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.

BACKGROUND: Fatty acids are important components of the oocyte microenvironment, exhibiting both pro-inflammatory and lipotoxic effects as well as anti-inflammatory effects depending on the presence and quantity of unsaturated bonds. Changes in the lipid profile of follicular fluid in obese patients may be a mechanism that leads to a decrease in oocyte competence and pregnancy rate in assisted reproductive technology programs. However, the available literature data is fragmentary and contradictory, which dictates the need for further research. AIM: The aim of this study was to investigate the lipid profile of follicular fluid in patients undergoing assisted reproductive technology programs with ovarian stimulation depending on the body mass index using MALDI (matrix-assisted laser desorption/ionization) mass spectrometry. METHODS: This study involved patients undergoing infertility treatment using assisted reproductive technology in a short protocol using gonadotropin-releasing hormone antagonists. The levels of essential saturated and unsaturated fatty acids were analyzed in the follicular fluid of the first aspirated follicle using MALDI mass spectrometry. RESULTS: Two study groups were formed out of 138 patients: patients with normal body mass index (n = 38) and patients with overweight and obesity (n = 76). The latter were characterized by higher levels of myristic and stearic acids and lower levels of oleic acid in the follicular fluid. The level of myristic acid in the follicular fluid positively correlated with the body mass index, while the level of oleic acid negatively correlated with this parameter. CONCLUSION: The data obtained indicate that overweight and obesity are associated with increased levels of saturated myristic and stearic acids in the follicular fluid, which negatively impact folliculogenesis, as well as a decrease in the level of monounsaturated oleic acid in the follicular fluid, which is able to compensate for the lipotoxic effects of saturated fatty acids.

Introduction. Conventional semen analysis offers limited insights into a man’s reproductive potential and does not always adequately explain male infertility. According to the literature, about 15% of infertile patients have normal sperm parameters. Sperm DNA fragmentation may be one of the causes of male infertility. According to the latest edition of the WHO guideline for the examination and processing of ejaculate (WHO, 2021), the research method and threshold for sperm DNA fragmentation remains in the examination laboratory. Objective. Therefore, the aim of this study was to determine sperm DNA fragmentation threshold for men of North-Western region of Russia. Material and methods. A retrospective study of the results of routine sperm analysis and sperm DNA fragmentation rate were performed in 1731 patients from ART department from January 2019 to November 2023. Additionally, the anamnestic data of the patients were analyzed. Results. Univariate analysis showed that the sperm DNA fragmentation rate dependeds on the patient’s age (p<0.001), the number of days of abstinence (p<0.001), as well as such sperm parameters as motility (p<0.001) and morphology (p=0.002). To determine the threshold value of the sperm DNA fragmentation, ROC analysis was performed. The sperm DNA fragmentation rate pronounced predictable in relation to the motility (PR <32%) and morphology (< 4%) of spermatozoa. The averaging of the obtained threshold results approaches 15%. Comparative analysis between the results, taking into account the threshold value, showed that the ejaculate volume in the group with sperm DNA fragmentation <15% was statistically lower than in the group ≥15% (3.39±1.54 ml and 3.55±1.5% ml, respectively; p=0.049). The motility and proportion of morphologically normal spermatozoa in the “TUNEL<15%” group were higher (55.8±16% versus 52.9±17.5%; p<0.001; 4.41±3.44% versus 4.07±3.23%; p=0.037). The age of patients and the number of days of abstinence in the “TUNEL<15%” group were lower (36±6.2 years versus 37±6.7; p=0.001; 3.74±1.73 days and 4.12±2.7 days; p=0.006). Conclusions. The study showed a relationship between sperm DNA fragmentation and such sperm parameters as morphology and motility. A reference value for the sperm DNA fragmentation rate is provided — 15%.

A modern approach to predicting the quality of embryos in ART programs is the search for promising biomarkers in follicular fluid. Objective. To determine the composition of follicular fluid and blood serum in women with infertility and evaluate its effect on the quality of embryos. Material and methods. A pilot prospective cohort study was conducted on 26 patients aged 27 to 44 years who were undergoing infertility treatment at the ART department of the D.O. Ott Research Institute from June 2023 to September 2023. Inclusion criteria: primary and secondary infertility. Exclusion criteria: contraindications to the use of ART methods. Samples of follicular fluid (FF) and blood serum obtained on the day of follicle puncture were examined for 26 indicators: vitamins B12, B9, vitamin D, magnesium, phosphorus, iron, copper, zinc, total calcium, homocysteine, glucose, total protein, albumin, urea, uric acid, creatinine, cholesterol, LDL, ferritin, transferrin, latent iron-binding capacity, total iron-binding capacity, transferrin iron saturation coefficient, cystatin C, C-reactive protein, albumin-globulin ratio. The patients were divided into two groups depending on the obtained embryos: group 1 — with optimal embryo quality; group 2 — with embryos of suboptimal quality. Results. The levels of homocysteine, vitamin B12, glucose, creatinine, total protein, albumin, LDL, cholesterol, transferrin, ferritin, iron, total iron-binding capacity, transferrin saturation coefficient with iron, magnesium, copper, zinc, total calcium between FF and blood serum are statistically significantly different (p<0.0001). In serum, the median of the listed substances is higher than in FF. Also, a statistically significant difference was observed between the levels in the FF and in the blood: vitamin D, cystatin C, CRP, albumin-globulin ratio, urea (p< 0.0001) and phosphorus (p=0.04). In the FF, the median of the listed substances is higher than in the blood. As a result of the analysis of FF and serum parameters in women with infertility, depending on the quality of the embryos, a statistically significant difference was obtained in the follicular fluid for homocysteine (p=0.004), uric acid (p=0.015), comparing the serum/FF ratio for folic acid (p=0.004), vitamin D (p=0.007), glucose (p=0.009), phosphorus (p=0.016), cystatin C (p=0.036). Conclusion. The composition of the studied biochemical parameters in the FF was determined. Levels of homocysteine, uric acid in FF and assessment of the serum/FF ratio for folic acid, vitamin D, glucose, phosphorus, cystatin C can serve as biomarkers for predicting the quality of embryos.

BACKGROUND: Despite the widespread use of preimplantation genetic testing for aneuploidies, its high cost and limited applicability stimulate the search for non-invasive methods for assessing the chromosomal status of embryos. Morphokinetic parameters of embryonic development recorded using TIME-LAPSE incubators are considered potential prognostic markers of embryo “competence,” but their diagnostic value requires further study, particularly in the context of Russian domestic culture systems. AIM: The aim of this study was to conduct a comparative analysis of morphokinetic parameters of human donor embryos during culture in a TIME-LAPSE embryonic development incubator to identify temporal patterns associated with different chromosomal statuses. METHODS: This prospective observational study included donor embryos obtained by fertilization of donor oocytes with donor sperm between June 2023 and March 2025. Embryos were cultured in an EmbryoVisor incubator (Westtrade LTD, Russia) with recording of key morphokinetic events: time of pronuclei fading (tPNf); two to nine discrete cells (t2–t9); first evidence of compaction (tSc); morula formation (tM); initiation of blastulation (tSB); full blastocyst formation (tB); and expanded blastocyst formation (tEB). The chromosomal status of the embryos was determined by array comparative genomic hybridization (aCGH) after trophectoderm biopsy. Statistical analysis was performed using the Mann–Whitney, Kruskal–Wallis, and ANOVA tests. RESULTS: The development of 28 donor embryos was analyzed. Embryos from zygotes with abnormal ploidy (3PN/0PN) demonstrated earlier first divisions (t2–t6, p 0.05) than from those with normal ploidy (2PN). Euploid embryos reached the morula (tM) and blastocyst (tB) stages faster than aneuploid and mosaic embryos (p = 0.03 and p = 0.037, respectively). Embryos with the 46, XY karyotype had a shorter cc2b interval (p = 0.022) compared to those with the 46, XX karyotype. CONCLUSION: Morphokinetic parameters, particularly the time of early divisions and first evidence of compaction, are associated with the chromosomal status of embryos. The data obtained may be used to develop non-invasive algorithms for embryo selection.

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.

Background: The treatment strategy for patients with a history of multiple failures in in vitro fertilization cycles remains a subject of active research. Embryo transfer in cryocycles is known to be more successful than “fresh” embryo transfer, when ovarian stimulation, follicle puncture, fertilization, and embryo culture were performed but the overall efficiency of in vitro fertilization cycles decreases with maternal age and transfer of poor quality embryos. One of the key factors influencing the failure of high quality embryo implantation is the deficiency of the adhesive matrix, which ensures the interaction between the embryo and the endometrium. The implementation of protocols that include the use of hyaluronic acid-enriched transfer media in the process of embryo transfer is a promising direction for improving implantation outcomes. Special attention of researchers is paid to determining the indications for the use of this technique. Aim: The aim of this study was to evaluate the effect of hyaluronic acid-enriched transfer media on the pregnancy rate in cryocycles. Materials and methods: This retrospective cohort comparative study, included 609 women who underwent controlled ovarian stimulation and frozen-thawed embryo transfer. Results: The main outcomes of frozen-thawed embryo transfer cycles were analyzed, taking into account the quality of the transferred embryos and the use of hyaluronic acid-enriched transfer media. The latter was not shown to be associated with an increase in biochemical and clinical pregnancy rates in women under 35 years of age with a history of repeated implantation failures or after transfer of poor quality embryos. However, in patients over 40 years of age with poor quality embryos, the use of hyaluronic acid-enriched transfer media was effective, since it increased the clinical pregnancy rate. As expected, our data showed an increase in the clinical pregnancy rate in women with high quality embryos on the day of transfer, regardless of the patient’s age. At the same time, in patients with a thin (thickness less than 7 mm) endometrium or a history of miscarriage, the use of hyaluronic acid-enriched transfer media was also associated with an increase in the clinical pregnancy rate and live birth during embryo transfer in cryocycles. Conclusions: The data obtained have shown that the use of hyaluronic acid-enriched transfer media in cryocycles may be recommended for women over 40 years of age, regardless of the quality of the transferred embryos, as well as for women of any reproductive age with a thin endometrium and a history of miscarriage when transferring good quality embryos.

BACKGROUND: The developing embryo secretes a wide range of cytokines that are essential for establishing a “dialogue” between the embryo and the endometrium, as well as for autocrine effects on the embryo. Non-invasive assessment of the embryo using the study of its secretome is of great interest in reproductive medicine, as it can be used for early selection of the most viable embryos in assisted reproductive technology programs. AIM: The aim of this study was to determine the prognostic significance of cytokine levels in media after embryo culture and their impact on endothelial function in achieving clinical pregnancy in assisted reproductive technology protocols, as well as the effect of these media on endothelial function. METHODS: The presence of cytokines [interleukin-1b, -4, -5, -6, -8, -10, growth factors (granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor)] in embryo-conditioned culture media was determined in patients undergoing infertility treatment using assisted reproductive technology. To detect cytokine levels below the sensitivity threshold, we studied the effect of conditioned culture media on the proliferation and migration of endothelial cells. RESULTS: The patients were divided into 2 groups: in group 1 (n = 34), no cytokines were found, whereas in group 2 (n = 14), at least one of the studied interleukins or growth factors was detected. Patients in group 2 had more oocyte-cumulus complexes, 2PN2PB zygotes, embryos on day 3 of culture, embryos of quality ≥ 6B on day 3 of culture, blastocysts, and blastocysts of quality ≥ 3BB. Additionally, the effective doses of gonadotropins per oocyte and per embryo were lower in group 2. The clinical pregnancy rate was also higher in group 2. We found no differences in endothelial cell proliferation and migration parameters. CONCLUSION: A higher concentration of cytokines in media after embryo culture may indicate greater implantation potential; therefore, cytokine assessment in culture media could be used as an additional non-invasive method for embryo evaluation.

Background: The introduction of time-lapse incubators into assisted reproductive technology practices provides a detailed examination of human pre-implantation embryo development. The continuous time-lapse filming technology is used to determine prognostic markers of embryo viability and implantation potential based on morphokinetic parameters. At present, the main time intervals for morphokinetic events during the pre-implantation phase of human embryo development have been documented primarily in embryos from infertile patients, with limited data available concerning the development of donor embryos. In this regard, it becomes relevant to follow the early development of such embryos and describe the embryonic development timeline using time-lapse technology. Aim: The aim of this study was to determine the time intervals of critical events in the pre-implantation development of human donor embryos. Materials and methods: The material for the study was 18 donor embryos obtained after fertilization of donor oocytes with donor sperm. The embryos were cultured for 140 hours in an EmbryoVisor time-lapse incubator (Westtrade Ltd., Russia). Results: The video image analysis of diploid donor embryo development showed that the disappearance of both pronuclei occurred at 22.2 (21.0–25.4) hours post fertilization, the 2-cell embryo stage was observed at 24.5 (23.4–27.3) hours post fertilization, the 4-cell embryo stage at 35.9 (34.6–38.6) hours post fertilization, and the 8-cell embryo stage at 52.8 (49.0–58.8) hours post fertilization. Morula formation occurred at 86.0 (76.9–95.4) hours post fertilization, and complete blastocyst formation was recorded at 107.0 (99.1–114.3) hours post fertilization. Triploid embryos tended to have a delay in the cleavage stage and a shorter compaction phase, yet generally developed within similar timeframes as diploid embryos. Conclusions: The analysis of video recordings obtained after culturing donor embryos in the time-lapse incubator allows for comparing the morphokinetic parameters of pre-implantation development of the donor embryos, taking into account their ploidy. The checkpoints in the development of pre-implantation embryos from the zygote stage to blastocyst formation are characterized. A tendency is noted for earlier disappearance of pronuclei, a delay at the cleavage stage from four to eight cells, and a shorter compaction stage in the donor embryos with impaired ploidy. Various anomalies in the development of such embryos are also described. Special attention should be paid, perhaps, to embryos that do not fit into the established development intervals, exhibit anomalies such as reverse cleavage, direct division from one to three cells, and excessive fragmentation, or stop developing at one point or another, since this may indicate anomalies in the embryo’s genome such as, for example, aneuploidy. Timely identification of these deviations may lead to the exclusion of embryos with morphokinetic abnormalities from transfer, thereby favoring the selection of normally developing embryos to enhance implantation success and promote ongoing pregnancies. An increase in the sample of donor embryos under study, information on their genetic status and the clinical results of pregnancy after embryo transfer, and further accumulation of data will augment the ability to predict embryo implantation potential without the use of invasive methods.

BACKGROUND: The development of new systems for the cultivation of embryos of model objects and the creation of embryonic models based on stem cells allow for describing the events of early embryogenesis in more detail. However, these models have a number of limitations, endorsing only indirect extrapolation of the knowledge gained to events occurring in vivo. Despite certain limitations, including those related to some differences between embryos developing in vivo and in vitro, implantation conditions in vivo and in vitro, and limited time for culturing human embryos, the study of post-implantation development has great prospects. AIM: The aim of this study was to evaluate the viability and potential for the development of human embryos from the blastocyst stage during their prolonged coculture with the endometrium in culture media designed to support the growth to the blastocyst stage. MATERIALS AND METHODS: Embryos obtained through assisted reproductive technology were cultured from the blastocyst stage to day 19st of development in culture media designed to support the growth to the blastocyst stage, in the presence of the endometrium. On day 19st, embryos and endometrial fragments were assessed under an inverted microscope using Hoffman modulation contrast. RESULTS: During visual assessment under an inverted microscope on day 19st of development and days 13–14st of coculture with the endometrium, viable developing embryos without signs of degradation were recorded, both freely located in a mass of undifferentiated cells and in direct contact with the endometrium. CONCLUSIONS: The data obtained indicate the ability of the embryo to further develop in the presence of the endometrium from the blastocyst stage to day 19st in in culture media that supports the growth to the blastocyst stage. This may serve as an experimental model for both evaluating endometrial receptivity in vitro and studying intercellular communication during implantation.

The effectiveness of the use of a modified intrauterine gel form of granulocyte colony stimulating factor in the complex treatment of Ascherman syndrome is evaluated. The study involved 31 patients with a history of Ascherman syndrome. For 6 months, the patients underwent weekly intrauterine administration of 1 ml of gel with a concentration of granulocyte colony stimulating factor of 9 million IU/ml using an intrauterine catheter. Monthly M-echo was evaluated on the 19th–22nd day of the menstrual cycle, as well as the volume of menstrual blood loss according to the Pictorial Blood Loss Assessment Chart. After the treatment was completed, a control hysteroscopy was performed. There is a statistically significant increase in M-echo from 2. 61 ± 1. 71 to 6. 52 ± 1. 23 mm (p 0. 001). At the same time, the proportion of patients with M-echo 6 and 7 mm increased from 6. 45 and 0% to 80. 65 and 51. 61, respectively, over the 6 months of the study. There is also a statistically significant increase in menstrual blood loss from 24. 9 ± 2. 6 to 53. 5 ± 3. 4 points on the Pictorial Blood Loss Assessment Chart (p 0. 001). A response to therapy was achieved in 17 patients, among them 12 patients were diagnosed with pregnancy that occurred spontaneously (17. 65%) or as a result of in vitro fertilization protocol (52. 9%). The data obtained indicate that intrauterine administration of a gel with granulocyte colony stimulating factor promotes endometrial regeneration in patients suffering from Ascherman syndrome, which is expressed in an increase in M-echo and the volume of menstrual blood loss. Thus, adjuvant use of a hydrogel with granulocyte colony stimulating factor and hyaluronic acid is an effective treatment option in restoring reproductive function and preventing recurrence of adhesiogenesis after surgical treatment of Ascherman syndrome. In general, the proposed gel form with granulocyte colony stimulating factor allows us to reduce the frequency of administration of the drug, which theoretically can increase both the compliance of patients primarily by reducing the number of invasive procedures, and reduce treatment costs.

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: The adipokines leptin, adiponectin, and ghrelin are expressed not only in adipose tissue but also in the hypothalamic-pituitary-gonadal axis organs and the uterus. They regulate the gonadotropin-releasing hormone and gonadotropin secretion, steroidogenesis, folliculogenesis, and implantation, and serve as promising markers of assisted reproductive technology program outcomes. AIM: The aim of this study was to evaluate the dynamics of blood adipokine levels and their ratios during the controlled ovarian hyperstimulation in assisted reproductive technology programs depending on the onset of clinical pregnancy. MATERIALS AND METHODS: This study involved 51 patients undergoing infertility treatment using assisted reproductive technology in a short protocol with gonadotropin-releasing hormone antagonists. Depending on the onset of clinical pregnancy, two study groups were formed: non-pregnant (group 1, n = 22) and pregnant (group 2, n = 29). Blood leptin, adiponectin, and ghrelin levels were assessed using enzyme immunoassay at three points: assisted reproductive technology protocol start-up, the day of oocyte pick-up, and the day of embryo transfer. RESULTS: On the day of oocyte pick-up, patients in group 1 had higher leptin levels (5.47 ± 1.92 vs. 3.76 ± 0.75 ng/ml; p = 0.0004), leptin/adiponectin ratios (11.45 ± 4.50 vs. 4.73 ± 1.08; p 0.001), and leptin/ghrelin ratios (0.43 ± 0.17 vs. 0.24 ± 0.07; p 0.001). On the day of embryo transfer, patients in group 1 had higher levels of leptin (6.37 ± 2.13 vs. 3.29 ± 1.21 ng/ml; p 0.001) and adiponectin (0.75 ± 0.22 vs. 0.60 ± 0.09 ng/ml; p = 0.001), as well as ratios of leptin/adiponectin (9.06 ± 3.73 vs. 5.59 ± 2.32; p 0.001), leptin/ghrelin (0.48 ± 0.15 vs. 0.20 ± 0.07; p 0.001), and adiponectin/ghrelin (0.06 ± 0.02 vs. 0.04 ± 0.01; p 0.001). CONCLUSIONS: The data obtained indicate that blood adipokine levels change during the controlled ovarian hyperstimulation in assisted reproductive technology protocols depending on clinical pregnancy and can be used to predict its onset.

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.

BACKGROUND: According to reports, the efficiency of in vitro fertilization and intra cytoplasmic sperm injection protocols is decreased in patients positive for various autoantibodies, as opposed to autoantibody negative patients. However, there are contradictory data indicating no autoantibody effect on the outcome of infertility treatment using assisted reproductive technology. AIM: The aim of this study was to evaluate the embryological outcome and clinical efficiency of infertility treatment in in vitro fertilization and intra cytoplasmic sperm injection protocols in women in the presence of reproductively significant autoantibodies. MATERIALS AND METHODS: This prospective study enrolled 90 infertile patients undergoing assisted reproductive technology treatment. The follicular fluid obtained on the day of oocyte retrieval was evaluated for a wide autoantibody panel using commercial ELISA kits. The main group (n = 52) included women with autoantibody levels determined in the follicular fluid of more than three standard deviations from the mean values determined among all patients. The comparison group consisted of 38 women with autoantibody levels of less than three standard deviations from all subjects. The intergroup comparative analysis included clinical and anamnestic data, hormonal parameters, ovarian reserve, embryological data, and in vitro fertilization and intra cytoplasmic sperm injection efficiency. RESULTS: Reliably lower ovarian reserve parameters (anti-Mullerian hormone levels 1.9 (1.4; 4.0) vs. 3.3 (2.2; 6.5) ng/ml; p = 0.005; number of antral follicles 8.5 (6.0; 12.0) vs. 11.0 (9.0; 17.0); p = 0.003) have been noted in the main group relatively to the comparison group. The autoantibodies to thyroid peroxidase and cardiolipin content in the follicular fluid has been shown to be negatively associated with the number of two-pronuclear zygotes, the presence of autoantibodies to aromatase correlating negatively with the fertilization rate. Furthermore, the follicular fluid levels of autoantibodies to thyroid peroxidase (105 IU/ml) and cardiolipin (5.1 IU/ml) are reliably associated with a higher frequency of a suboptimal response to previous controlled ovarian stimulation, a lower incidence of high quality embryos on days 3 and 4 of in vitro cultivation, a decreased number of top-quality blastocysts, and the clinical efficiency of in vitro fertilization and intra cytoplasmic sperm injection programs. CONCLUSIONS: The presence of reproductively significant autoantibodies is an independent risk factor for reducing the total efficiency of assisted reproductive technology treatment.