Differential Urinary Proteomic Analysis of High-Risk Cervical Intraepithelial Neoplasia.

Human papillomavirus (HPV)-associated lesions and malignancies exhibit alterations in the composition and functionality of the extracellular matrix (ECM) that represent the complex molecular pathways present between infection and disease. A total of 20 urine samples were used, including from 10 patients with cervical intraepithelial neoplasia grade 3 (CIN3) and 10 healthy controls to perform the label-free quantitative analysis using the nano-HPLC and ESI-MS ion trap mass analyzer and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) fast screening. Among 476 identified/quantified proteins, 48 were significantly changed (log2-fold change ≥1.0 or ≤-1.0, -log10 (bbinominal, p-value ≥ 1.3), of which were 40 proteins (down-regulated) and 8 proteins (up-regulated) in CIN3, in comparison to healthy controls. The biological function and key pathway enrichment of the gene set using gen set enrichment analysis (GSEA) were analyzed. The ECM-receptor interaction pathway (NES = -1.64, p = 0.026) was down-regulated by 13 proteins (HSPG2, COL6A1, COL6A3, SPP1, THBS1, TNC, DAG1, FN1, COMP, GP6, VTN, SDC1, and CD44; log2 FC range from -0.03 to -1.48) for the CIN3 group in the KEGG database. The MALDI-TOF/MS screening showed the difference of protein profiles between the control and CIN3 groups, i.e., using the scatter plot with a well-separated shape, as well as effectively distinguishing both groups (control and CIN3) using genetic algorithms (GA) with cross-validation (51.56%) and recognition capability (95.0%). Decreased levels of ECM-receptor interaction proteins may cause disturbances in the interactions of cells with the ECM and play an important role in the development and progression of cervical cancer.

Small Non-Coding RNAs as New Biomarkers to Evaluate the Quality of the Embryo in the IVF Process.

The increased interest in assisted reproduction through in vitro fertilization (IVF) leads to an urgent need to identify biomarkers that reliably highly predict the success of pregnancy. Despite advances in diagnostics, treatment, and IVF approaches, the 30% success rate of IVF seems insurmountable. Idiopathic infertility does not have any explanation for IVF failure especially when a patient is treated with a healthy competitive embryo capable of implantation and development. Since appropriate intercellular communication is essential after embryo implantation, the emergence of the investigation of embryonic secretome including short non-coding RNA (sncRNA) molecules is crucial. That's why biomarker identification, sncRNAs secreted during the IVF process into the blastocyst's cultivation medium, by the implementation of artificial intelligence opens the door to a better understanding of the bidirectional communication between embryonic cells and the endometrium and so the success of the IVF. This study presents a set of promising new sncRNAs which are revealed to predictively distinguish a high-quality embryo, suitable for an embryo transfer in the IVF process, from a low-quality embryo with 86% accuracy. The identified exact combination of miRNAs/piRNAs as a non-invasively obtained biomarker for quality embryo determination, increasing the likelihood of implantation and the success of pregnancy after an embryo transfer.

Impact of post-Chernobyl radiation on flow cytometry parameters of human sperm.

OBJECTIVE: The aim of this study was to analyse the long-term radiation effects on human sperm. METHODS: In total, 104 samples of male donors from 2 regions of Ukraine were tested. Group 1 consisted of 32 donors from the Ivano-Frankivsk region, group 2 included 72 volunteers from the Zhytomyr region. The average age of donors in both groups was 35 ± 6 years (range 24-49). To assess the level of apoptosis, membrane mitochondrial potential, concentration of reactive oxygen species, and ploidy of sperm, flow cytometry was performed. RESULTS: The individual equivalent dose of group 1 was < 0.4 mSv and of group 2 ≥ 0.4 mSv. Live spermatozoa with signs of apoptosis were significantly higher (p < 0.05) in group 2 in comparison to group 1 (15.6% and 11.2%, respectively). Spermatozoa without violating integrity were 73.2% in group 1 and approximately 16% higher than the indices of group 2. The percentage of dead necrotic spermatozoa was twice as high in men with a predicted equivalent dose of ≥ 0.4 mSv than in comparison group. A higher percentage of spermatozoa with low mitochondrial membrane potential, di- and tetraploid was found in group 2. CONCLUSIONS: An equivalent individual dose of ≥ 0.4 mSv can cause a decrease in mitochondrial potential, an increase in the production of spermatozoa with pathological ploidy, as well as to provoke increasing apoptosis in cells.

Pregnancy outcome prediction after embryo transfer based on serum human chorionic gonadotrophin concentrations.

OBJECTIVE: The aim of the study was to evaluate the predictive value of the human chorionic gonadotropin (hCG) concentration on the 14th and 16th post-ovulation day after embryo transfer/cryoembryo transfer as well as the dynamics of its increase with respect to the outcome of pregnancy. MATERIALS AND METHODS: In total, 130 embryo transfers and cryoembryo transfers in women aged 22 to 38 years who experienced a single embryo transfer or single cryoembryo transfer with confirmed pregnancy (hCG level over 15 IU/l on 14th post-ovulation day - D14) were selected. The input parameters (hCG D14, hCG D16, hCG D16-D14, hCG D16/D14 and positivity of at least 2.5-fold increase in hCG D16 compared to hCG D14) were evaluated by regression analysis in relation to the outcome parameters (bio-chemical pregnancy, clinical pregnancy, clinical pregnancy terminated by abortion up to 12 weeks of gestation, clinical pregnancy terminated by childbirth). RESULTS: Single concentrations of hCG D14 and D16, as well as the difference between these concentrations, were a statistically significant indicator of the prediction of bio-chemical pregnancy (P = 0.000215, P = 0.000227 and P = 0.000421). Contrary to expectations, the proportion of hCG D16 and D14 concentrations did not show statistical significance for either parameter, as well as the fulfilment of the condition of at least a 2.5fold increase in hCG D16 compared to D14. None of the studied input parameters was confirmed as a statistically significant marker for the prediction of miscarriage in the whole group of patients. However, in the group of confirmed clinical pregnancies, the serum concentration of hCG D16 (P = 0.0248) and the difference between concentrations D16 and D14 (P = 0.0185) were confirmed as a positive predictor of the progression of pregnancy until delivery. CONCLUSIONS: Single hCG concentrations are a good prognostic factor for predicting the outcome of pregnancy, but the determination of the cut-off limit is limited by inter-laboratory deviation as well as by timing of blood collection for hCG determination on the exact post-ovulatory day. The results of individual studies are therefore difficult to use in clinical practice. The dynamics of hCG concentrations appear to be a more reliable predictor of pregnancy outcome. In our cohort, we confirmed the statistical significance of the difference in hCG concentration between the 16th and 14th post-ovulation day not only for the prediction of bio-chemical pregnancy, but also as a predictor of the progression of clinical pregnancy into childbirth. To determine the optimal values of this difference, it is necessary to evaluate a larger group of patients. Conversely, the statistical significance of the proportion of hCG concentrations between the 16th and 14th post-ovulation day was not  confirmed.

Effect of body condition and season on yield and quality of in vitro produced bovine embryos.

The aim of our study was to examine the effects of cow's body condition score (BCS; scale 1-5) and season on the quality of bovine in vitro produced embryos. The proportion of good quality oocytes (Q1 and Q2) was higher (P < 0.05) in the BCS 2 (57.60%) and BCS 3 (60.90%) groups compared with the BCS 1 (43.60%) group. There were no statistical differences in embryo cleavage and blastocyst rate among the BCS groups. The highest total cell number (TCN, DAPI stain) of blastocysts (P < 0.05), recorded in BCS 1 (122.27 ± 6.90) in comparison with BCS 2 (101.8 ± 3.60) or BCS 3 (105.44 ± 3.70) groups, was related to higher dead cell (DCI, TUNEL) index in this group (7.07%) when compared with BCS 2 (6.54%) or BCS 3 (6.06%), respectively. The yield of good quality oocytes during spring was lower (P < 0.05) compared with the summer season. There were significant differences (P < 0.05) in maturation and cleavage rates between autumn and summer (73.42%, 76.2% vs. 85.0%, 41.8%, respectively). The highest (P < 0.01) blastocyst rate was noted during spring and summer months. Significant difference (P < 0.05) in the TCN among spring (99.38 ± 3.90), autumn (110.1 ± 4.58) or summer (108.96 ± 3.52) was observed. The highest proportion of embryos with the best (grade I) actin cytoskeleton (phalloidin-TRITC) quality was noted during the summer months. Our results indicate that body condition affects the initial quality of oocytes, but does not affect embryo cleavage, blastocyst rate and actin quality. This finding may suggest that development in vitro can mask the influence of BCS. The season affects yield and quality of blastocysts in the way that the autumn period is more favorable for embryo development.

ER-α agonist induces conversion of fibroblasts into myofibroblasts, while ER-ß agonist increases ECM production and wound tensile strength of healing skin wounds in ovariectomised rats.

Oestrogen deprivation is one of the major factors responsible for many age-related processes, including poor wound healing in women. Previously, it has been shown that oestrogens have a modulatory effect in different wound-healing models. Therefore, in this study, the effect of selective oestrogen receptor (ER) agonists (PPT - ER-α agonist, DPN - ER-ß agonist) on excisional and incisional wound-healing models was compared in ovariectomised rats in vivo as well as on human dermal fibroblasts (HDF) and human umbilical endothelial cells (HUVEC) in vitro. In the in vivo study, 4 months after either ovariectomy or sham ovariectomy, Sprague-Dawley rats were randomly divided into four groups and subjected to two incisional and excisional wounds: (i) control - sham operated, vehicle-treated; (ii) ovariectomised, vehicle-treated; (iii) ovariectomised, PPT treated; (iv) ovariectomised, DPN treated. In the in vitro study, HDFs and HUVECs were used. After treatment with ER agonists, cells were processed for immunocytochemistry and gelatin zymography. Our study shows that stimulation of ER-α leads to the differentiation of fibroblasts into myofibroblasts both in vivo and in vitro. On the other hand, the formation of extracellular matrix was more prominent, and wound tensile strength (TS) was increased when ER-ß was stimulated. In contrast, stimulation of ER-α led to a more prominent increase in the expression of MMP-2 and decrease in wound TS. New information is presented in this investigation concerning oestrogen replacement therapy (ERT) in different wound-healing models. This study demonstrates that the ERT should be both wound and receptor-type specific.

[The role of estrogens in regulations of biological processes involved into wound healing--a review]. / Uloha estrogénov v regulácii biologických procesov zapojených do hojenia rán--literárny prehl'ad.

Since the average age of female population in developed countries has increased, women live up to one-third of their life in the post-menopausal period during which altered wound healing frequently occurs. It has been shown that estrogens and estrogen receptors play a key role in the regulation of processes involved in tissue repair and regeneration. Hence, for better orientation in this area of biomedical research and clinical practice the role of estrogens and their receptors in wound healing was described in this review.

Concentration of trace elements in human semen and relation to spermatozoa quality.

In this study the concentrations of trace elements such as lead, cadmium, iron, nickel, copper and zinc in the human semen (n = 47), occurrence of pathological spermatozoa, and correlations of these elements to pathological forms were investigated. For each sample of human spermatozoa at least 500 spermatozoa were evaluated. Metal contents were determined by the voltametric method and flame absorption spectrophotometry method. The concentrations of trace elements in human semen were: lead 1.49 +/- 0.40 mg x kg(- 1), cadmium 0.13 +/- 0.15 mg x kg(- 1), iron 2.59 +/- 0.21 mg x kg(- 1), nickel 0.40 +/- 0.07 mg x kg(- 1), copper 0.28 +/- 0.06 mg x kg(- 1), and zinc 153.93 +/- 67.08 mg x kg(- 1), respectively. The total percentage of pathological spermatozoa was 41.61 +/- 9.80% with predominancy of broken flagellum, flagellum torso and separated flagellum. In relation to trace elements the analysis showed correlation between copper and lead (r = -0.47), nickel and iron (r = 0.36), lead and flagellum ball (r = -0.39), cadmium and large heads (r = 0.37) and between iron and other forms of pathological spermatozoa (r = -0.32). Results of this study describe possible effects of trace elements on the spermatozoa quality in normal human sperm.

Postsurgical administration of estradiol benzoate decreases tensile strength of healing skin wounds in ovariectomized rats.

BACKGROUND: The most effective method of increasing the level of estrogen in the wounds of post-menopausal women undergoing routine surgical procedures is by long-term preoperative administration. However, in the case of acute surgery or trauma, the most effective method of increasing the level of estrogen is administration immediately pre- or postsurgery. This study, therefore, was aimed at assessing the effect of postsurgical administration of estradiol benzoate on wound healing in ovariectomized (OVX) Sprague Dawley rats. MATERIALS AND METHODS: Three months prior to the wound healing experiment, 16 rats were anesthetized and underwent ovariectomy, while the other eight rats were sham operated. Two parallel full thickness skin incisions and two round full thickness skin excisions were performed on the dorsum of each rat. Dose of 10 microg/d of estradiol benzoate was administered to eight OVX rats for 6 d postoperatively, whereas the other animals received a placebo. After 6 d, all animals were sacrificed and samples removed for biomechanical and histological evaluation. RESULTS: The mean wound tensile strength of OVX estrogen treated rats (9.54 +/- 3.24 g/mm(2)) was significantly lower compared with vehicle-treated OVX animals (14.57 +/- 4.12 g/mm(2)) as well as with control rats subjected to sham-OVX surgery (11.71 +/- 3.33 g/mm(2)). Nevertheless, the histological evaluation in OVX estrogen treated rats showed a significantly increased process of neo-angiogenesis associated with slightly decreased collagen deposition. CONCLUSION: Our results indicate that the question of the clinical significance of this type of hormone replacement therapy remains open and requires further research.