We report on the telomere length (TL) changes in metaphase chromosomes throughout preimplantation development of human embryos-a period of genome-wide epigenetic reprogramming. Using semiquantitative fluorescence in situ hybridization, we measured relative TLs in the metaphase chromosomes of 69 preimplantation embryos from the zygote up to and including the blastocyst stage. Relative TLs increased significantly from zygote to the 2-5-cell stage, remained almost unchanged at the stages of 2-5 and 6-12-cells and decreased by the blastocyst stage. Concurrently with relative TL decrease at the blastocyst stage, an increase in interindividual TL variability occurred. The zygote-inherited, but not newly synthesized chromatids maintained parent-specific telomeres (longer in paternal compared to maternal chromosomes) up to and including the 2-5-cell stage, with a follow-up TL equalization in 6-12-cell embryos. The extent of interchromatid TL asymmetry-a phenomenon potentially linked to telomere lengthening through recombination-was assessed by TL ratios between sister chromatids and showed similar patterns across all stages of preimplantation development. The longer telomere is presumably located in the highly hydroxymethylated sister chromatid of hemihydroxymethylated chromosomes, i.e., those having higher 5-hydroxymethylcytosine content in one sister chromatid than in the other due to global epigenetic reprogramming in early embryogenesis. To conclude, our study suggests that in human preimplantation development telomeres are reprogrammed in conjunction with genome-wide epigenetic reprogramming of an embryo. By the blastocyst stage, when epigenetic reprogramming comes to an end, parent-specific TLs are also completely reprogrammed and every embryo develops its own unique TL pattern.

In the present study, we aimed to investigate intratumoral karyotype diversity as well as the estrogen/progesterone effect on the cytogenetic profile of uterine leiomyomas (ULs). A total of 15 UL samples obtained from 15 patients were cultured in the media supplemented with estrogen and/or progesterone and without adding hormones. Conventional cytogenetic analysis of culture samples revealed clonal chromosomal abnormalities in 11 out of 15 ULs. Cytogenetic findings were presented by simple and complex chromosomal rearrangements (64% and 36% of cases, respectively) verified through FISH and aCGH. In most ULs with complex chromosomal rearrangements, the breakpoints did not feature clusterization on a single chromosome but were evenly distributed across rearranged chromosomes. The number of breakpoints showed a strong positive correlation with the number of rearranged chromosomes. Moreover, both abovementioned parameters were in a linear dependency from the number of karyotypically different clones per UL. This suggests that complex chromosomal rearrangements in ULs predominantly originate through sequential events rather than one hit. The results of UL cytogenetic analysis depended on the presence of estrogen and/or progesterone in the culture medium. The greatest variety of cytogenetically different cell clones was detected in the samples cultured without hormone supplementation. Their counterparts cultured with progesterone supplementation showed a sharp decrease in clone number, whereas such a decrease induced by estrogen or estrogen-progesterone supplementation was insignificant. These findings suggest that estrogen-progesterone balance is crucial for forming a UL cytogenetic profile, which, in turn, may underlie the unique response of the every karyotypically abnormal UL to medications.

BACKGROUND: The search for new criteria for semen quality based on the evaluation of the structural and functional state of the sperm genome remains a relevant task in reproductive medicine. AIM: This work aimed to assess DNA integrity and the content of 5-hydroxymethylcytosine (5hmC) in the same ejaculated spermatozoa obtained from patients with normozoospermia and pathozoospermia. METHODS: The study included semen samples from 63 patients with normozoospermia (n = 33) and pathozoospermia (n = 30). Microscopic slides were prepared from the samples, on which fragmented DNA was first detected using the TUNEL assay, followed by digital image acquisition. Subsequently, 5hmC was detected by indirect immunofluorescence, and digital images of the same microscopic fields were acquired again. In total, 126,000 spermatozoa were analyzed (2000 per sample). RESULTS: A substantial proportion of spermatozoa (72.8%–94.2%) in all samples showed no DNA integrity violations and exhibited a low (background) level of 5hmC. The proportions of spermatozoa exhibiting DNA fragmentation, increased hydroxymethylation, or both characteristics simultaneously were 0.05%–13.8%, 0.15%–11.5%, and 0.99%–13.38%, respectively. The proportion of spermatozoa with DNA fragmentation and/or DNA hyperhydroxymethylation did not differ between patients with normozoospermia and those with pathozoospermia. DNA fragmentation and DNA hyperhydroxymethylation in ejaculated spermatozoa were found to be interdependent, and their coexistence in gametes was nonrandom. CONCLUSION: The nonrandom coexistence of DNA fragmentation and DNA hyperhydroxymethylation in spermatozoa, along with the interdependence of these features, indicates a common trigger, most likely oxidative stress. However, the presence of additional factors leading to DNA damage or altered hydroxymethylation levels may explain the less than complete overlap of these features in spermatozoa. The assessment of DNA fragmentation and hydroxymethylation levels in spermatozoa appears to be a promising approach for evaluating semen quality and identifying potential causes of idiopathic infertility.

Chromosomal abnormalities of the embryo are the most common cause of first-trimester pregnancy loss. In this single-center study, we assessed the frequency and the spectrum of chromosomal abnormalities in miscarriages for each year of maternal age from 23 to 44. Cytogenetic data were obtained by conventional karyotyping of 7118 miscarriages in women with naturally conceived pregnancies. Chromosomal abnormalities were identified in 67.25% of miscarriages. The total incidence of chromosomal abnormalities increased with maternal aging; however, its average change for a one-year increase in maternal age differed between age spans, equaling 0.704% in the span from 23 to 37 years and 2.095% in the span from 38 to 44 years. At the age of 38 years, the incidence rate surged sharply by 14.79% up to 79.01% and then increased progressively up to 94% in 44-year-old women. The spectrum of chromosomal abnormalities in miscarriages was the same for each year of maternal age from 23 to 44 years. However, the proportions of particular chromosomal abnormalities differed between karyotypically abnormal miscarriages in younger and older women. The proportions of trisomy 16, polyploidy, monosomy X, mosaic aneuploidies, and structural rearrangements decreased with increasing maternal age. In contrast, the proportions of multiple aneuploidies and regular trisomies 13, 15, 18, 21, and 22 showed an upward trend with maternal aging. To summarize, despite the increase in the total incidence of chromosomal abnormalities in miscarriages with maternal aging, the rate of change differs for younger and older women, being three times lower in the former than in the latter. Moreover, the proportion of some abnormalities in karyotypically abnormal miscarriages shows a steady growth, whereas the proportion of others becomes increasingly low with maternal aging, most probably due to the age-dependent prevalence of different molecular and cellular defects.

BACKGROUND: The search for markers of disorders leading to miscarriage with normal embryonic karyotype is an important clinical and diagnostic problem, especially in pregnancies conceived with assisted reproductive technologies. AIM: Analysis of chorionic cells ploidy in naturally and assisted reproductive technologies conceived pregnancies. MATERIALS AND METHODS: A total of 52 chorion samples were included in the study. The samples were divided into groups depending on the developmental status of pregnancy (progressing/arrested), the way of conception (natural/assisted reproductive technologies) and karyotype (normal/trisomy 16). The ploidy of chorionic cells was studied using fluorescence in situ hybridization on interphase nuclei preparations. A total of 50,657 interphase nuclei were analyzed. RESULTS: Along with predominant diploid cells, polyploid cells were detected in all chorionic samples. Their frequency varied among samples from 0.1 to 8.22%. Polyploid cells comprised mainly tetraploid cells which were detected in all samples; triploid cells were also detected in 45 samples, and octoploid cells — in 5 samples. The highest total frequency of all polyploid cell types was found in chorion from assisted reproductive technologies-conceived arrested pregnancies, and the lowest — in chorion from progressing pregnancies. Frequency of tetraploid cells demonstrated the same pattern. Frequency of triploid cells was not associated with a developmental status of pregnancy and the way of conception. However, in chorion samples with trisomy on chromosome 16 in naturally conceived arrested pregnancies, a tendency towards a decrease in the frequency of triploid cells was noted. CONCLUSIONS: An elevated frequency of polyploid cells in chorion may indicate placentation abnormalities, leading to miscarriage even in the absence of embryonic karyotype anomalies. Therefore, an increase in somatic polyploidization in chorion may be considered a promising diagnostic marker of disorders in the placenta formation and functioning.

BACKGROUND: The studies on how sex steroid hormones affect growth of uterine leiomyoma cells with chromosomal abnormalities is highly relevant for development of personalized tumor therapy. AIM: To study in vitro the isolated and combined effects of estrogen and progesterone on uterine leiomyoma cells with chromosomal aberrations — deletions in 7q. MATERIALS AND METHODS: The study was performed on 15 uterine leiomyomas, excised from 15 women of 26–44 years of age who were not treated with hormones. Uterine leiomyoma cells were cultured in hormone-free medium, in the medium supplemented with estrogen, progesterone or both hormones. The chromosome preparations were made and stained with QFH/AcD to perform conventional karyotyping and fluorescence in situ hybridization (FISH) to accurately describe chromosomal rearrangements. The frequency of uterine leiomyoma cells with chromosomal aberrations was assessed by interphase FISH. RESULTS: Deletions in 7q were identified in 6 out of 15 karyotyped uterine leiomyomas; four of them had one clone with deletion in 7q whereas two others comprised two clones with 7q deletions of different length. The frequency of cells carrying deletions in 7q greatly varied in uterine leiomyoma samples cultured in hormone-free medium: from 3.5% to 93.6%. Exposure of cell cultures to estrogen and progesterone resulted in a fold change frequency increase in some of the uterine leiomyomas and decrease in the others. The most significant changes in the frequency of cells with deletions in 7q were registered in response to the isolated estrogen and, to a lesser extent, to progesterone exposure; less significant changes were observed after combined hormonal effect. CONCLUSIONS: In uterine leiomyomas with deletions in 7q, the frequency of abnormal cells may either increase or decrease in response to estrogen and progesterone in vitro supplementation. The isolated effect of estrogen or progesterone on the frequency of uterine leiomyoma cells with deletion in 7q is more pronounced compared to the combined one.

BACKGROUND: The study of telomere length and influencing factors in early human development has both fundamental and applied importance. AIM: A comparative assessment of telomere length in the compartments of human blastocysts, and the analysis of the telomere length association with the quality of blastocysts, genetic imbalance and the maternal age. MATERIALS AND METHODS: The study was performed on trophectoderm and inner cell mass samples of 41 human blastocysts, 26 of which were genetically imbalanced according to preimplantation genetic testing and verification of its results. The microscope slides were prepared for further telomere detection in interphase nuclei by quantitative fluorescence in situ hybridization (Q-FISH). RESULTS: Telomeres in trophectoderm were longer than in inner cell mass, with their length varied from blastocyst to blastocyst. Telomere length in either trophectoderm or inner cell mass did not differ between genetically balanced and imbalanced blastocysts. There was a tendency towards a decrease in telomere length in the blastocyst compartments with increasing maternal age, however, a statistically significant correlation was not confirmed. The telomere length in the inner cell mass, but not in the trophectoderm, was associated with blasocysts’ quality based on the Gardner grade: medium quality blastocysts had longer telomeres than high quality blastocysts. CONCLUSIONS: Long telomeres in trophectoderm may be necessary for implantation and subsequent placentation. Telomere length can be considered among modifiers of the effects of karyotype abnormalities and other negative factors: the inheritance by an embryo of long telomeres apparently gives it a developmental advantage even when genetically imbalanced or has poor morphology. Implantation seems to be an important checkpoint for negative selection of embryos with “unsuccessful” combinations of telomere length, karyotype, and morphology.

BACKGROUND: Epigenetic genome reprogramming is an important determinant of human embryo development. However, its mechanisms remain poorly elucidated, especially in genetically unbalanced embryos. AIM: The aim of this study is the analysis of DNA methylation and hydroxymethylation levels in trophectoderm and inner cell mass of genetically balanced and unbalanced human blastocysts. MATERIALS AND METHODS: Twenty-two IVF-derived human blastocysts were enrolled in the study; of these blastocysts, 15 were genetically unbalanced and 7 — genetically balanced. Detection of 5-methylcytosine and 5-hydroxymethylcytosine was performed on trophectoderm and inner cell mass nuclei by indirect immunofluorescence. RESULTS: In genetically unbalanced blastocysts, the DNA methylation level was elevated in both compartments. The DNA hydroxymethylation level, in contrast, was elevated only in inner cell mass, whereas trophectoderm cells retained the same level as in genetically balanced embryos. These changes equalized the inner cell mass and trophectoderm DNA hydroxymethylation levels in genetically unbalanced blastocysts, while in genetically balanced ones the 5-hydroxymethylcytosine content in inner cell mass lagged behind that in trophectoderm. CONCLUSIONS: Genetic imbalance is associated with differential epigenetic changes in trophectoderm and inner cell mass cells of human blastocysts: DNA methylation level increases in both compartments while DNA hydroxymethylation level increases only in inner cell mass. The trophectoderm cells in genetically unbalanced blastocysts retain the same hydroxymethylation level as in genetically balanced ones, suggesting a possible explanation of the ability of karyotypically abnormal embryos to implant.

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.

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.

Background. The epigenome of gametes is formed under the control of the developmental programme and the influence of environmental factors. How cytosine oxidation patterns are formed and altered in human spermatogenesis remains obscure so far. The aim of the study was to assess 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) patterns in human spermatogenic cells and spermatozoa. Materials and Methods. The study was performed on testicular biopsy samples of 10 azoospermic patients and ejaculate samples of 5 sperm donors and 8 patients from infertile couples. The microscope slides were prepared for further indirect immunofluorescence to detect 5fC and 5caC and FISH to determine spermatogenic cell ploidy. Results. 5fC and 5caC were undetectable in mitotic and meiotic chromosomes of spermatogenic cells, and was present exclusively in some spermatogonia and spermatid interphase nuclei as well as in some ejaculated spermatozoa. The frequency of spermatozoa with 5fC and 5caC varied in a wide range and was higher in patients than in sperm donors (p=0,007, p=0,028). The increase in frequency of spermatozoa with 5fC and 5caC was accompanied with the decrease in frequency of morphologically normal and progressively motile spermatozoa. Conclusions. 5fC and 5caC are differentially distributed in human spermatogenic cells and spermatozoa. The immunocytochemically detected increase of 5fC and 5caC in individual spermatozoa is most likely induced by oxidative stress caused by effects of internal and external factors rather than developmental programme. The evaluation of 5fC and 5caC in spermatozoa can be potentially used as an additional criterion of ejaculate quality.

We studied some features of blood and lymph microcirculation in the brain, heart, and liver of female rats with developed alcoholic cardiomyopathy. In female rats after 24-week forced consumption of 10% ethanol solution, the size and inotropic function of the heart were measured by echocardiography. Microcirculation in the brain, myocardium, and liver was assessed by laser Doppler flowmetry using LAKK-OP2 and LAZMA-D computerized laser analyzers. Using spectral wavelet analysis, we determined the absolute and normalized to total perfusion amplitudes of microcirculation oscillations reflecting various regulatory mechanisms. Intact animals served as controls. In rats of the experimental group, alcoholic cardiomyopathy completely developed. Under these conditions, the index of microcirculation in the brain, myocardium, and liver significantly decreased. At the same time, there was a redistribution in the brain between shunting and nutritive blood flow in favor of the latter. In the myocardium and liver, this ratio did not change.

AIM:To evaluate intraoperative changes in renal microcirculation during percutaneous nephrolithotomy (PCNL), as well as its dynamics in the early postoperative period.MATERIALS AND METHODS:A total of 240 patients treated in the Urology Clinic of the Saratov State Medical University in 2021-2022 were included in the study. All patients underwent PCNL. In the first group (n=105) the standard PCNL through 30 Ch access was done. In the second group (n=135), the procedure was performed through an access of 16 Ch. Intraoperatively, intrapelvic pressure was evaluated according to the authors method, which consists in direct measurement in the collecting system during the procedure, allowing for a faster and more accurate assessment. Prior to surgery, Doppler mapping of the renal blood flow was performed, and indirect registration of the microcirculation index (MCI) was done directly on the operating table using laser Doppler flowmetry (LDF). The diagnostic study was performed at the point of intersection of the 12th rib and the psoas muscle, both on the ipsilateral and contralateral side. In addition, during the procedure, a registration of MI of the mucosa of the calyceal fornix accessible in the direct vision through the access tract for 4 minutes was carried out twice.RESULTS:The index of microcirculation (IM) in the fornix of the upper calyx before the fragmentation of the stone in the 1st group of patients was 26.67+/-4.7 pf.u. compared to 25.4+/-5.9 pf.u. in the second group. At the same time, the value recorded on the skin was 13.08+/-1.2 pf.u. in the first group compared to 13.1+/-0.77 pf.u. in the second group (p>0.05). During the initial registration, the PM immediately after stone fragmentation was 19.5 +/- 1.2 pf.u, while on the overlying skin it was 11.2 +/- 0.9 pf.u. In the contralateral kidney area, IM was 10.2+/-0.9 pf.u. In the case of an intraoperative increase in intrapelvic pressure, IM was 22.3+/-1.6 pf.u. compared to 12.1+/-0.7 pf.u on the skin. The dynamics of IM on the skin tended to further decrease and returned to normal values of 10.3 +/- 0.7 pf.u on the 3rd day. When intraoperative intrapelvic pressure exceeded the normal value, IM by the 5th day was 10.1+/-0.4 pf.u. When determining the correlation of IM with RI of the ipsilateral kidney, a direct moderate correlation was revealed (r=+0.516).CONCLUSION:The measurement of microcirculation in the intra- and postoperative period allows to assess changes in the intrarenal microcirculation both directly and indirectly. This method can be used as an additional tool for assessing obstructive changes and the activity of pyelonephritis. A significant correlation between IM and RI indicates that functional changes in the renal and skin microcirculation tend to develop simultaneously.

We present a clinical case of giant uterine fibroids in this research, with the peculiarity of surgical treatment and the course of the postoperative period described. A series of genetic studies specific to uterine fibroids was performed, namely, mutations in the MED12 gene, overexpression of the HMGA2 gene, and chromosomal imbalance. We did not detect mutations of exon 2 of the MED12 gene and an increase in the expression of the HMGA2 gene in the patients myomatous node sample. The molecular karyotype arr(22)1 (chromosome 22 monosomy) was established by comparative genomic hybridization in the tissue of giant uterine fibroids.

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.