Changes in the Transcription of Proliferation- and Apoptosis-Related Genes in Embryos in Women of Different Ages under the Influence of Extracellular Vesicles from Donor Follicular Fluid In Vitro.

We studied the influence of extracellular vesicles from the follicular fluid of a young donor on gene expression (MKI67, MYBL2, CCNB1, CCND1, CCNE1, CALM2, BAX, NDRG1, TP53I3, VEGF, VCAN, HAS2, CTSL2, PIBF1, RPL37, PFKP, GPX3, and AQP3) in embryos of women of different ages. According to nanoparticle tracking analysis data, the concentration of extracellular vesicles was 3.75±0.47×1011 particles/ml and the mean particle size was 138.78±9.90 nm. During co-culturing of the follicular fluid extracellular vesicles with blastocysts of young women, we observed significantly increased expression of mRNA for genes CTSL2, CCND1, CCNE1, VEGF and reduced expression of BAX gene mRNA in comparison with embryos in women of late reproductive age. We hypothesized that addition of extracellular vesicles of the oocyte follicular fluid from a young donor to the culture medium of embryos could slow down apoptosis process typical of blastocyst cells in women above 36 years.

Influence of Extracellular Vesicles from the Follicular Fluid of Young Women and Women of Advanced Maternal Age with Different miRNA Profiles on Sperm Functional Properties.

We studied the effect of co-culturing of extracellular vesicles in the follicular fluid of young women and women of advanced maternal age on sperm motility. Vesicles were obtained by differential centrifugation. The sperm fraction was isolated from the seminal fluid of 18 patients (age 28-36 years). The spermatozoa were incubated with vesicles (1:2 ratio) for 60 or 120 min at 37°C in a CO2 incubator. A fraction of spermatozoa incubated without vesicles served as the control. After the incubation, the sperm samples were sedimented by centrifugation, fixed in 2.5% glutaraldehyde, and analyzed by transmission electron microscopy. RNA was isolated from the follicular fluid vesicles by column method followed by cDNA synthesis in a reaction mixture according to miScript II RT Kit protocol (Qiagen). After 60-min incubation with extracellular vesicles from the follicular fluid of women of advanced maternal age, the sperm motility and hyperactivation slightly changed in comparison with the group where incubation was performed with follicular fluid vesicles from young women and control group. Follicular fluid miRNA profiles in women of different ages varied, which suggests different functional compositions and effects of follicular fluid vesicles of different age groups on sperm characteristics. Transmission electron microscopy revealed differences in the interaction of follicular fluid vesicles from women of different age groups with spermatozoa. Further study of the effect of extracellular vesicles from the follicular fluid and analysis of their transcriptomic, proteomic, and metabolomic composition on sperm mobility and fertilizing ability will improve the effectiveness of assisted reproductive technology programs in patients with male infertility.

Influence of Extracellular Vesicles of the Follicular Fluid on Morphofunctional Characteristics of Human Sperm.

We studied the effect of extracellular vesicles of the follicular fluid on morphofunctional characteristics of human spermatozoa using CASA (computer-assisted sperm analysis) analytical system. The vesicles were obtained by sequential centrifugation at different rotational speeds and frozen at -80°C in the Sydney IVF Gamete Buffer medium. The sperm fraction was isolated from the seminal fluid of 21 patients aged 27-36 years by differential centrifugation in a density gradient. The precipitate was suspended in Sydney IVF Gamete Buffer to a concentration of 106/ml and incubated with vesicles (1:2) at 37°C in a CO2 incubator for 30 min and 1 h. Sperm fraction incubated without vesicles served as the control. After incubation, some sperm samples were centrifuged at 700g for 5 min and fixed in 2.5% glutaraldehyde in 0.1 M buffer for transmission electron microscopy. After 30-min and 1-h incubation, the progressive and total sperm motility improved, the curvilinear and linear velocity of spermatozoa did not change significantly. Incubation with vesicles significantly changed the trajectory of sperm movement, which can attest to an increase in their hyperactivation and, probably, fertilizing capacity. Analysis of the effect of extracellular vesicles of follicular fluid on sperm motility will help to improve the effectiveness of assisted reproductive technology programs with male infertility factor by improving sperm characteristics in patients with asthenozoospermia and increasing the fertilizing ability of the sperm.

Laser Microtomography for IVF Oocyte of Human.

For in vitro fertilization technology, the quality of oocytes has a direct impact on the egg fertilization and developmental competence of early embryo. The morphological criteria are used for the estimation of oocyte quality before its fertilization in vitro. To date, only one method is known to determine the maturity of oocyte. This is the routine observation with a light microscope. The aim of this article was to adapt the noninvasive quantitative laser scanning microtomography (QLSM) for the investigation of morphological features of a human oocyte in vitro. This approach was used to accumulate the Z-stack gallery of optical sections of IVF oocyte. The layer-by-layer acquisition allows the fine cytoplasmic structure imaging. Applying the QLSM Z-stack of optical sections, the cellular volume was calculated with quantitative 3D reconstruction of a human oocyte. The volume value and intracellular structure were used as novel criteria to assess the oocyte state after the stress evoked by cryopreservation procedure.

Transcription profile analysis of the endometrium revealed molecular markers of the personalized 'window of implantation' during in vitro fertilization.

To determine the most informative markers for assessing the functional state of endometrium during the 'window of implantation' and creating a model for assessment of the readiness of endometrium for embryo implantation. Forty-seven women with tubal infertility and a successful IVF pregnancy participated in the study. Pipelle endometrial sample was performed during the supposed 'window of implantation' in natural cycle with subsequent histological study, and transcriptional profile of genes GPX3, PAEP, DPP4, TAGLN, HABP2, IMPA2, AQP3, HLA-DOB, MSX1, POSTN determined by real-time quantitative polymerase chain reaction (qRT-PCR). Differences in the level of mRNA expression of all the studied genes in the receptive endometrium were found in comparison to the prereceptive one, which allowed us to classify two functional states of the endometrium. The results of histological examination responded to the stage of maturation of the endometrium in 78.7% of cases. Receptive endometrial status can be determined based on the integral evaluation of mRNA expression level of 4 PAEP, DPP4, MSX1, and HLA-DOB genes. The model for determining a personalized `window implantation' is offered for practical application in ART.

[Metabolomic profiling in culture media of day-5 human embryos]. / Profilirovanie metabolitov v pitatel'nykh sredakh piatidnevnykh émbrionov cheloveka.

The aim of this study was to determine the changes of metabolomic profiles in embryonic culture media (ECM) for the evaluation of quality and implantation potential of human embryos. ECM (n=163) were collected on day 5 before transfer or cryopreservation. Some embryos were used in preimplantation genetic screening for detection of aneuploidy karyotypes. Samples were subdivided into groups according to embryo morphological classification (by Gardner), genetic analysis and implantation data. ECM were extracted with methanol, precipitates were separated by centrifugation and metabolite production of individual embryo was analysed by LC-MS (the positive ion mode). After peak detection and retention time alignment, data were analysed using the PCA algorithm. MS fingerprinting analysis of embryo culture medium showed significant differences between morphologically divided groups. Intragroup comparisons did not reveal differences between subclasses. Genetic screening of embryos revealed 33 aneuploid karyotypes. It was shown that chromosome number did not affect the metabolite profiles comparing with the normal group. The culture media of embryos that were positive or negative for successful implantation showed specific signatures that allowed to distinguish embryos with different outcomes.The characterization of ECMs by LC-MS may facilitate more accurate selection of the best embryo for the implantation, improving single-embryo transfer and thus eliminating the risk and undesirable effects of multiple pregnancies.

[The role of inherited thrombophilia and undifferentiated connective tissue dysplasia syndrome in the pathogenesis of female infertility: A clinical and morphological study].

UNLABELLED: Impaired endometrial receptivity is a major cause of reproductive losses in in vitro fertilization (IVF) cycles given a normal embryo. Its causes may be associated with many diseases, including inherited thrombophilia (IT) and undifferentiated connective tissue dysplasia syndrome (uCTDS). However, endometrial receptivity remains little studied. OBJECTIVE: to investigate the morphological and immunohistochemical substrates of impaired endometrial receptivity in women with uCTDS, IT, and their concurrence. SUBJECTS AND METHODS: Antibodies against ER, PgR, LIF, VEGF, and PAI-1 were used to morphologically and immunohistochemically examine pipelle endometrial biopsy specimens taken from 141 women in the implantation window (on days 6-7 after ovulation). In accordance with their clinical diagnoses, the patients were divided into 4 groups: 1) 13 patients with uCTDS; 2) 100 with IT; 3) 19 with uCTDS and IT; 4) 9 healthy surrogate mothers (a control group). In the examined groups, a total of 145 (90.1%) out of all the IVF protocols were unsuccessful. In the remaining 16 (9.9%) patients without exception, miscarriage started at less than 10 weeks' gestation. RESULTS: In the implantation window, the endometrium was immature in 101 (83.1%) women and corresponded to late proliferation or early secretion phases; 102 (84.3%) women were also found to have no mature pinopodes, pointing to the fact that the endometrial receptivity was very low. Immunohistochemical examination revealed the lower expression of the receptivity marker LIF in the endometrial surface epithelium and its higher expression in the stroma in the study groups (p < 0.05 for the uCTDS and uCTDS+IT groups) and the higher expression of PAI-1 and VEGF in the epithelium, stroma, and endothelium in the study groups than in the control group (p < 0.05), suggesting the intensity of neoangiogenetic processes and impaired fibrinolysis in these patients. CONCLUSION: uCTDS and IT are risk factors of impaired endometrial receptivity in the pathogenesis of infertility. The manifestations of impaired endometrial receptivity in this case are a decrease in mature pinopodes in the surface epithelium; focal stromal sclerosis; and redistribution of the receptivity marker LIF from the surface epithelium to the stroma, which may be used for diagnosis, prediction, and the development of targeted therapy.

[Clinical and experimental substantiation of application of composite prosthesis to prosthetic repair of hiatal hernias].

RESEARCH OBJECTIVE: Improve the results of surgical treatment of hiatal hernia through the use of prosthetic techniques hiatal size adjustment. MATERIAL AND METHODS: The pilot study on 24 laboratory animals with use composite and PTFE of prosthesis is executed. Results are used in clinical practice when performing fitting a prosthetic hiatal hernia repair. RESULTS: The composite prosthesis is reliably fixed to a diaphragm by connecting fabric sprouting it and doesn't tend to shift. PTFE implant encapsulated with surrounding implant scar, dosage and do not germinate connective tissue. The use of composite prostheses in the clinic for correction hiatal hernia significantly reduces the rate of recurrence of hernia. CONCLUSION: The most optimum implant for prosthetic repair of hiatal hernia is the composite prosthesis.

Protamine and fertilin mRNA: potential biomarkers of assisted reproductive technology outcomes.

We studied the relationship between the levels of protamines 1 and 2 (PRM1 and PRM2) and fertilin-ß (ADAM-2) mRNA expression and outcomes of infertility treatment using assisted reproductive technologies was studied. Analysis of the relationships between the outcomes of in vitro fertilization and embryo transfer and profiles of the expression of seminal genes PRM1, PRM2, ADAM-2 mRNA, evaluated by reverse transcription quantitative PCR was carried out in 79 couples. Significant differences in the expression of seminal PRM1, PRM2, ADAM-2 mRNA were detected in couples with different outcomes of in vitro fertilization and embryo transfer. The levels of seminal gene expression are potential predictors of the efficiency of in vitro fertilization and embryo transfer.

[The ovarian hyperstimulation syndrome in extracorporeal oocyte fertilization and the transfer of cleavage-stage embryos]. / Sindrom giperstimuliatsii iaichnikov pri ékstrakorporal'nom oplodotvorenii ootsitov i perenose drobiashchikhsia émbrionov.

Major clinical characteristics of ovarian hyperstimulation syndrome are described and criteria of predicting the course of this disease and measures to prevent it presented. Statistical data on the presence of this syndrome in various forms during various schemes of superovulation stimulation are offered. Optimal schemes for the treatment of the condition are suggested.

[Morphologic characteristics of the development of embryos in the preimplantation phase of the process of extracorporeal fertilization]. / Morfologicheskie osobennosti razvitiia pre'dimplantatsionnykh émbrionov v programme ékstrakorpora'lnogo oplodotvoreniia.

The authors presented results of the assessment of morphological parameters and human embryo division rate in preimplantation when various schemes of superovulation stimulation were used in the routine for extracorporeal fertilization. A great number of embryos with favourable morphologic signs were obtained due to the routine. It was demonstrated that over 50% of all the embryos employed in various schemes of superovulation stimulation were at the stage of four and more blastomeres by the moment of their implantation to the uterine cavity 44-48 hrs after the in vitro insemination. A significant increment in the rate of embryo preimplantation development in vitro was revealed during superovulation stimulation with clomifen citrate in combination with an inductor versus the stimulation with the inductor alone. There was no difference in the frequency of conception induced by the transfer of embryos in all the groups exposed to simulation of superovulation.