Olga A. Efimova

Placental microRNA expression in pregnancies complicated by superimposed pre‑eclampsia on chronic hypertension

Pre-eclampsia (PE) is a complication of pregnancy that affects 5–8% of women after 20 weeks of gestation. It is usually diagnosed based on the de novo onset of hypertension and proteinuria. Preexisting hypertension in women developing PE, also known as superimposed PE on chronic hypertension (SPE), leads to elevated risk of maternal and fetal mortality. PE is associated with an altered microRNA (miRNA) expression pattern in the placenta, suggesting that miRNA deregulation is involved in the pathogenesis of PE. Whether and how the miRNA expression pattern is changed in the SPE placenta remains unclear. The present study analyzed the placental miRNA expression profile in pregnancies complicated by SPE. miRNA expression profiles in SPE and normal placentas were investigated using an Ion Torrent sequencing system. Sequencing data were processed using a comprehensive analysis pipeline for deep miRNA sequencing (CAP-miRSeq). A total of 22 miRNAs were identified to be deregulated in placentas from patients with SPE. They included 16 miRNAs previously known to be associated with PE and 6 novel miRNAs. Among the 6 novel miRNAs, 4 were upregulated (miR-518a, miR-527, miR-518e and miR-4532) and 2 downregulated (miR-98 and miR-135b) in SPE placentas compared with controls. The present results suggest that SPE is associated with specific alterations in the placental miRNA expression pattern, which differ from alterations detected in PE placentas, and therefore, provide novel targets for further investigation of the molecular mechanisms underlying SPE pathogenesis.

Frequency and Spectrum of MED12 Exon 2 Mutations in Multiple Versus Solitary Uterine Leiomyomas From Russian Patients.

Uterine leiomyomas (ULs) are common benign tumors affecting women of different ethnicities. A large proportion of UL has mutations in MED12. Multiple and solitary ULs usually manifest with different severities, suggesting that their origin and growth pattern may be driven by different molecular mechanisms. Here, we compared the frequency and the spectrum of MED12 exon 2 mutations between multiple (n=82) and solitary (n=40) ULs from Russian patients. Overall, we detected MED12 exon 2 mutations in 51.6% (63/122) of ULs. The frequency of MED12 exon 2 mutations was almost two-fold higher in samples from the multiple UL patients than in those from the solitary UL patients – 61% (50/82) versus 32.5% (13/40). The increased MED12 exon 2 mutation frequency in the multiple ULs was not accompanied by significant alterations in the spectrum of mutation categories, which included missense mutations, deletions, splicing defects, and multiple (double/triple) mutations. Each mutation category had a unique mutation set, comprising both frequent and rarely encountered mutations, which did and did not overlap between the studied groups, respectively. We conclude that in contrast to the solitary ULs, the multiple ULs predominantly originate through MED12-associated mechanisms. The nature of these mechanisms seems to be similar in solitary and multiple ULs, as they contain similar mutations. In multiple UL patients, they are likely to be nonsporadic, indicating the existence of specific factors predisposing to multiple UL development. These data suggest that to clearly understand UL pathogenesis, solitary and multiple tumors should probably be analyzed as separate sets.

Genome-wide 5-hydroxymethylcytosine patterns in human spermatogenesis are associated with semen quality

We performed immunofluorescent analysis of DNA hydroxymethylation and methylation in human testicular spermatogenic cells from azoospermic patients and ejaculated spermatozoa from sperm donors and patients from infertile couples. In contrast to methylation which was present throughout spermatogenesis, hydroxymethylation was either high or almost undetectable in both spermatogenic cells and ejaculated spermatozoa. On testicular cytogenetic preparations, 5-hydroxymethylcytosine was undetectable in mitotic and meiotic chromosomes, and was present exclusively in interphase spermatogonia Ad and in a minor spermatid population. The proportions of hydroxymethylated and non-hydroxymethylated diploid and haploid nuclei were similar among samples, suggesting that the observed alterations of 5-hydroxymethylcytosine patterns in differentiating spermatogenic cells are programmed. In ejaculates, a few spermatozoa had high 5-hydroxymethylcytosine level, while in the other ones hydroxymethylation was almost undetectable. The percentage of highly hydroxymethylated (5-hydroxymethylcytosine-positive) spermatozoa varied strongly among individuals. In patients from infertile couples, it was higher than in sperm donors (P<0.0001) and varied in a wider range: 0.12-21.24% versus 0.02-0.46%. The percentage of highly hydroxymethylated spermatozoa correlated strongly negatively with the indicators of good semen quality - normal morphology (r=-0.567, P<0.0001) and normal head morphology (r=-0.609, P<0.0001) - and strongly positively with the indicator of poor semen quality: sperm DNA fragmentation (r=0.46, P=0.001). Thus, the immunocytochemically detected increase of 5hmC in individual spermatozoa is associated with infertility in a couple and with deterioration of sperm parameters. We hypothesize that this increase is not programmed, but represents an induced abnormality and, therefore, it can be potentially used as a novel indicator of semen quality.We performed immunofluorescent analysis of DNA hydroxymethylation and methylation in human testicular spermatogenic cells from azoospermic patients and ejaculated spermatozoa from sperm donors and patients from infertile couples. In contrast to methylation which was present throughout spermatogenesis, hydroxymethylation was either high or almost undetectable in both spermatogenic cells and ejaculated spermatozoa. On testicular cytogenetic preparations, 5-hydroxymethylcytosine was undetectable in mitotic and meiotic chromosomes, and was present exclusively in interphase spermatogonia Ad and in a minor spermatid population. The proportions of hydroxymethylated and non-hydroxymethylated diploid and haploid nuclei were similar among samples, suggesting that the observed alterations of 5-hydroxymethylcytosine patterns in differentiating spermatogenic cells are programmed. In ejaculates, a few spermatozoa had high 5-hydroxymethylcytosine level, while in the other ones hydroxymethylation was almost undetectable. The percentage of highly hydroxymethylated (5-hydroxymethylcytosine-positive) spermatozoa varied strongly among individuals. In patients from infertile couples, it was higher than in sperm donors (P<0.0001) and varied in a wider range: 0.12-21.24% versus 0.02-0.46%. The percentage of highly hydroxymethylated spermatozoa correlated strongly negatively with the indicators of good semen quality – normal morphology (r=-0.567, P<0.0001) and normal head morphology (r=-0.609, P<0.0001) – and strongly positively with the indicator of poor semen quality: sperm DNA fragmentation (r=0.46, P=0.001). Thus, the immunocytochemically detected increase of 5hmC in individual spermatozoa is associated with infertility in a couple and with deterioration of sperm parameters. We hypothesize that this increase is not programmed, but represents an induced abnormality and, therefore, it can be potentially used as a novel indicator of semen quality.

Oxidized form of 5-methylcytosine—5-hydroxymethylcytosine: a new insight into the biological significance in the mammalian genome

In this review, we summarize data on 5-hydroxymethylcytosine—a modification of cytosine with a recently discovered epigenetic effect. We discuss the biochemical mechanisms of 5-hydroxymethylcytosine generation and further modification in the mammalian genome, analyze the role of 5-hydroxymethylcytosine in the epigenetic reprogramming during mammalian gametogenesis and early embryogenesis as well as in the regulation of gene expression. We also show recent data on the diseases and the adverse environmental factors, linked to the DNA hydroxymethylation disruptions.

Catechol-O-methyltransferase Val158Met polymorphism is associated with increased risk of multiple uterine leiomyomas either positive or negative for MED12 exon 2 mutations

Aims To study the possible association of catechol-O-methyltransferase (COMT) Val158Met polymorphism with multiple and solitary uterine leiomyomas (ULs) and to check whether the COMT Val/Val genotype is associated with MED12 exon 2 mutations in fibroids. Methods The COMT Val158Met allele and genotype frequencies were compared between age-matched women with ULs (n=104) and controls (n=59). Patients with UL were subcategorised by diagnosis of solitary (n=59) or multiple (n=45) fibroids and by the presence of somatic MED12 exon 2 mutations in at least one fibroid (n=32) or in neither fibroid (n=26). The association of COMT Val/Val genotype with the presence of any ULs, solitary/multiple ULs and ULs positive/negative for MED12 exon 2 mutations was evaluated by χ2 tests using a dominant genotype model (G/G vs G/A+A/A) and expressed as ORs and 95% CIs. Results The COMT Val/Val genotype frequency did not differ between the patients with UL and the controls (28.8% vs 18.6%, p=0.149, OR 1.77; CI 0.81 to 3.86). However, it was significantly higher in the patients who had multiple UL compared with the solitary UL (40% vs 20.3%, p=0.028, OR 2.61; CI 1.09 to 6.24) and to the controls (40% vs 18.6%, p=0.016, OR 2.91; CI 1.20 to 7.06). No association of the COMT Val/Val genotype with UL-specific MED12 exon 2 mutations was found (p=0.662, OR 0.77; CI 0.23 to 2.53). Conclusions Women with COMT Val/Val genotype are at high risk of developing multiple uterine fibroids either positive or negative for MED12 exon 2 mutations. These data are important to design new strategies for UL prophylaxis and treatment.

A comparative cytogenetic analysis of miscarriages following natural conception and assisted reproductive technologies

The present paper summarizes results of cytogenetic study of 679 chorionic samples from miscarriages following natural conception and assisted reproductive technologies. Frequency and spectrum of karyotype pathology and its correlation with maternal age and term of gestation are analyzed. The results are compared and discussed with relevant other studies

The Evolution of Ideas on the Biological Role of 5-methylcytosine Oxidative Derivatives in the Mammalian Genome

In this review, we summarize the data on 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine— cytosine modifications which are produced by TET-mediated oxidation of 5-methylcytosine in DNA. We show the biochemistry of modified cytosine, as well as methods for its global and location analysis. We also highlight the milestones in the evolution of ideas on the biological role of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in the mammalian genome from their discovery in 2009 to the present time.

Genomic distribution of 5-formylcytosine and 5-carboxylcytosine in human preimplantation embryos: PENDINA et al.

5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) are derivatives of 5-methylcytosine (5mC). Sequential enzymatic oxidation of 5mC produces 5-hydroxymethylcytosine (5hmC), 5fC, and 5caC. Similarly to 5hmC, 5fC and 5caC serve as transient intermediates in active DNA demethylation which is a crucial step in epigenetic reprogramming of mammalian preimplantation embryos. This article is protected by copyright. All rights reserved.

Differential DNA Hydroxymethylation in Human Uterine Leiomyoma Cells Depending on the Phase of Menstrual Cycle and Presence of MED12 Gene Mutations

Using immunofluorescence with specific antibodies, we analyzed DNA hydroxymethylation in uncultured cells from 25 human uterine leiomyomas considering the menstrual cycle phase during surgery and the presence of MED12 gene mutations. It was found that each tumor node had specific DNA hydroxymethylation level that did not depend on the presence of mutations in MED12 gene, but depended on the phase of menstrual cycle. The degree of DNA hydroxymethylation was significantly lower in cells of leiomyomas excised during the luteal phase compared to the follicular phase (p=0.0431). Hormonal status changing at various phases of menstrual cycle is a factor affecting DNA hydroxymethylation in leiomyoma cells.