Validation and incorporation of digital entheses into a preliminary GLobal OMERACT Ultrasound DActylitis Score (GLOUDAS) in psoriatic arthritis.

OBJECTIVES: The main objective was to generate a GLobal OMERACT Ultrasound DActylitis Score (GLOUDAS) in psoriatic arthritis and to test its reliability. To this end, we assessed the validity, feasibility and applicability of ultrasound assessment of finger entheses to incorporate them into the scoring system. METHODS: The study consisted of a stepwise process. First, in cadaveric specimens, we identified enthesis sites of the fingers by ultrasound and gross anatomy, and then verified presence of entheseal tissue in histological samples. We then selected the entheses to be incorporated into a dactylitis scoring system through a Delphi consensus process among international experts. Next, we established and defined the ultrasound components of dactylitis and their scoring systems using Delphi methodology. Finally, we tested the interobserver and intraobserver reliability of the consensus- based scoring systemin patients with psoriatic dactylitis. RESULTS: 32 entheses were identified in cadaveric fingers. The presence of entheseal tissues was confirmed in all cadaveric samples. Of these, following the consensus process, 12 entheses were selected for inclusion in GLOUDAS. Ultrasound components of GLOUDAS agreed on through the Delphi process were synovitis, tenosynovitis, enthesitis, subcutaneous tissue inflammation and periextensor tendon inflammation. The scoring system for each component was also agreed on. Interobserver reliability was fair to good (κ 0.39-0.71) and intraobserver reliability good to excellent (κ 0.80-0.88) for dactylitis components. Interobserver and intraobserver agreement for the total B-mode and Doppler mode scores (sum of the scores of the individual abnormalities) were excellent (interobserver intraclass correlation coefficient (ICC) 0.98 for B-mode and 0.99 for Doppler mode; intraobserver ICC 0.98 for both modes). CONCLUSIONS: We have produced a consensus-driven ultrasound dactylitis scoring system that has shown acceptable interobserver reliability and excellent intraobserver reliability. Through anatomical knowledge, small entheses of the fingers were identified and histologically validated.

Model of micro-metastases of breast cancer cells in ovarian tissue: Cryopreservation of ZR-75-1 and MDA-MB-231 cells with increased speed of warming increases malignancy.

In medicine, ovarian tissue cryopreservation exists for fertility preservation of cancer patients. In fact, ovarian tissue frozen for subsequent thawing and re-transplantation can be contaminated with cancer cells. Therefore, investigations on the effect of cryopreservation on the post-thawed viability of such cells are relevant. Speed of warming is a key parameter of cell cryopreservation. However, the data about comparative viability of cancer cells cryopreserved with different parameters of warming are limited. The aim of our investigations was to assess the malignancy of cryopreserved cancer cells after conventional cooling followed by relatively slow and quick speed of warming. In vitro cultured breast cancer cells of lines ZR-75-1 and MD0MD-231 in form of compacted fragments (as a model of solid tumors) were frozen following a protocol usually used for freezing of ovarian tissue (6 % ethylene glycol+6 % glycerol+0.15 M sucrose, -0.3 °C/min). Cells were warmed by two routine regimes of warming: at 37 °C ("slow" warming) and 100 °C ("quick" warming). Biological properties of cells were investigated: viability, proliferation rate, 2D- and 3D-migration, transmembrane movement and invasion. Quick warming at 100 °C in comparison with slow warming at 37 °C exhibited significantly higher cell survival for MDA-MB-231 cells: 70.1 % vs. 63.2 % and for ZR-75-1 86.8 % vs. 82.9 %, respectively. The cell motility including 2D movement and 3D transmembrane migration were higher after quick thawing at 100 °C. Invasive abilities of cells after cryopreservation were higher than that of fresh (non-treated cells). Both thawing regimes showed a similar rate of cell proliferation. Cryopreservation procedures, and especially this one with quick thawing, increase malignancy of ZR-75-1 and MDA-MB-231 breast cancer cells and risk of metastasis.

RNA Transcripts in Human Ovarian Cells: Two-Time Cryopreservation Does Not Affect Developmental Potential.

Sometimes, for medical reasons, when a frozen tissue has already thawed, an operation by re-transplantation may be cancelled, and ovarian tissues should be re-frozen for transplantation next time. Research about the repeated cryopreservation of ovarian cells is rarely reported. It has been published that there is no difference in the follicle densities, proportions of proliferation of early preantral follicles, appearance of atretic follicles, or ultrastructural quality of frozen-thawed and re-frozen-rethawed tissue. However, the molecular mechanisms of a repeated cryopreservation effect on the developmental potential of ovarian cells are unknown. The aim of our experiments was to investigate the effect of re-freezing and re-thawing ovarian tissue on gene expression, gene function annotation, and protein-protein interactions. The morphological and biological activity of primordial, primary, and secondary follicles, aimed at using these follicles for the formation of artificial ovaries, was also detected. Second-generation mRNA sequencing technology with a high throughput and accuracy was adopted to determine the different transcriptome profiles in the cells of four groups: one-time cryopreserved (frozen and thawed) cells (Group 1), two-time cryopreserved (re-frozen and re-thawed after first cryopreservation) cells (Group 2), one-time cryopreserved (frozen and thawed) and in vitro cultured cells (Group 3), and two times cryopreserved (re-frozen and re-thawed after first cryopreservation) and in vitro cultured cells (Group 4). Some minor changes in the primordial, primary, and secondary follicles in terms of the morphology and biological activity were detected, and finally, the availability of these follicles for the formation of artificial ovaries was explored. It was established that during cryopreservation, the CEBPB/CYP19A1 pathway may be involved in regulating estrogen activity and CD44 is crucial for the development of ovarian cells. An analysis of gene expression in cryopreserved ovarian cells indicates that two-time (repeated) cryopreservation does not significantly affect the developmental potential of these cells. For medical reasons, when ovarian tissue is thawed but cannot be transplanted, it can be immediately re-frozen again.

Comparative Transcriptomic Analyses for the Optimization of Thawing Regimes during Conventional Cryopreservation of Mature and Immature Human Testicular Tissue.

Cryopreservation of human testicular tissue, as a key element of anticancer therapy, includes the following stages: saturation with cryoprotectants, freezing, thawing, and removal of cryoprotectants. According to the point of view existing in "classical" cryobiology, the thawing mode is the most important consideration in the entire process of cryopreservation of any type of cells, including cells of testicular tissue. The existing postulate in cryobiology states that any frozen types of cells must be thawed as quickly as possible. The technologically maximum possible thawing temperature is 100 °C, which is used in our technology for the cryopreservation of testicular tissue. However, there are other points of view on the rate of cell thawing, according to how thawing should be carried out at physiological temperatures. In fact, there are morphological and functional differences between immature (from prepubertal patients) and mature testicular tissue. Accordingly, the question of the influence of thawing temperature on both types of tissues is relevant. The purpose of this study is to explore the transcriptomic differences of cryopreserved mature and immature testicular tissue subjected to different thawing methods by RNA sequencing. Collected and frozen testicular tissue samples were divided into four groups: quickly (in boiling water at 100 °C) thawed cryopreserved mature testicular tissue (group 1), slowly (by a physiological temperature of 37 °C) thawed mature testicular tissue (group 2), quickly thawed immature testicular tissue (group 3), and slowly thawed immature testicular tissue (group 4). Transcriptomic differences were assessed using differentially expressed genes (DEG), the Kyoto Encyclopedia of Genes and Genomes (KEGG), gene ontology (GO), and protein-protein interaction (PPI) analyses. No fundamental differences in the quality of cells of mature and immature testicular tissue after cryopreservation were found. Generally, thawing of mature and immature testicular tissue was more effective at 100 °C. The greatest difference in the intensity of gene expression was observed in ribosomes of cells thawed at 100 °C in comparison with cells thawed at 37 °C. In conclusion, an elevated speed of thawing is beneficial for frozen testicular tissue.

The EFSUMB Guidelines and Recommendations for Musculoskeletal Ultrasound - Part I: Extraarticular Pathologies.

The first part of the guidelines and recommendations for musculoskeletal ultrasound, produced under the auspices of the European Federation of Societies for Ultrasound in Medicine and Biology (EFSUMB), provides information about the use of musculoskeletal ultrasound for assessing extraarticular structures (muscles, tendons, entheses, ligaments, bones, bursae, fasciae, nerves, skin, subcutaneous tissues, and nails) and their pathologies. Clinical applications, practical points, limitations, and artifacts are described and discussed for every structure. After an extensive literature review, the recommendations have been developed according to the Oxford Centre for Evidence-based Medicine and GRADE criteria and the consensus level was established through a Delphi process. The document is intended to guide clinical users in their daily practice.

The EFSUMB Guidelines and Recommendations for Musculoskeletal Ultrasound - Part II: Joint Pathologies, Pediatric Applications, and Guided Procedures.

The second part of the Guidelines and Recommendations for Musculoskeletal Ultrasound (MSUS), produced under the auspices of EFSUMB, following the same methodology as for Part 1, provides information and recommendations on the use of this imaging modality for joint pathology, pediatric applications, and musculoskeletal ultrasound-guided procedures. Clinical application, practical points, limitations, and artifacts are described and discussed for every joint or procedure. The document is intended to guide clinical users in their daily practice.

Cryoprotectants-Free Vitrification and Conventional Freezing of Human Spermatozoa: A Comparative Transcript Profiling.

INTRODUCTION: Spermatozoa cryopreservation is an important technique to preserve fertility for males. This study aimed at exploring the stability of epigenetics information in human spermatozoa, manipulated by two different technologies, freezing and vitrification. METHODS: Spermatozoa samples were distributed into three groups: 1. Fresh spermatozoa (control group), 2. Frozen spermatozoa, 3. Vitrified spermatozoa. Epigenetic differences of fresh and cryopreserved spermatozoa were evaluated using high-throughput RNA sequencing. RESULTS: Differentially expressed genes (DEGs) in frozen (1103 genes) and vitrified (333 genes) spermatozoa were evaluated. The bioinformatical analysis identified 8 and 15 significant pathways in groups of frozen and vitrified spermatozoa, respectively. The majority of these pathways are most relevant to immune and infectious diseases. The DEGs of the fertilization process are not detected during vitrification. The freezing process induces more down-regulation of genes and is relevant to apoptosis changes and immune response. CONCLUSION: Cryopreservation of human spermatozoa is an epigenetically safe method for male fertility preservation. Cryoprotectant-free vitrification can induce more minor biological changes in human spermatozoa, in comparison with conventional freezing.

Aseptic capillary vitrification of human spermatozoa: Cryoprotectant-free vs. cryoprotectant-included technologies.

The protocol of aseptic cryoprotectant-free vitrification on human spermatozoa is well documented. However, data about the effect of permeable cryoprotectants at this procedure is limited. Presented study aimed to test the aseptic capillary vitrification technologies using permeable cryoprotectant-included or cryoprotectant-free media. Thirty-two normal samples were included and analyzed after vitrification in three different media and thawing. Three treatment groups were formed: Group 1, basic medium; Group 2, basic medium with 0.25 M sucrose; Group 3, basic medium with glycerol. Before plunging into liquid nitrogen, capillaries were filled by 10 µl of spermatozoa suspension and isolated from liquid nitrogen by location in hermetically closed 0.25 ml straws. Progressive motility, plasma membrane integrity, total motility/viability after 24, 48 and 72 h in vitro culture, apoptosis and mitochondrial membrane potential (ΔΨm) were determined after thawing at 42 °C. Progressive motility of spermatozoa in groups 1, 2, 3 was 24.9 ± 1.7%, 34.5 ± 2.8% and 34.0 ± 1.4%, respectively (P1-2,3<0.05). The plasma membrane integrity of spermatozoa in groups 2 and 3 (48.4 ± 2.9% and 45.5 ± 3.9%, respectively) was higher than in Group 1 (33.3 ± 2.1%, P < 0.05). After 24 h, 48 h and 72 h in vitro culture, the total motility and viability of spermatozoa in Group 1 was significantly lower than Group 2 and Group 3. The apoptosis rate in Group 3 (44.5 ± 3.0%) and Group 2 (47.7 ± 4.1%) were lower than in Group 1 (52.5 ± 4.4%; P < 0.05). ΔΨm rates in Group 3 and Group 2 were higher than in Group 1 (P < 0.05) with no statistical differences between this parameter in Group 2 and Group 3 (P > 0.1). In conclusion, supplementation of medium for aseptic capillary technology for cryoprotectant-free vitrification of human spermatozoa by permeable cryoprotectant does not improve the quality of spermatozoa after warming.

New method for cryoprotectant-free freezing of human oligoasthenoteratozoospremic spermatozoa with high-molecular polymer.

Data about cryoprotectant-free cryopreservation of human ICSI spermatozoa are limited. The aim of this investigation was to compare two technologies for cryopreservation of spermatozoa from men with oligoasthenoteratozoospermia: standard conventional freezing with 5% glycerol (freezing in glycerol) and cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone (PVP) (PVP-freezing). Capillaries with spermatozoa were cooled in vapor and then plunginged into liquid nitrogen. Head-, midpiece- and tail-abnormality of spermatozoa, mitochondrial membrane potential (MMP) and DNA fragmentation rates after cryopreservation were evaluated. After warming of spermatozoa, fertilization of oocytes (ICSI) was performed. It was detected the lower rate of morphological abnormalities of PVP-frozen spermatozoa in comparison with cells frozen with glycerol (34.6 ± 4.1% vs. 20.7 ± 4.7%, respectively) (P < 0.05). Quality of cells with high MMP after warming in spermatozoa frozen with glycerol was lower than in PVP-frozen spermatozoa (34.7 ± 4.2 vs. 54.5 ± 4.2%, respectively) (P < 0.05). It was established that the DNA fragmentation rate in PVP-frozen spermatozoa was significantly lower in comparison with spermatozoa frozen with glycerol (23.1 ± 2.5% vs. 38.8 ± 3.0%, respectively) (P < 0.05). After fertilization (ICSI) of oocytes, it was established that cleavage and blastulation rates were higher in oocytes after fertilization with PVP-frozen spermatozoa than with spermatozoa frozen with glycerol. Fertilization-, development to 8-blastomeres-, and blastocyst-rates were for PVP-frozen and spermatozoa frozen with glycerol, respectively: 94.4 ± 7.8 vs. 82.2 ± 6.2% (P > 0.1 with tendency to increasing), 90.0 ± 4.6 vs. 69.5 ± 5.1% (P < 0.05), and 45.4 ± 4.1% vs. 30.9 ± 3.3% (P < 0.05). It was concluded that permeable cryoprotectant-free freezing with 5% high-molecular-weight (360 kDa) polyvinylpyrrolidone can be applied successfully for cryopreservation of human oligoasthenoteratozoospremic spermatozoa.

New method of FACS analyzing and sorting of intact whole ovarian fragments (COPAS) after long time (24 h) cooling to 5 °C before cryopreservation.

As recently announced by the American Society for Reproductive Medicine (ASRM), human ovarian tissue cryopreservation is an established option for fertility preservation in prepubertal girls and young women undergoing gonadotoxic treatments for cancer as well as some autoimmune diseases. Proper ovarian tissue assessment before and after cryopreservation is essential to increase success rates. Ovarian fragments from 16 patients were divided into small pieces in form of cortex with medulla, and randomly divided into the following two groups. Pieces of Group 1 (n = 16) were frozen immediately after operation, thawed and just after thawing their quality was analyzed. Group 2 pieces (n = 16) after operation were cooled to 5 °C for 24 h, then frozen after 24 h pre-cooling to 5 °C, thawed and just after thawing their quality was analyzed. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development and viability of follicles (Calcein-AM and Propidium Iodide) using complex object parametric analyzer and sorter machine (COPAS). Positive effect of cooling of cells to low supra-zero temperatures on their future development after re-warming has been observed. New flow cytometry- technique is suitable for the evaluation and sorting of cryopreserved whole human whole intact ovarian fragments. Long time (24 h) cooling of ovarian tissue to 5 °C before cryopreservation has a trend of a cell viability increasing.

Increasing of malignancy of breast cancer cells after cryopreservation: molecular detection and activation of angiogenesis after CAM-xenotransplantation.

BACKGROUND: Ovarian tissue cryopreservation has a wide range of cancerous indications. Avoiding relapse becomes a specific concern that clinicians frequently encounter. The data about the comparative viability of cancer cells after cryopreservation are limited. This study aimed to evaluate the effect of cryopreservation on breast cancer cells. METHODS: We used in-vitro cultured ZR-75-1 and MDA-MB-231 cell lines. Cell samples of each lineage were distributed into the non-intervened and cryopreserved groups. The cryopreservation procedures comprised programmed slow freezing followed by thawing at 100 °C, 60 s. Biological phenotypes and the related protein markers were compared between the two groups. The EVOS FL Auto 2 Cell Image System was used to monitor cell morphology. Cell proliferation, motility, and penetration were characterized by CCK-8, wound-healing, and transmembrane assay, respectively. The expression of Ki-67, P53, GATA3, E-cadherin, Vimentin, and F-Actin was captured by immunofluorescent staining and western blotting as the proxy measurements of the related properties. The chorioallantoic membrane (CAM) xenotransplantation was conducted to explore angiogenesis induced by cancer cells. RESULTS: After 5 days in vitro culture, the cell concentration of cryopreserved and non-intervened groups was 15.7 × 104 vs. 14.4 × 104cells/ml, (ZR-75-1, p > 0.05), and 25.1 × 104 vs. 26.6 × 104 cells/ml (MDA-MB-231, p > 0.05). Some cryopreserved ZR-75-1 cells presented spindle shape with filopodia and lamellipodia and dissociated from the cell cluster after cryopreservation. Both cell lines demonstrated increased cell migrating capability and invasion after cryopreservation. The expression of Ki-67 and P53 did not differ between the cryopreserved and non-intervened groups. E-cadherin and GATA3 expression downregulated in the cryopreserved ZR-75-1 cells. Vimentin and F-actin exhibited an upregulated level in cryopreserved ZR-75-1 and MDA-MB-231 cells. The cryopreserved MDA-MB-231 cells induced significant angiogenesis around the grafts on CAM with the vascular density 0.313 ± 0.03 and 0.342 ± 0.04, compared with that of non-intervened cells of 0.238 ± 0.05 and 0.244 ± 0.03, p < 0.0001. CONCLUSIONS: Cryopreservation promotes breast cancer cells in terms of epithelial-mesenchymal transition and angiogenesis induction, thus increasing metastasis risk.

Long-term (24h) cooling of ovarian fragments in the presence of permeable cryoprotectants prior to freezing: Two unsuccesful IVF-cycles and spontaneous pregnancy with baby born after re-transplantation.

Cancer is the second major cause of death in the world. The problem of post-cancer infertility plays a significant role, because chemotherapy can be gonadotoxic. Cryopreservation of ovarian tissue before cancer therapy with re-implantation after convalescence is the potential key solution to this problem. The aim of this study was to test the viability of cryopreserved human ovarian cortex after long-term cooling in culture medium composed of permeable cryoprotectants. Ovarian fragments from sixteen patients were randomly divided into two groups. After the operation, tissue pieces assigned to both groups were cooled to 5 °C for 22-24 h, frozen and thawed. Group 1 pieces (n = 32) were cooled before cryopreservation in the standard culture medium, and Group 2 pieces (n = 32) were cooled in the freezing medium (culture medium+6% ethylene glycol+6% dimethyl sulfoxide+0.15 M sucrose). Freezing was performed in standard 5 ml cryo-vials with ice formation at -9 °C, cooling from -9 to -34 °C at a rate of -0.3 °C/min and plunging at -34 °C into liquid nitrogen. After thawing in a 100 °C (boiling) water bath, the removal of cryoprotectants was performed in 0.5 M sucrose with 20 min exposure in sucrose and 30 min stepping rehydration. The effectiveness of the pre-freezing cooling of tissue was evaluated by the development of follicles (histology). Six months after the autotransplantation, oocytes from the twenty-seven-year old, hormonally stimulated patient were retrieved and fertilized with her partner sperm through the intracytoplasmic spermatozoa injection (ICSI). For groups 1 and 2, 93.5 ± 1.9% and 96.4 ± 2.0% of the preantral follicles, respectively, were morphologically normal (P > 0.1) (with a tendency toward increasing in quality in Group 2). Six months after the auto-transplantation, two ICSI cycles resulted in the gathering and transplantation of high quality embryos, but no pregnancy had been established. Thirteen months after the auto-transplantation, the patient became spontaneously pregnant and delivered a healthy baby girl at term. Long-term (24 h) cooling of ovarian tissue to 5 °C before cryopreservation in the presence of permeable cryoprotectants simplifies the protocol of cryopreservation and has a tendency of increasing of the cells viability after thawing.

Banking of human ovarian tissue potentially contaminated by cancer cells: experimental model for study of cryo-stability of these cells.

Auto-transplantation of cryopreserved ovarian tissue for cancer survivors comes with the primary concern of the possible existence of cancer cells in the transplanting tissue. Lineal cancer cells are presented by industry in form of separated cells (suspensions). Data about experimental models for evaluation of a cryopreservation effect on viability of compacted lineal cancer cells is currently limited. This study aims to develop a suitable experimental model for cryobiological investigations of compacted cancer cells obtained after in vitro culture of a cell suspension. Suspended lineal breast cancer cells (ZR-75-1 and MDA-MB-231) were in vitro cultured in AIM V medium for formation of monolayer. Evaluation of the cell viability was performed by healing assay, transmembrane cell migration, invasion assay and immunofluorescent test of F-actin. It was established the possibility of formation of monolayer from viable cancer cells, scarification of monolayer of these cells and formation of compacted fragments. It is described also a behaviour of compacted cells during cryopreservation (saturation by permeable cryoprotectants, thawing and removal of cryoprotectants). The described method can be used for cryobiological investigations of lineal suspended cancer cells in compacted form as a model of tissues contaminated by malignant cells and solid tumors.

Conventional freezing vs. cryoprotectant-free vitrification of epididymal (MESA) and testicular (TESE) spermatozoa: Three live births.

Data of cryoprotectant-free vitrification of human testicular and epididymal spermatozoa are limited. The aim of this investigation was to compare two aseptic technologies of TESE (testicular) and MESA (epididymal) spermatozoa cryopreservation: standard conventional freezing with the use of cryoprotectants and cryoprotectant-free vitrification. Sperm motility, capacitation-like changes, acrosome reaction and the mitochondrial membrane potential of frozen (5% glycerol, -10 °C/min) and vitrified (Human Tubal Fluid + 1% Human Serum Albumin+0.25 M sucrose, plunging into liquid nitrogen of capillaries with spermatozoa isolated from liquid nitrogen (aseptic method) were compared. The quality of the cryoprotectant-free vitrified MESA- and TESE-spermatozoa was higher than that of spermatozoa conventionally frozen with permeable cryoprotectants. Intracellular sperm injection (ICSI) was performed with vitrified spermatozoa. We report the birth of three healthy babies from two women following ICSI with motile MESA- and TESE-spermatozoa vitrified without cryoprotectants. This is the first report of full-term pregnancies and babies born after ICSI with epididymal and testicular spermatozoa vitrified without cryoprotectants. In conclusion, cryoprotectant-free vitrification can be successfully applied for the cryopreservation of motile TESE- and MESA-spermatozoa.

Vitrification of human pronuclear oocytes by direct plunging into cooling agent: Non sterile liquid nitrogen vs. sterile liquid air.

In fact, a full sterilization of commercially-produced liquid nitrogen contaminated with different pathogens is not possible. The aim of this study was to compare the viability of human pronuclear oocytes subjected to cooling by direct submerging of open carrier in liquid nitrogen versus submerging in clean liquid air (aseptic system). One- and three-pronuclei stage embryos (n = 444) were cryopreserved by direct plunging into liquid nitrogen (vitrified) in ethylene glycol (15%), dimethylsulphoxide (15%) and 0.2M sucrose. Oocytes were exposed in 20, 33, 50 and 100% vitrification solution for 2, 1 and 1 min, and 30-50 s, respectively at room temperature. Then first part of oocytes (n = 225) were directly plunged into liquid nitrogen, and second part of oocytes (n = 219) into liquid air. Oocytes were thawed rapidly at a speed of 20,000 °C/min and then subsequently were placed into a graded series of sucrose solutions (0.5, 0.25, 0.12 and 0.06M) at 2.5 min intervals and cultured in vitro for 3 days. In both groups, the rate of high-quality embryos (Grade 6A: 6 blastomeres, no fragmentation; Grade 8A: 8 blastomeres, no fragmentation; Grade 8A compacting: 8 blastomeres, beginning of compacting) was noted. The rates of high-quality embryos developed from one-pronuclear oocytes vitrified by cooling in liquid nitrogen and liquid air were: 39.4% ± 0.6 and 38.7% ± 0.8, respectively (P > 0.1). These rates for three-pronuclear oocytes were: 45.8 ± 0.8% and 52.0 ± 0.7%, respectively (P < 0.05). In conclusion, vitrification by direct submerging of oocytes in clean liquid air (aseptic system) is a good alternative for using of not sterile liquid nitrogen.

Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate.

BACKGROUND: Phosphatidylserine is the phospholipid component which plays a key role in cell cycle signaling, specifically in regards to necrosis and apoptosis. When a cell affected by some negative factors, phosphatidylserine is no longer restricted to the intracellular side of membrane and can be translocated to the extracellular surface of the cell. Cryopreservation can induce translocation of phosphatidylserine in response to hypoxia, increasing intracellular Ca2+, osmotic disruption of cellular membranes, generation of reactive oxygen species and lipid peroxidation. As such the aim of this study was to test the level of phosphatidylserine translocation in frozen human medulla-contained and medulla-free ovarian tissue fragments. METHODS: Ovarian fragments from twelve patients were divided into small pieces of two types, medulla-free cortex (Group 1, n = 42, 1.5-3.0 × 1.5-3.0 × 0.5-0.8 mm) and cortex with medulla (Group 2, n = 42, 1.5-3.0 × 1.5-3.0 × 1.5-2.0 mm), pre-cooled after operative removal to 5 °C for 24 h and then conventionally frozen with 6 % dimethyl sulfoxide, 6 % ethylene glycol and 0.15 M sucrose in standard 5-ml cryo-vials. After thawing at +100 °C and step-wise removal of cryoprotectants in 0.5 M sucrose, ovarian pieces were xenografted to SCID mice for 45 days. The efficacy of tissues cryopreservation, taking into account the presence or absence of medulla, was evaluated by the development of follicles (histology with hematoxylin-eosin) and through the intensity of translocation of phosphatidylserine (FACS with FITC-Annexin V and Propidium Iodide). RESULTS: For Groups 1 and 2, the mean densities of follicles per 1 mm3 were 9.8, and 9.0, respectively. In these groups, 90 and 90 % preantral follicles appeared morphologically normal. However, FACS analysis showed a significantly decreased intensity of translocation of phosphatidylserine (FITC-Annexin V positive) after cryopreservation of tissue with medulla (Group 2, 59.6 %), in contrast with tissue frozen without medulla (Group 1, 78.0 %, P < 0.05). In Groups 1 and 2 it was detected that 21.6 and 40.0 % cells were viable (FITC-Annexin V negative, Propidium Iodide negative). CONCLUSION: The presence of medulla in ovarian pieces is beneficial for post-thaw development of cryopreserved human ovarian tissue.

The diagnostic value of ultrasound in the assessment of soft tissue masses in rheumatology practice - a case series of 4 patients.

A great number of studies have proved the added value of musculoskeletal ultrasound (MSUS) in the diagnosis, assessment of disease activity and treatment response in both inflammatory and degenerative rheumatic diseases. However, it is a frequent scenario that rheumatologists should also assess patients with various soft tissue masses, referred to their practices. In such cases, MSUS could be a valuable and precise tool that helps in the evaluation and triage of these lesions. Hereafter, we describe a case series, where MSUS played a decisive role in the diagnostic process and allowed for prompt patients' management.

Ultrasound description and follow up of painful cervical interspinous bursitis in a Polymyalgia Rheumatica patient - a case report.

We present a Polymyalgia Rheumatica (PMR) case with active Cervical Interspinous Bursitis (CIB) causing debilitat-ing neck pain as the most intensive symptom of the disease as reported by the patient. CIB was diagnosed and followed by Musculoskeletal Ultrasound (MSUS). MSUS examination of patient's posterior cervical region reviled well demarcated an-/ hypoechoic lesions around and cranially of the spinous processes of the sixth and seventh cervical vertebra. The initial detailed sonographic characteristics of the CIB are described, as well as the evolution of lesions size and extent with the treatment and patient's clinical improvement. To our knowledge this is the rst detailed sonographic description of CIB in PMR.

An ultrasound study of the long posterior sacroiliac ligament in healthy volunteers and in patients with noninflammatory sacroiliac joint pain.

AIM: To describe the sonoanatomy of the long posterior sacroiliac ligament (LPSL) in healthy volunteers and to assess by ultrasound the LPSL in patients with noninflammatory sacroiliac joint pain (SIP). MATERIAL AND METHODS: We assessed 64 LPSLs of 32 healthy controls and 40 LPSLs of 40 patients with unilateral noninflammatory SIP and a positive Fortin finger test. LPSLs in both groups were assessed for the presence of alterations in their structure, continuity and echogenicity and their thickness was measured in three predefined points. All patients were examined in prone position following a strict scanning protocol. RESULTS: Detailed sonoanatomy description and measurement of the LPSL in healthy volunteers are provided (length: 31.32±4.79 mm, width: 8.14±1.28 mm, thickness: 2.05±0.55 mm; 1.64±0.41 mm and 1.51±0.42 mm at the iliac and sacral entheses and in its middle part, respectively). The LPSLs were found to be significantly thicker in the SIP group, with an optimum criterion value of >2.0 mm in its middle part to identify pathologically thickened ligaments. In addition, LPSLs inthe SIP group presented significantly more often hypoechogenicity/altered fibrillar structure (57.5% vs.16%) and/or periligamentous edema (72.8% vs 28%). The combination of either altered structure or periligamentous edema, with thickening of theligament's body showed the best diagnostic accuracy (sensitivity and specificity 83.9% and 94.7% for the first combination and 100% and 84.6% for the second combination) to identify LPSL pathology in noninflammatory SIP. CONCLUSIONS: LPSL could be assessed by ultrasound and sonopathological lesions could be identified in patients with SIP.

Epigenetic Alterations in Cryopreserved Human Spermatozoa: Suspected Potential Functional Defects.

BACKGROUND: Gene set enrichment analysis (GSEA) was conducted on raw data, and alternative splicing (AS) events were found after mRNA sequencing of human spermatozoa. In this study, we aimed to compare unknown micro-epigenetics alternations in fresh and cryopreserved spermatozoa to evaluate the effectivity of cryopreservation protocols. METHODS: Spermatozoa were divided into three groups: fresh spermatozoa (group 1), cryoprotectant-free vitrified spermatozoa (group 2), and conventionally frozen spermatozoa (group 3). Nine RNA samples (three replicates in each group) were detected and were used for library preparation with an Illumina compatible kit and sequencing by the Illumina platform. RESULTS: Three Gene Ontology (GO) terms were found to be enriched in vitrified spermatozoa compared with fresh spermatozoa: mitochondrial tRNA aminoacylation, ATP-dependent microtubule motor activity, and male meiotic nuclear division. In alternative splicing analysis, a number of unknown AS events were found, including functional gene exon skipping (SE), alternative 5' splice sites (A5SS), alternative 3' splice sites (A3SS), mutually exclusive exon (MXE), and retained intron (RI). CONCLUSIONS: Cryopreservation of spermatozoa from some patients can agitate epigenetic instability, including increased alternative splicing events and changes in crucial mitochondrial functional activities. For fertilization of oocytes, for such patients, it is recommended to use fresh spermatozoa whenever possible; cryopreservation of sperm is recommended to be used only in uncontested situations.