sSMC. The obtained data raise the question of the instability of the sSMC and, as a consequence, the possible presence of additional rearrangements, which, in traditional cytogenetic analysis of patients with PKS, are usually described as i(12)(p10).

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.

[Evolutionary Aspects of Genomic Imprinting].

Genomic imprinting is an epigenetic phenomenon that differentiates maternal and paternal copies of genes in the genome and causes monoallelic expression depending on parental origin. Imprinting is an evolutionary puzzle, as it bears the costs of diploidization without its advantages, namely, protection from recessive mutations. The aim of this review is to answer the question of why genomic imprinting arose and became fixed in the evolution of angiosperms, insects, marsupials, and placental mammals.

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.

[Multiple epimutations in imprinted genes in the human genome and congenital disorders].

The current knowledge on multiple epimutations in imprinted genes and their impact on the disturbances of human prenatal development and the emergence of hereditary diseases are reviewed and analyzed. Genetic mechanisms regulating the epigenetic state of imprinted loci in the human genome are discussed.

[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.

[Multilocus epimutations of imprintome in the pathology of embryo development].

Genomic imprinting is one of the most significant epigenetic phenomena, which is involved in the support of eutherians and human embryo development. Molecular mechanisms of imprinting disturbance in the pathology of pre- and postnatal ontogeny are related to a considerable degree to aberrant DNA methylation of imprinted genes. At present time data about multiple abnormalities of DNA methylation arising simultaneously in several imprinted loci are accumulated. This fact brings up the problem of interpretation of imprintome structural and functional organization, as well as interaction of imprinted genes. At present study DNA methylation analysis of 51 imprinted genes in placental tissues of human spontaneous abortions was performed. The presence of several epimutations affected from four to 12 imprinted genes was observed in each embryo. Majority of epimutations (78%) had a postzygotic origin. It was shown for the first time that the total incidence of abnormal DNA methylation of maternal and paternal alleles of imprinted genes, which lead to suppression of embryo development, is significantly higher than the incidence of epimutations, which can lead to stimulation of ontogenesis processes. This fact supports at the epigenetic level the "sex conflict" hypothesis, which explains the appearance of monoallelic imprinted genes expression in the evolution of mammals.

[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.

[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.

[Epimutations of imprinting genes in the human genome: classification, causes, association with hereditary pathology].

Genomic imprinting is an epigenetic phenomenon characterized by monoallelic expression of the genes depending on their parental origin. The molecular basis of this expression is covalent modifications of DNA and histones that are formed during maturation of germline cells. Abnormalities of the establishment of genome imprinting during gametogenesis or its maintenance at various stages of development, caused by aberrant epigenetic modifications of the chromatin, predominantly disturbance of DNA methylation state, are a form of mutational variability of imprinted genomic loci. In this review, we consider the spectrum of epimutations of imprinted genes, present their classification, and discuss possible causes of their appearance and their role in etiology of hereditary human diseases.

[Epimutations of the KCNQ1OT1 imprinting center of chromosome 11 in early human embryo lethality].

Disturbance of the epigenetic status of the H19 and KCNQ1OT1 imprinting centers of chromosome 11 and the CDKN1C imprinted gene in early human embryolethality have been studied. This is the first study to detect hypomethylation of KCNQ1OT1 in the maternal homolog in placental tissues from some (9.5%) spontaneous abortions with impaired cell proliferation during the first trimester of pregnancy. Tissue specificity of the aberrant methylation status of the imprinting center has been found. A hypothesis on the postimplantation origin of epimutations in somatic cells of the developing embryo is put forward. The selective role of epimutations of imprinted genes in early human ontogeny as compared to uniparental chromosome inheritance is considered; estimation of the epigenetic risk entailed in using assisted reproductive technologies is discussed.

[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.

[Evaluation of the role of uniparental disomy in early embryolethality of man]. / Otsenka roli odnoroditel'skoi disomii v rannei émbrional'noi letal'nosti cheloveka.

We carried out systematic studies of the contribution of uniparental disomy for eight human chromosomes, 2, 9, 11, 15, 16, 19, 20, and 21, to the etiology of spontaneous mortality of human embryos. Most of these chromosomes have regions with orthologous imprinted genes syntenic with those on mouse chromosomes, the disturbed expression of which is related to embryolethality in mice. Screening of uniparental disomy in spontaneous 5- to 16-week abortuses was performed by evaluation of the pattern of inheritance of alleles of polymorphic microsatellite loci located in the studied chromosomes. A total of 100 human embryos with cytogenetically determined normal karyotype were studied, in which arrest at the early stages of intrauterine development was determined by ultrasound examination of pregnant women. During this study, 13 embryos were discarded due to karyotype anomalies or nonpaternity. No cases of uniparental disomy were found among the 87 studied abortuses for any of chromosomes studied. The analysis of the results of this study and four other studies concerning the search for uniparental disomy in dead embryos and fetuses did not reveal its elevated frequency in spontaneous abortuses as compared to the theoretically expected value based on evaluation of the probable combination of meiotic errors in human gametes. The data we obtained suggest that, first, uniparental disomies for human chromosomes that have regions with orthologous imprinted genes syntenic with mouse chromosomes do not contribute noticeably to the death of human embryos at the early developmental stages and, second, the mechanisms underlying embryolethality as a result of disturbed expression of imprinted loci differ markedly in mammals evolutionarily remote from one other.

[Maternal cell contamination of cultures of spontaneous abortion fibroblasts: importance for cytogenetic analysis of embryonic lethality]. / Kontaminatsiia kul'tur fibroblastov spontannykh abortusov kletkami materi: znachenie dlia tsitogeneticheskogo analiza prichin émbional'noi letal'nosti.

The results of standard cytogenetic analysis of the long-term cultures of embryonic fibroblasts of 478 first-trimester spontaneous abortions were retrospectively reviewed. In 16% of embryos with cytogenetically confirmed karyotype 46,XX, the Y chromosome was found by molecular genetic methods. Prior to obtaining the chromosome preparations, the cell cultures of Y chromosome-carrying embryos were maintained for a longer period than the cultures of embryos without the Y chromosome. Thus, a late entry of a culture into the logarithmic growth phase serves as marker of maternal cell contamination. We developed a mathematic model for assessment of karyotype incidence and the "sex ratio" of spontaneous abortions, taking into account risk of maternal cell contamination in extraembryonic tissue cultures. Thus estimated, the incidence of chromosomal abnormalities in the studied sample increased from 54.6 to 60.3% and the expected sex ratio increased from 0.66 to 1.02 in abortions with normal karyotype. Using molecular analysis of inheritance of polymorphic DNA markers of six autosomes (2, 11, 16, 19, 20, and 21), the proposed model was tested on 60 embryos with karyotype 46,XX and their parents. Numerical chromosome abnormalities were revealed in uncultured tissues of seven abortions (11.7%), including four without the Y chromosome, which is in a good agreement with the expected incidence of karyotype abnormalities (8.3%) predicted by our model. In view of this, estimating risk of maternal cell contamination in embryonic cell cultures seems necessary for correctly assessing the effect of natural selection in humans, for understanding the mechanisms that determine the sex ratio, and for evaluating the precision of prenatal cytogenetic diagnosis of chromosomal abnormalities.

[A search for mutations in the DIA1 gene in case of hereditary methemoglobinemia type I in the iakut population]. / Poisk mutatsii v gene DIA1 pri nasledstvennoi metgemoglobinemii I tipa v iakutskoi populiatsii.

In the patients with enzymopenic hereditary methemoglobinemia type I, a disease widely distributed on the territory of Yakutia, a search for the mutations in exons 3 and 4 of the DIA1 gene encoding NADH-cytochrome b5 reductase was carried out. It was shown that Yakut patients have none of three missence mutations, Arg57Gln, Leu72Pro, and Val105Met, described in case of this disease in the neighboring populations, Chinese and Japanese, inhabiting the territories south of Yakutia.