A View on Uterine Leiomyoma Genesis through the Prism of Genetic, Epigenetic and Cellular Heterogeneity.

Uterine leiomyomas (ULs), frequent benign tumours of the female reproductive tract, are associated with a range of symptoms and significant morbidity. Despite extensive research, there is no consensus on essential points of UL initiation and development. The main reason for this is a pronounced inter- and intratumoral heterogeneity resulting from diverse and complicated mechanisms underlying UL pathobiology. In this review, we comprehensively analyse risk and protective factors for UL development, UL cellular composition, hormonal and paracrine signalling, epigenetic regulation and genetic abnormalities. We conclude the need to carefully update the concept of UL genesis in light of the current data. Staying within the framework of the existing hypotheses, we introduce a possible timeline for UL development and the associated key events-from potential prerequisites to the beginning of UL formation and the onset of driver and passenger changes.

Telomere Length in Human Spermatogenic Cells as a New Potential Predictor of Clinical Outcomes in ART Treatment with Intracytoplasmic Injection of Testicular Spermatozoa.

Predicting the clinical outcomes of intracytoplasmic sperm injection (ICSI) cycles that use the testicular spermatozoa of azoospermic patients presents a challenge. Thus, the development of additional approaches to assessing the competence of a testicular-sperm-derived embryo without causing damage to gametes or the embryo is necessary. One of the key parameters in determining such developmental competence is telomere length (TL). We aimed to analyze TLs in spermatogenic cells from the testicular biopsy samples of azoospermic patients and determine how this parameter influences embryo competence for pre- and post-implantation development. Using Q-FISH, we studied the TL of the chromosomes in spermatogonia and spermatocytes I from the TESE biopsy samples of 30 azoospermic patients. An increase in TL was detected during the differentiation from spermatogonia to spermatocytes I. The patients' testicular spermatozoa were used in 37 ICSI cycles that resulted in 22 embryo transfers. Nine pregnancies resulted, of which, one was ectopic and eight ended in birth. The analysis of embryological outcomes revealed a dependence between embryo competence for development to the blastocyst stage and the TL in spermatogenic cells. The TLs in spermatogonia and spermatocytes I in the testicular biopsy samples were found to be higher in patients whose testicular sperm ICSI cycles resulted in a birth. Therefore, the length of telomeres in spermatogenic cells can be considered as a potential prognostic criterion in assessing the competence of testicular-sperm-derived embryos for pre- and post-implantation development. The results of this study provide the basis for the development of a laboratory test for the prediction of testicular sperm ICSI cycle outcomes.

Uterine Leiomyomas with an Apparently Normal Karyotype Comprise Minor Heteroploid Subpopulations Differently Represented in vivo and in vitro.

In the present study, we aimed to check whether uterine leiomyomas (ULs) with an apparently normal karyotype in vitro comprise "hidden" cell subpopulations with numerical chromosome abnormalities (heteroploid cells). A total of 32 ULs obtained from 32 patients were analyzed in the study. Each UL was sampled for in vivo and in vitro cytogenetic studies. Karyotyping was performed on metaphase preparations from the cultured UL samples. A normal karyotype was revealed in 20 out of the 32 ULs, of which 9 were selected for further study based on the good quality of the interphase preparations. Then, using interphase FISH with centromeric DNA probes, we analyzed the copy number of chromosomes 7 and 16 in 1,000 uncultured and 1,000 cultured cells of each selected UL. All of the ULs included both disomic cells representing a predominant subpopulation and heteroploid cells reaching a maximum frequency of 21.6% (mean 9.8%) in vivo and 11.5% (mean 6.1%) in vitro. The spectrum of heteroploid cells was similar in vivo and in vitro and mostly consisted of monosomic and tetrasomic cells. However, their frequencies in the cultured samples differed from those in the uncultured ones: while the monosomic cells decreased in number, the tetrasomic cells became more numerous. The frequency of either monosomic or tetrasomic cells both in vivo and in vitro was not associated with the presence of MED12 exon 2 mutations in the tumors. Our results suggest that ULs with an apparently normal karyotype consist of both karyotypically normal and heteroploid cells, implying that the occurrence of minor cell subpopulations with numerical chromosome abnormalities may be considered a characteristic of UL tumorigenesis. Different frequencies of heteroploid cells in vivo and in vitro suggest their dependence on microenvironmental conditions, thus providing a pathway for regulation of their propagation, which may be important for the UL pathogenesis.

Telomere Length in Chromosomally Normal and Abnormal Miscarriages and Ongoing Pregnancies and Its Association with 5-hydroxymethylcytosine Patterns.

The present study investigates telomere length (TL) in dividing chorionic cytotrophoblast cells from karyotypically normal and abnormal first trimester miscarriages and ongoing pregnancies. Using Q-FISH, we measured relative TLs in the metaphase chromosomes of 61 chorionic villous samples. Relative TLs did not differ between karyotypically normal samples from miscarriages and those from ongoing pregnancies (p = 0.3739). However, among the karyotypically abnormal samples, relative TLs were significantly higher in ongoing pregnancies than in miscarriages (p < 0.0001). Relative TLs were also significantly higher in chorion samples from karyotypically abnormal ongoing pregnancies than in those from karyotypically normal ones (p = 0.0018) in contrast to miscarriages, where relative TL values were higher in the karyotypically normal samples (p = 0.002). In the karyotypically abnormal chorionic cytotrophoblast, the TL variance was significantly lower than in any other group (p < 0.05). Assessed by TL ratios between sister chromatids, interchromatid TL asymmetry demonstrated similar patterns across all of the chorion samples (p = 0.22) but significantly exceeded that in PHA-stimulated lymphocytes (p < 0.0001, p = 0.0003). The longer telomere was predominantly present in the hydroxymethylated sister chromatid in chromosomes featuring hemihydroxymethylation (containing 5-hydroxymethylcytosine in only one sister chromatid)-a typical sign of chorionic cytotrophoblast cells. Our results suggest that the phenomena of interchromatid TL asymmetry and its association to 5hmC patterns in chorionic cytotrophoblast, which are potentially linked to telomere lengthening through recombination, are inherent to the development programme. The TL differences in chorionic cytotrophoblast that are associated with karyotype and embryo viability seem to be determined by heredity rather than telomere elongation mechanisms. The inheritance of long telomeres by a karyotypically abnormal embryo promotes his development, whereas TL in karyotypically normal first-trimester embryos does not seem to have a considerable impact on developmental capacity.

Telomere Length in Metaphase Chromosomes of Human Triploid Zygotes.

The human lifespan is strongly influenced by telomere length (TL) which is defined in a zygote-when two highly specialised haploid cells form a new diploid organism. Although TL is a variable parameter, it fluctuates in a limited range. We aimed to establish the determining factors of TL in chromosomes of maternal and paternal origin in human triploid zygotes. Using Q-FISH, we examined TL in the metaphase chromosomes of 28 human triploid zygotes obtained from 22 couples. The chromosomes' parental origin was identified immunocytochemically through weak DNA methylation and strong hydroxymethylation in the sperm-derived (paternal) chromosomes versus strong DNA methylation and weak hydroxymethylation in the oocyte-derived (maternal) ones. In 24 zygotes, one maternal and two paternal chromosome sets were identified, while the four remaining zygotes contained one paternal and two maternal sets. For each zygote, we compared mean relative TLs between parental chromosomes, identifying a significant difference in favour of the paternal chromosomes, which attests to a certain "imprinting" of these regions. Mean relative TLs in paternal or maternal chromosomes did not correlate with the respective parent's age. Similarly, no correlation was observed between the mean relative TL and sperm quality parameters: concentration, progressive motility and normal morphology. Based on the comparison of TLs in chromosomes inherited from a single individual's gametes with those in chromosomes inherited from different individuals' gametes, we compared intraindividual (intercellular) and interindividual variability, obtaining significance in favour of the latter and thus validating the role of heredity in determining TL in zygotes. A comparison of the interchromatid TL differences across the chromosomes from sets of different parental origin with those from PHA-stimulated lymphocytes showed an absence of a significant difference between the maternal and paternal sets but a significant excess over the lymphocytes. Therefore, interchromatid TL differences are more pronounced in zygotes than in lymphocytes. To summarise, TL in human zygotes is determined both by heredity and parental origin; the input of other factors is possible within the individual's reaction norm.

Environmental Epigenetics and Genome Flexibility: Focus on 5-Hydroxymethylcytosine.

Convincing evidence accumulated over the last decades demonstrates the crucial role of epigenetic modifications for mammalian genome regulation and its flexibility. DNA methylation and demethylation is a key mechanism of genome programming and reprogramming. During ontogenesis, the DNA methylome undergoes both programmed changes and those induced by environmental and endogenous factors. The former enable accurate activation of developmental programs; the latter drive epigenetic responses to factors that directly or indirectly affect epigenetic biochemistry leading to alterations in genome regulation and mediating organism response to environmental transformations. Adverse environmental exposure can induce aberrant DNA methylation changes conducive to genetic dysfunction and, eventually, various pathologies. In recent years, evidence was derived that apart from 5-methylcytosine, the DNA methylation/demethylation cycle includes three other oxidative derivatives of cytosine-5-hydroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxylcytosine. 5hmC is a predominantly stable form and serves as both an intermediate product of active DNA demethylation and an essential hallmark of epigenetic gene regulation. This makes 5hmC a potential contributor to epigenetically mediated responses to environmental factors. In this state-of-the-art review, we consolidate the latest findings on environmentally induced adverse effects on 5hmC patterns in mammalian genomes. Types of environmental exposure under consideration include hypnotic drugs and medicines (i.e., phenobarbital, diethylstilbestrol, cocaine, methamphetamine, ethanol, dimethyl sulfoxide), as well as anthropogenic pollutants (i.e., heavy metals, particulate air pollution, bisphenol A, hydroquinone, and pentachlorophenol metabolites). We put a special focus on the discussion of molecular mechanisms underlying environmentally induced alterations in DNA hydroxymethylation patterns and their impact on genetic dysfunction. We conclude that DNA hydroxymethylation is a sensitive biosensor for many harmful environmental factors each of which specifically targets 5hmC in different organs, cell types, and DNA sequences and induces its changes through a specific metabolic pathway. The associated transcriptional changes suggest that environmentally induced 5hmC alterations play a role in epigenetically mediated genome flexibility. We believe that knowledge accumulated in this review together with further studies will provide a solid basis for new approaches to epigenetic therapy and chemoprevention of environmentally induced epigenetic toxicity involving 5hmC patterns.

Inter-Cell and Inter-Chromosome Variability of 5-Hydroxymethylcytosine Patterns in Noncultured Human Embryonic and Extraembryonic Cells.

5-hydroxymethylcytosine (5hmC) is an oxidative derivative of 5-methylcytosine (5mC). Recent studies have revealed a sharp difference in the levels of 5hmC in 2 opposite DNA strands of a given chromosome and a chromosome-wide cell-to-cell variability in mammalian cells. This asymmetric 5hmC distribution was found in cultured cells, which may not fully mimic in vivo epigenetic processes. We have checked whether inter-chromosome and inter-cell variability of 5hmC patterns is typical for noncultured human cells. Using indirect immunofluorescence, we analyzed the localization of 5hmC and its co-distribution with 5mC on direct preparations of mitotically active cells from human embryonic lung and chorionic cytotrophoblast samples. We demonstrated 3 types of chromosomes according to the 5hmC accumulation pattern: hydroxymethylated (5hmC in both sister chromatids), hemihydroxymethylated (5hmC in only 1 sister chromatid), and nonhydroxymethylated ones. Each accumulation type was not specific to any particular chromosome, resulting in different 5hmC patterns between homologous chromosomes, among chromosomes within each metaphase plate, among metaphases in one tissue, and between the tissues. The 5mC distribution was stable: chromosomes were methylated in R-bands and, especially in embryonic lung cells, in the heterochromatic regions 1q12, 9q12, and 16q11.2. Our results provide the first evidence of inter-cell and inter-chromosome variability of 5hmC patterns in human noncultured embryonic and extraembryonic cells.

Chromosome hydroxymethylation patterns in human zygotes and cleavage-stage embryos.

We report the sequential changes in 5-hydroxymethylcytosine (5hmC) patterns in the genome of human preimplantation embryos during DNA methylation reprogramming. We have studied chromosome hydroxymethylation and methylation patterns in triploid zygotes and blastomeres of cleavage-stage embryos. Using indirect immunofluorescence, we have analyzed the localization of 5hmC and its co-distribution with 5-methylcytosine (5mC) on the QFH-banded metaphase chromosomes. In zygotes, 5hmC accumulates in both parental chromosome sets, but hydroxymethylation is more intensive in the poorly methylated paternal set. In the maternal set, chromosomes are highly methylated, but contain little 5hmC. Hydroxymethylation is highly region specific in both parental chromosome sets: hydroxymethylated loci correspond to R-bands, but not G-bands, and have well-defined borders, which coincide with the R/G-band boundaries. The centromeric regions and heterochromatin at 1q12, 9q12, 16q11.2, and Yq12 contain little 5mC and no 5hmC. We hypothesize that 5hmC may mark structural/functional genome 'units' corresponding to chromosome bands in the newly formed zygotic genome. In addition, we suggest that the hydroxymethylation of R-bands in zygotes can be treated as a new characteristic distinguishing them from G-bands. At cleavages, chromosomes with asymmetrical hydroxymethylation of sister chromatids appear. They decrease in number during cleavages, whereas totally non-hydroxymethylated chromosomes become numerous. Taken together, our findings suggest that, in the zygotic genome, 5hmC is distributed selectively and its pattern is determined by both parental origin of chromosomes and type of chromosome bands - R, G, or C. At cleavages, chromosome hydroxymethylation pattern is dynamically changed due to passive and non-selective overall loss of 5hmC, which coincides with that of 5mC.

A comparative cytogenetic study of miscarriages after IVF and natural conception in women aged under and over 35 years.

PURPOSE: To compare the frequency and the spectrum of karyotype abnormality in the first trimester miscarriages in women aged under and over 35 years, who conceived naturally (NC) and who conceived through in vitro fertilization (IVF). METHODS: Comparative analysis of cytogenetic data obtained by karyotyping of miscarriages in patients who conceived naturally, and who conceived through IVF. Patients were subcategorized by their age: <35 years (NC, n = 173; IVF, n = 108) and ≥ 35 years (NC, n = 107; IVF, n = 111). RESULTS: A total of 499 miscarriage karyotypes was analyzed. The spectrum and the relative proportions of different cytogenetic categories in karyotypically abnormal miscarriages differed neither between the NC and IVF patients aged <35 years, nor between the NC and IVF patients aged ≥ 35 years. In the patients aged <35 years, the incidence of abnormal miscarriage karyotype was lower in the IVF group (37.04 % vs 62.43%). In the patients aged ≥ 35 years, the incidence of miscarriages with cytogenetic pathology did not differ between the NC and the IVF group (75.70 % vs 58.56%). The lowest frequency of karyotypically abnormal miscarriages (29.82%) was detected in the young IVF-treated patients at <7 weeks of gestation. CONCLUSIONS: IVF does not increase the risk of a pregnancy loss because of abnormal embryonic karyotype, nor does it increase the preponderance for any specific type of cytogenetic abnormality in both patients aged under and over 35 years. In young IVF-treated women early pregnancy loss is generally caused by non-cytogenetic factors. Identification of a cytogenetically normal spontaneous abortion is clinically significant and reinforces the importance of developing an appropriate diagnosis and treatment strategies for IVF patients in order to reduce the risk of euploid pregnancy loss.

Re-Examination of PGT-A Detected Genetic Pathology in Compartments of Human Blastocysts: A Series of 23 Cases.

Background: In recent years, preimplantation genetic testing for aneuploidies (PGT-A) has become widespread in assisted reproduction. However, contrary to expectations, PGT-A does not significantly improve the clinical outcomes of assisted reproductive technologies. One of the underlying reasons is the discordance between the PGT-A results and the true chromosomal constitution of the blastocyst. In this case series, we re-examined the PGT-A results in trophectoderm (TE) re-biopsies and in the two isolated blastocyst compartments-the TE and the inner cell mass (ICM). Methods: This study enrolled 23 human blastocysts from 17 couples who were referred for assisted reproduction. The blastocysts were unsuitable for uterine transfer due to the chromosomal imbalance revealed by PGT-A using array comparative genomic hybridization (aCGH) (n = 11) or next-generation sequencing (NGS) (n = 12). The re-examination of the PGT results involved two steps: (1) a TE re-biopsy with subsequent aCGH and (2) blastocyst separation into the TE and the ICM with a subsequent cell-by-cell analysis of each isolated compartment by fluorescence in situ hybridization (FISH) with the DNA probes to chromosomes 13, 16, 18, 21, and 22 as well as to the PGT-A detected imbalanced chromosomes. Results: In 8 out of 23 cases, the PGT-A results were concordant with both the re-biopsy and the isolated TE and ICM analyses. The latter included the diagnoses of full non-mosaic aneuploidies (five cases of trisomies and two cases of monosomies). In one case, the results of PGT-A, aCGH on the TE re-biopsy, and FISH on the isolated TE showed Xp tetrasomy, which contrasted with the FISH results on the isolated ICM, where this chromosomal pathology was not detected. This case was classified as a confined mosaicism. In 4 out of 23 cases, the results were partially discordant. The latter included one case of trisomy 12, which was detected as non-mosaic by PGT-A and the re-biopsy and as mosaic by FISH on the isolated TE and ICM. This case was classified as a true mosaicism with a false negative PGT-A result. In 11 out of 23 cases, the re-examination results were not concordant with the PGT-A results. In one of these discordant cases, non-mosaic tetraploidy was detected by FISH in the isolated TE and ICM, whereas the PGT-A and the TE re-biopsy failed to detect any abnormality, which advocated for their false negative result. In two cases, the re-examination did not confirm full aneuploidies. In eight cases, full or partial mosaic aneuploidies as well as chaotic mosacism were not confirmed in the isolated TE nor the isolated ICM. Thus, in 47.8% of cases, the PGT-A results did not reflect the true chromosomal constitution of a blastocyst. Conclusions: The PGT results may have different prognostic value in the characterization of the chromosomal constitution of a blastocyst. The detected non-mosaic aneuploidies have the highest prognostic value. In stark contrast, most PGT-identified mosaic aneuploidies fail to characterize the true chromosomal constitution of a blastocyst. Once detected, a differential diagnosis is needed.

Prenatal Detection of Trisomy 2: Considerations for Genetic Counseling and Testing.

We report on the case of prenatal detection of trisomy 2 in placental biopsy and further algorithm of genetic counseling and testing. A 29-year-old woman with first-trimester biochemical markers refused chorionic villus sampling and preferred targeted non-invasive prenatal testing (NIPT), which showed low risk for aneuploidies 13, 18, 21, and X. A series of ultrasound examinations revealed increased chorion thickness at 13/14 weeks of gestation and fetal growth retardation, a hyperechoic bowel, challenging visualization of the kidneys, dolichocephaly, ventriculomegaly, increase in placental thickness, and pronounced oligohydramnios at 16/17 weeks of gestation. The patient was referred to our center for an invasive prenatal diagnosis. The patient's blood and placenta were sampled for whole-genome sequencing-based NIPT and array comparative genomic hybridization (aCGH), respectively. Both investigations revealed trisomy 2. Further prenatal genetic testing in order to confirm trisomy 2 in amniocytes and/or fetal blood was highly questionable because oligohydramnios and fetal growth retardation made amniocentesis and cordocentesis technically unfeasible. The patient opted to terminate the pregnancy. Pathological examination of the fetus revealed internal hydrocephalus, atrophy of brain structure, and craniofacial dysmorphism. Conventional cytogenetic analysis and fluorescence in situ hybridization revealed chromosome 2 mosaicism with a prevalence of trisomic clone in the placenta (83.2% vs. 16.8%) and a low frequency of trisomy 2, which did not exceed 0.6% in fetal tissues, advocating for low-level true fetal mosaicism. To conclude, in pregnancies at risk of fetal chromosomal abnormalities that refuse invasive prenatal diagnosis, whole-genome sequencing-based NIPT, but not targeted NIPT, should be considered. In prenatal cases of trisomy 2, true mosaicism should be distinguished from placental-confined mosaicism using cytogenetic analysis of amniotic fluid cells or fetal blood cells. However, if material sampling is impossible due to oligohydramnios and/or fetal growth retardation, further decisions should be based on a series of high-resolution fetal ultrasound examinations. Genetic counseling for the risk of uniparental disomy in a fetus is also required.

Cytogenomic Profile of Uterine Leiomyoma: In Vivo vs. In Vitro Comparison.

We performed a comparative cytogenomic analysis of cultured and uncultured uterine leiomyoma (UL) samples. The experimental approach included karyotyping, aCGH, verification of the detected chromosomal abnormalities by metaphase and interphase FISH, MED12 mutation analysis and telomere measurement by Q-FISH. An abnormal karyotype was detected in 12 out of 32 cultured UL samples. In five karyotypically abnormal ULs, MED12 mutations were found. The chromosomal abnormalities in ULs were present mostly by complex rearrangements, including chromothripsis. In both karyotypically normal and abnormal ULs, telomeres were ~40% shorter than in the corresponding myometrium, being possibly prerequisite to chromosomal rearrangements. The uncultured samples of six karyotypically abnormal ULs were checked for the detected chromosomal abnormalities through interphase FISH with individually designed DNA probe sets. All chromosomal abnormalities detected in cultured ULs were found in corresponding uncultured samples. In all tumors, clonal spectra were present by the karyotypically abnormal cell clone/clones which coexisted with karyotypically normal ones, suggesting that chromosomal abnormalities acted as drivers, rather than triggers, of the neoplastic process. In vitro propagation did not cause any changes in the spectrum of the cell clones, but altered their ratio compared to uncultured sample. The alterations were unique for every UL. Compared to its uncultured counterpart, the frequency of chromosomally abnormal cells in the cultured sample was higher in some ULs and lower in others. To summarize, ULs are characterized by both inter- and intratumor genetic heterogeneity. Regardless of its MED12 status, a tumor may be comprised of clones with and without chromosomal abnormalities. In contrast to the clonal spectrum, which is unique and constant for each UL, the clonal frequency demonstrates up or down shifts under in vitro conditions, most probably determined by the unequal ability of cells with different genetic aberrations to exist outside the body.

Age and Serum AMH and FSH Levels as Predictors of the Number of Oocytes Retrieved from Chromosomal Translocation Carriers after Controlled Ovarian Hyperstimulation: Applicability and Limitations.

We studied the impact of age and the serum anti-Müllerian hormone (AMH)/follicle-stimulating hormone (FSH) levels on the number of cumulus-oocyte complexes (COCs) retrieved from female reciprocal and Robertsonian translocation carriers after controlled ovarian hyperstimulation (COH). The number of COCs retrieved after COH was retrospectively analyzed in female translocation carriers and 46,XX partners of male translocation carriers from 100 couples. The median number of COCs varied from nine to 16 and did not differ among subgroups of women categorized by age, presence and type of a translocation. The number of COCs correlated negatively with the woman's age in both the reciprocal and the Robertsonian translocation carriers, while in 46,XX women no correlation was detected. The number of COCs did not differ between the reciprocal and the Robertsonian translocation carriers aged either <35 or ≥35 years. In translocation carriers, the number of COCs correlated with the serum AMH level only in the younger-age subgroups; the correlation was strong positive in reciprocal and moderate positive in Robertsonian translocation carriers. The 46,XX women aged both <35 and ≥35 years showed similar moderate positive correlations. Across all subgroups, the number of COCs correlated moderately negatively with the serum FSH level only in Robertsonian translocation carriers aged <35 years. Our results suggest that chromosomal translocations per se do not increase the risk of poor oocyte retrieval outcome after COH. In translocation carriers, oocyte retrieval outcome depends to a large extent on their age. The serum AMH level strongly predicts oocyte retrieval outcomes only in young reciprocal translocation carriers, while the serum FSH level has a moderate predictive value in young Robertsonian translocation carriers.

On the Complexity of Mechanisms and Consequences of Chromothripsis: An Update.

In the present review, we focus on the phenomenon of chromothripsis, a new type of complex chromosomal rearrangements. We discuss the challenges of chromothripsis detection and its distinction from other chromoanagenesis events. Along with already known causes and mechanisms, we introduce aberrant epigenetic regulation as a possible pathway to chromothripsis. We address the issue of chromothripsis characteristics in cancers and benign tumours, as well as chromothripsis inheritance in cases of its occurrence in germ cells, zygotes and early embryos. Summarising the presented data on different phenotypic effect of chromothripsis, we assume that its consequences are most likely determined not by the chromosome shattering and reassembly themselves, but by the genome regions involved in the rearrangement.

Reproductive History of a Woman With 8p and 18p Genetic Imbalance and Minor Phenotypic Abnormalities.

We report on the phenotype and the reproductive history of an adult female patient with an unbalanced karyotype: 8p23 and 18p11.3 terminal deletions and 8p22 duplication. The indication for karyotyping of the 28-year-old patient was a structural rearrangement in her miscarriage specimen: 45,ХХ,der(8;18)t(8;18)(p23;p11.3). Unexpectedly, the patient had the same karyotype with only one normal chromosome 8, one normal chromosome 18, and a derivative chromosome, which was a product of chromosomes 8 and 18 fusion with loss of their short arm terminal regions. Fluorescence in situ hybridization revealed that derivative chromosome was a pseudodicentric with an active centromere of chromosome 8. Array comparative genomic hybridization confirmed 8p and 18p terminal deletions and additionally revealed 8p22 duplication with a total of 43 OMIM annotated genes being affected by the rearrangement. The patient had minor facial and cranial dysmorphia and no pronounced physical or mental abnormalities. She was socially normal, had higher education and had been married since the age of 26 years. Considering genetic counseling, the patient had decided to conceive the next pregnancy through in vitro fertilization (IVF) with preimplantation genetic testing for structural chromosomal aberrations (PGT-SR). She underwent four IVF/PGT-SR cycles with a total of 25 oocytes obtained and a total of 10 embryos analyzed. Only one embryo was balanced regarding chromosomes 8 and 18, while the others were unbalanced and demonstrated different combinations of the normal chromosomes 8 and 18 and the derivative chromosome. The balanced embryo was transferred, but the pregnancy was not registered. After four unsuccessful IVF/PGT-SR cycles, the patient conceived naturally. Non-invasive prenatal testing showed additional chromosome 18. The prenatal cytogenetic analysis of chorionic villi revealed an abnormal karyotype: 46,ХХ,der(8;18)t(8;18)(p23;p11.3)mat,+18. The pregnancy was terminated for medical reasons. The patient has a strong intention to conceive a karyotypically normal fetus. However, genetic counseling regarding this issue is highly challenging. Taking into account a very low chance of balanced gametes, emotional stress caused by numerous unsuccessful attempts to conceive a balanced embryo and increasing age of the patient, an IVF cycle with a donor oocyte should probably be considered.

Whole­exome sequencing in Russian children with non­type 1 diabetes mellitus reveals a wide spectrum of genetic variants in MODY­related and unrelated genes.

The present study reports on the frequency and the spectrum of genetic variants causative of monogenic diabetes in Russian children with non­type 1 diabetes mellitus. The present study included 60 unrelated Russian children with non­type 1 diabetes mellitus diagnosed before the age of 18 years. Genetic variants were screened using whole­exome sequencing (WES) in a panel of 35 genes causative of maturity onset diabetes of the young (MODY) and transient or permanent neonatal diabetes. Verification of the WES results was performed using PCR­direct sequencing. A total of 38 genetic variants were identified in 33 out of 60 patients (55%). The majority of patients (27/33, 81.8%) had variants in MODY­related genes: GCK (n=19), HNF1A (n=2), PAX4 (n=1), ABCC8 (n=1), KCNJ11 (n=1), GCK+HNF1A (n=1), GCK+BLK (n=1) and GCK+BLK+WFS1 (n=1). A total of 6 patients (6/33, 18.2%) had variants in MODY­unrelated genes: GATA6 (n=1), WFS1 (n=3), EIF2AK3 (n=1) and SLC19A2 (n=1). A total of 15 out of 38 variants were novel, including GCK, HNF1A, BLK, WFS1, EIF2AK3 and SLC19A2. To summarize, the present study demonstrates a high frequency and a wide spectrum of genetic variants causative of monogenic diabetes in Russian children with non­type 1 diabetes mellitus. The spectrum includes previously known and novel variants in MODY­related and unrelated genes, with multiple variants in a number of patients. The prevalence of GCK variants indicates that diagnostics of monogenic diabetes in Russian children may begin with testing for MODY2. However, the remaining variants are present at low frequencies in 9 different genes, altogether amounting to ~50% of the cases and highlighting the efficiency of using WES in non­GCK­MODY cases.

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.

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.

Placental microRNA expression in pregnancies complicated by superimposed pre­eclampsia on chronic hypertension.

Pre-eclampsia (PE) is a complication of pregnancy that affects 5­8% 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 (CAP­miRSeq). 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 (miR­518a, miR­527, miR­518e and miR­4532) and 2 downregulated (miR­98 and miR­135b) 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.