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.

Extrusion of Neutrophil Extracellular Traps (NETs) Negatively Impacts Canine Sperm Functions: Implications in Reproductive Failure.

Reproductive failure in dogs is often due to unknown causes, and correct diagnosis and treatment are not always achieved. This condition is associated with various congenital and acquired etiologies that develop inflammatory processes, causing an increase in the number of leukocytes within the female reproductive tract (FRT). An encounter between polymorphonuclear neutrophils (PMNs) and infectious agents or inflammation in the FRT could trigger neutrophil extracellular traps (NETs), which are associated with significantly decreased motility and damage to sperm functional parameters in other species, including humans. This study describes the interaction between canine PMNs and spermatozoa and characterizes the release of NETs, in addition to evaluating the consequences of these structures on canine sperm function. To identify and visualize NETs, May-Grünwald Giemsa staining and immunofluorescence for neutrophil elastase (NE) were performed on canine semen samples and sperm/PMN co-cultures. Sperm viability was assessed using SYBR/PI and acrosome integrity was assessed using PNA-FITC/PI by flow cytometry. The results demonstrate NETs release in native semen samples and PMN/sperm co-cultures. In addition, NETs negatively affect canine sperm function parameters. This is the first report on the ability of NETs to efficiently entrap canine spermatozoa, and to provide additional data on the adverse effects of NETs on male gametes. Therefore, NETs formation should be considered in future studies of canine reproductive failure, as these extracellular fibers and NET-derived pro-inflammatory capacities will impede proper oocyte fertilization and embryo implantation. These data will serve as a basis to explain certain reproductive failures of dogs and provide new information about triggers and molecules involved in adverse effects of NETosis for domestic pet animals.

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.

Autophagy is activated in human spermatozoa subjected to oxidative stress and its inhibition impairs sperm quality and promotes cell death.

STUDY QUESTION: Does oxidative stress (OS) activate autophagy in human sperm? SUMMARY ANSWER: Human spermatozoa subjected to OS activate an autophagic response. WHAT IS KNOWN ALREADY: Autophagy is a regulated pathway of lysosomal degradation which helps eukaryotic cells to maintain or restore homeostasis, being a cellular stress response mechanism. OS is a main cause of impaired sperm function and is linked to male infertility; however, whether OS activates autophagy in human spermatozoa is unknown. STUDY DESIGN, SIZE, DURATION: Human spermatozoa were exposed separately to ionomycin and hydrogen peroxide in order to induce OS. An untreated control group was included. Sperm cells under OS were then exposed to chloroquine in order to block autophagy. An untreated control and a control incubated only with the OS inducer were included in each experimental setting. PARTICIPANTS/MATERIALS, SETTING, METHODS: For this study, semen samples from normozoospermic donors were used and motile sperm cells were selected by the swim up technique. First, the generation of OS under our experimental conditions was demonstrated by analyzing sperm parameters including viability, reactive oxygen species (ROS) production, mitochondrial membrane potential (ΔΨm) motility and thiol oxidation. Then, proteins involved in autophagy, including the microtubule-associated protein light chain 3 (LC3), particularly LC3-I and LC3-II, autophagy-related 5 (ATG5) and autophagy-related 16 (ATG16) proteins as well as the phosphorylated form of AMP-activated protein kinase (pAMPK) were evaluated in spermatozoa exposed to OS and compared to the untreated control. Finally, the impact of autophagy blocking by chloroquine treatment on sperm quality, metabolic parameters, including glycolysis and oxidative phosphorylation, as well as the cell death markers phosphatidylserine externalization and caspase activation was analyzed. Sperm quality parameters, cell death markers and autophagy-related proteins were analyzed by flow cytometry. Motility was evaluated by the computer-assisted sperm analysis system and metabolic parameters were analyzed using an extracellular flux analyzer. MAIN RESULTS AND THE ROLE OF CHANCE: Exposure to ionomycin and hydrogen peroxide promotes OS resulting in increased ROS production and decreased viability, ΔΨm and motility, while increasing thiol oxidation. These alterations were accompanied by a decrease in LC3-I, indicating that autophagy was activated upon OS exposure. Ionomycin also caused an increase in LC3-II, ATG5, ATG16 and pAMPK content. Autophagy blocking of sperm exposed to OS caused deterioration in sperm quality and metabolic parameters as well as an increase in cell death markers. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The study was carried out in vitro using motile sperm from normozoospermic donors; tests on sperm from infertile patients were not carried out. The autophagy blocking plus OS might generate a non-specific response to a highly stressful situation leading to the induction of cell death. WIDER IMPLICATIONS OF THE FINDINGS: Human spermatozoa subjected to OS activate an autophagic response and its blockage results in increased oxidative damage and commits spermatozoa to cell death. These results suggest a crucial role of autophagy as a stress response by male gametes, which contributes to maintaining the functionality and lifespan of ejaculated sperm cells. Detection of autophagy activation in sperm cells ex vivo could be included in semen analysis as a marker of OS, especially in men displaying high levels of seminal ROS. Novel strategies that aim to activate this cellular stress response could improve sperm quality/functionality under natural ejaculate conditions in which increased ROS levels are expected. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Fondo Nacional de Investigación Científica y Tecnológica, Chile (ANID/FONDECYT, Grant number 11170758 to P.U.); the Comisión Nacional de Investigación Científica y Tecnológica, Chile (ANID/CONICYT, Grant number PAI79160030 to P.U.) and the Dirección de Investigación, Universidad de La Frontera. The authors disclose no potential conflicts of interest.

SOCE-inhibitor reduced human sperm-induced formation of neutrophil extracellular traps.

Human spermatozoa activate neutrophil extracellular traps (NETs) in vitro. NETosis is an efficient mechanism through which polymorphonuclear neutrophils (PMN) capture sperm in vitro. The objective of this study was to establish the role of store-operated Ca+2 entry (SOCE) in human sperm-triggered NETs and its impact on sperm integrity and oocyte binding capacity. PMN isolated from donors were exposed to spermatozoa isolated from normozoospermic donors using the swim-up technique and were divided into the following groups: (1) sperm, (2) PMN, (3) PMN + sperm, (4) PMN (pretreated with 2-APB, SOCE inhibitor) + sperm, (5) (PMN + DNase) + sperm, and (6) (PMN + PMA) + sperm (positive control). NETs were quantified using PicoGreen® and visualised by scanning electron microscopy and immunofluorescence of extracellular DNA and neutrophil elastase. Plasma membrane, acrosome, and DNA integrity were analysed by flow cytometry, and oocyte binding was evaluated using the hemizona pellucida assay. Sperm-triggered NETosis negatively affected the sperm membrane and acrosome integrity and decreased the oocyte binding capacity. These effects were negated by an SOCE inhibitor, thus improving sperm function and achieving high oocyte binding capacity. The SOCE inhibitor significantly reduced NET formation compared with that in control PMN/sperm (P < 0.05). Collectively, these results advance the knowledge about the role of PMN in reproduction and will allow the development of strategies to block NET formation in situations of reduced fertilisation success.

Antioxidants and their effect on the oxidative/nitrosative stress of frozen-thawed boar sperm.

In swine, the use of frozen-thawed boar sperm for artificial insemination remains a suboptimal reproductive technology. Among the negative effects of cryopreservation on sperm cells, it is worth highlighting that cryopreservation causes irreversible alterations in motility and components of the sperm membrane as a result of dramatic changes in temperature (cooling/freezing curve) and osmolality. In addition, freeze-thawing may induce oxidative stress and increase the generation of reactive oxygen species (ROS) and nitrogen reactive species (RNS). While boar sperm cryopreservation has been reported to increase lipid peroxidation and the intracellular levels of hydrogen peroxide, less research on its impact on RNS has been conducted. Furthermore, previous studies have investigated the effects of supplementing cryopreservation media with antioxidants to counteract the deleterious effects of ROS and RNS. Antioxidants of synthetic origin or natural extracts have been used, with some showing noticeable and positive effects on functional sperm parameters both in vitro and in vivo. The aim of this review is to provide an update on the effect of different molecules with antioxidant capacity on the function of cryopreserved boar sperm.

Dental age in children with impacted maxillary canines.

OBJECTIVE: The aim of this study was to evaluate if delayed dental development is a cause of postponed care for patients with impacted maxillary canine (IMC). MATERIALS AND METHODS: This case-control study was based on 403,355 children and adolescents in Region Västra Götaland, Sweden. The subjects, who were in the age range of 9-16 years during the period of 2011-2013, underwent surgical exposure or removal of a maxillary canine. Demirjian's dental age assessment was carried out on panoramic radiographs. RESULTS: In total, 1028 patients, 514 with IMC and 514 age- and gender-matched controls, were enrolled. The patients with IMC exhibited a dental development delay of 0.2 years compared to the control group. In the impaction sub-groups, the female patients, patients in the chronological age group of 12-13 years, and patients with palatally positioned IMC had a significantly lower dental age than their paired-control subjects. CONCLUSIONS: Overall, the difference in dental age between patients with or without IMC is significant but small, and as such is likely of minor clinical relevance. Therefore, the timing of preventive care and treatment for patients with IMC should be the same as that for patients with normally erupting canines.

Multiparameter Flow Cytometry Assay for Analysis of Nitrosative Stress Status in Human Spermatozoa.

Male infertility is an increasing health problem, and oxidative/nitrosative stress plays an important role in the etiology of this condition. Nitrosative stress due to excessive levels of reactive nitrogen species (RNS) is associated with impaired male fertility. Flow cytometry may be a useful tool for semen evaluation, but the availability of multiparameter assays for analysis of sperm quality is limited. The present study standardized a multiparameter flow cytometry analysis for nitrosative stress status in human spermatozoa in a single assay. A suitable multicolor fluorochrome panel was designed and consisted of fluorescein-boronate to detect peroxynitrite, a highly RNS, propidium iodide to analyze viability, tetramethylrhodamine methyl ester perchlorate to detect mitochondrial membrane potential (MMP) and monobromobimane to analyze thiol oxidation. Proper positive and negative controls for each fluorochrome were used to establish the technique, and sperm cells of different qualities and spermatozoa subjected to cryopreservation were analyzed. The results showed that the controls clearly discriminated between the high and low fluorescence intensities for each fluorochrome. The analysis of sperm cells of different quality demonstrated that the assay properly detected differences in all parameters analyzed according to sperm quality. The results may be reported as the mean fluorescence intensity of each fluorochrome and the percentage of cells exhibiting different characteristics. In conclusion, a protocol was standardized to analyze nitrosative stress status, including peroxynitrite production, viability, MMP, and thiol oxidation, in a single analysis using flow cytometry. This protocol may be applied to research approaches and clinical andrology to improve the evaluation of sperm quality and provide a promising tool to increase the use of clinical flow cytometry. © 2020 International Society for Advancement of Cytometry.

Oxidative and nitrosative stress in frozen-thawed pig spermatozoa. II: Effect of the addition of saccharides to freezing medium on sperm function.

Saccharides have bioprotective properties, with a high capacity to preserve biological proteins and membranes during sperm cryopreservation. The aim of this study was to evaluate how replacing the lactose of cryopreservation media by sucrose (SUC) or trehalose (TRE) at concentrations of 0.2 M (SUC-1 and TRE-1) and 0.25 M (SUC-2 and TRE-2) affects frozen/thawed pig spermatozoa. The media used were composed of medium A (saccharide/egg yolk) and B (saccharide/egg yolk/glycerol), their osmolality being determined prior to freezing. Cell viability, membrane lipid disorder, acrosome integrity, mitochondrial membrane potential (MMP), lipid peroxidation, thiol group oxidation, total reactive oxygen species (ROS), peroxynitrite and superoxide anion (O2●-) were determined through flow cytometry; total motility (TM), progressive motility (PM) and kinetic parameters motility were determined immediately after thawing (T0) and again 30 (T30) and 60 (T60) minutes later. The SUC-2 and TRE-2 groups maintained viability significantly and presented fewer lipid membrane disorders, respectively, both with a significant increase in MMP. The production of O2●- and peroxynitrite was lower in the TRE-2 groups compared to the control (P < 0.05). Total motility at T0 was greater in the TRE-2 group (P < 0.05). Sperm kinetics was not affected by the treatment. The use of saccharides SUC and TRE at a concentration of 0.25 M improves sperm quality, so that both non-penetrating cryoprotectants can be utilized in pig sperm freezing media.

Rest days and storage of boar semen at 17°C: Effect on motility and sperm concentration.

Boar fertility is an important factor in farm production; it is therefore of interest to determine factors which reduce the fertilising capacity of semen samples stored at 17°C for use in intrauterine insemination. This work evaluated the effect of the number of rest days between each mounting of the boar, and the number of days that the semen was stored at 17°C, on sperm motility and semen concentration. We also analysed whether the boar's age influenced the sperm concentration. The results showed that only the total motility diminished as the storage time at 17°C increased (p < .05). A low negative correlation was observed between the variables' rest days and total and progressive motility. The sperm concentration presented no relation with rest days or the boar's age. The boars' rest days had no effect on motility and sperm concentration in the males studied, allowing them to be used with the frequencies described with no effect on these parameters.

Antioxidant effects of penicillamine against in vitro-induced oxidative stress in human spermatozoa.

Oxidative stress contributes importantly to the aetiology of male infertility, impairing sperm function. The protective effect of antioxidants on seminal parameters has been established, and the antioxidant penicillamine has shown beneficial effects; however, its protective effect on human spermatozoa exposed to oxidative stress has not been reported. The objective of this work was to evaluate the effect of penicillamine on human spermatozoa exposed in vitro to oxidative stress. First, the effect of penicillamine on spermatozoa from normozoospermic donors was evaluated. Then, the effect of penicillamine on spermatozoa exposed to oxidative stress induced separately by ionomycin and hydrogen peroxide (H2 O2 ) was analysed. An untreated control and a control treated only with the oxidative stress inducer were included. Reactive oxygen species (ROS) levels, viability, mitochondrial membrane potential (MMP) and motility were analysed. The results showed that penicillamine, added to the incubation medium, decreased the ROS levels induced by ionomycin and H2 O2 , and this effect was associated with better preservation of MMP, motility, and ATP levels. These results highlight the potential advantages of penicillamine supplementation of sperm culture medium, especially for semen samples with high ROS levels and also in circumstances where laboratory handling can cause an increase in ROS production.

Protective effect of the superoxide dismutase mimetic MnTBAP during sperm vitrification process.

Sperm cryopreservation is widely used in assisted reproduction and male infertility therapy; however, it induces oxidative stress affecting sperm quality. This work evaluated the effect of the antioxidant MnTBAP during vitrification steps in human spermatozoa. First, the effect of MnTBAP on viability and ROS production was evaluated. Then, the spermatozoa were vitrified in straws with the vitrification, warming and post-warming incubation media separately supplemented with MnTBAP. An untreated control was included. The sperm viability, ROS production, total and progressive motility were evaluated. The results showed that the direct exposure of spermatozoa to MnTBAP significantly decreases the ROS levels in comparison with the untreated control without affecting the viability. The supplementation of the vitrification medium with MnTBAP did not affect the parameters analysed. However, the supplementation of the warming and incubation post-warming media resulted in a decrease in ROS production and maintained viability and motility for 4 hr after warming with concentrations up to 100 µM of MnTBAP. Higher concentrations of MnTBAP caused a decrease in total motility. In conclusion, the use of MnTBAP during the warming or post-warming incubation media has beneficial effect decreasing ROS levels and maintaining the viability and motility during the vitrification procedure.

Increase of leucocyte-derived extracellular traps (ETs) in semen samples from human acute epididymitis patients-a pilot study.

PURPOSE: To study the effector mechanism against pathogens of polymorphonuclear neutrophils (PMN) and macrophages, called ETosis, involving the release of extracellular traps (ETs) in patients with acute epididymitis. To assess the different ET phenotypes present in semen samples and to identify correlations between ETosis and clinical parameters. MATERIALS AND METHODS: Samples from patients diagnosed with acute epididymitis were examined and compared with samples from uninfected controls. Biochemical analyses of seminal fluid included determination of peroxidase, α-glucosidase, fructose, and elastase levels. ETosis in semen was determined through presence of citrullinated histones, global histones, and extracellular DNA. Different ETosis phenotypes such as spread ETs, aggregated ETs, and diffuse ETs were identified by co-localisation of extruded DNA with myeloperoxidase and global histones. Anti-CD15+ and anti-CD68+ antibodies were used to identify different cell lines. RESULTS: Revealed a high number of ETs compared with the control group. The mean number of CD15+PMN and CD68+ macrophages was higher in the acute epididymitis group. ETosis increase in ejaculates correlated with clinical parameters such as enhancement of elastase concentrations and diminution of fructose in the semen. CONCLUSIONS: This work shows for the first time the presence of ETs and their components in semen from patients with acute epididymitis. The presence of infections is an important factor for induction of ETs in semen. Furthermore, the presence of ETosis in ejaculates is suggestive of developing infectious processes and might possibly have a diagnostic value.

LED-based red light photostimulation improves short-term response of cooled boar semen exposed to thermal stress at 37°C.

Pre-treatment of boar semen with a red light photostimulation procedure increases its "in vivo" fertilising ability. However, "in vitro" conducted studies shown contradictory results regarding the ability of photostimulated spermatozoa to react against strong stress and to achieve the capacitation status. The aim here was to determine the effect of photostimulation on the response to short-term moderate thermal stress of boar semen. Boar semen was exposed to red LED light regime emitting a 620-630 nm during 10 min of light, 10 min of rest and 10 min of light after 3 hr since semen was collected. An aliquot without photostimulation was included as a control. After the photostimulation, the sperm cells were incubated for 15 min at 37°C. Afterwards, motility, viability, intracellular Ca2+ level and production of reactive oxygen species (ROS) and peroxynitrite were analysed. The results showed that the photostimulated group maintained total motility throughout the time, whereas a significant decrease in total motility was observed in the nonphotostimulated control group. Furthermore, for kinetic parameters of motility, a significant increase was observed in LIN, STR and WOB in photostimulated spermatozoa. Peroxynitrite production was significantly increased in the photostimulated spermatozoa, whereas viability, ROS production and intracellular Ca2+ levels were not affected by photostimulation. In conclusion, photostimulation of commercial boar semen has a positive effect on motility of spermatozoa subjected to a short-term moderate thermal stress, which was concomitant with an increase in peroxynitrite production.

Prevalence of impacted maxillary canines-an epidemiological study in a region with systematically implemented interceptive treatment.

BACKGROUND: In dentistry, epidemiological studies are important for establishing high-quality care for the individual patient as well as for socio-economic reasons. OBJECTIVE: The aim of this epidemiological study was to evaluate the prevalence of impacted maxillary canines in a geographical region in which interceptive treatment is implemented systematically. Furthermore, the aim was to study the age and gender of the patients, and the location and surgical technique used for the impacted maxillary canine. METHOD: The study was based on 54 716 adolescents in the Region Västra Götaland, Sweden who were born in the period 1996-98. All patients in the three cohorts who had impacted maxillary canines treated with surgical exposure or surgical removal were identified in the dental record system used in the region. RESULTS: The prevalence of impacted maxillary canines when interceptive treatment was systematically implemented was 1.1% (N = 601). Overall, the cohorts of patients with impacted canines comprised 65% girls and 35% boys. Most of the canines were palatally impacted and the most common surgical technique was closed exposure. CONCLUSION: The prevalence of impacted maxillary canines in a geographical area in which interceptive treatment is systematically implemented is lower than that reported previously. The distributions of impacted canines with respect to gender and location are in accordance with those reported previously in similar studies.

Study on site-specific expression of bone formation and resorption factors in human dental follicles.

We sought to investigate site-specific expression of bone-regulatory factors expressed by human dental follicles and to compare the stimulated expression of tumour necrosis factor (ligand) superfamily, member 11/tumour necrosis factor receptor superfamily, member 11b (RANKL/OPG) in human dental follicle cells (HDFCs) from different patients. Analysis of bone-regulatory markers in follicles from 12 different study participants was performed using RT-qPCR and immunofluorescence; apical and coronal segments from each dental follicle were processed independently. Four additional dental follicles were used for cell cultures; HDFCs were precultured in osteogenic medium to initiate differentiation and thereafter cultured with 10-6 M forskolin (FSK) to activate the protein kinase cAMP (PKA/cAMP) signalling pathway and induce RANKL/OPG expression. We demonstrate that RANKL expression is significantly higher in the coronal part of follicles than in the apical part. High levels of collagen type 1 (COL1), alkaline phosphatase (ALP) and Gap-junction protein, alpha 1, 43 kDa (CX43) were expressed, whereas expression of Sp7 transcription factor (OSX), bone morphogenetic protein 2 (BMP2), colony-stimulating factor 1 (CSF-1), chemokine (C-C motif) ligand 2 (MCP1), and OPG was low in all samples. The immunofluorescence localization of CSF-1, MCP1, osteocalcin (OCN), RANKL, and BMP2 was not specific for either part of the follicles. In conclusion, a consistently high expression of CX43 suggests that gap-junction communication in HDFCs is essential for the eruption process. Furthermore, the induced expression of RANKL in HDFCs varies significantly between individuals and may relate to clinical variations in tooth eruption.

Cryopreservation induces mitochondrial permeability transition in a bovine sperm model.

When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 × 106 mL-1 and incubated for 4 h at 38 °C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca2+ level using FLUO3-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (ΔΨm) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by ΔΨm dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality.

Gene expression profiles in dental follicles from patients with impacted canines.

Animal studies suggest that the dental follicle (DF) plays a major role in tooth eruption. However, the role of the DF during tooth impaction and related root resorptions in adjacent teeth is not clear. The hypothesis for the present study is that expression of regulatory factors involved in the bone remodelling process necessary for tooth eruption may differ between dental follicles from teeth with different clinical situations. We have analysed the gene expression profiles in the DF obtained from impacted canines, with (N = 3) or without (N = 5) signs of root resorption, and from control teeth (normal erupting teeth, mesiodens) (N = 3). DF from 11 patients (mean age: 13 years) obtains at the time of surgical exposure of the tooth. Due to the surgical time point, all teeth were in a late developmental stage. Gene expression related to osteoblast activation/bone formation, osteoclast recruitment and activation was analysed by RTqPCR. Genes related to bone formation (RUNX2, OSX, ALP, OCN, CX43) were highly expressed in all the samples, but osteoclast recruitment/activation markers (OPG, RANKL, MCP-1, CSF-1) were negligible. No apparent patterns or significant differences in gene expression were found between impacted canines, with or without signs of root resorption, or when compared to control teeth. Our results suggest the DF regulation of osteoclastic activity is limited in the late pre-emergent stage of tooth development, irrespective if the tooth is normally erupting or impacted. We suggest that the follicle may have an important regulatory function for alveolar bone formation in the final eruption process and CX43-gap junction communication could be an important signalling pathway.

Effect of incubation temperature after devitrification on quality parameters in human sperm cells.

Sperm cryopreservation is common in assisted reproduction laboratories, providing a therapeutic option for several clinical conditions. This process has been optimized; however, the effect of post-thaw incubation temperature has been poorly studied. This work analyzed the effect of incubation temperature after devitrification on human sperm quality. Spermatozoa from normozoospermic donors were cryopreserved by vitrification. After devitrification, the spermatozoa were separated into two aliquots: (i) incubated at room temperature (RT, 22-25 °C) and (ii) incubated at 37 °C. Reactive oxygen species (ROS), viability, mitochondrial membrane potential (ΔΨM), phosphatidylserine externalization and motility were analyzed immediately after devitrification (control) and after 2, 4 and 6 h. Spermatozoa incubated at RT showed a conserved viability and ΔΨM compared to the control, while the incubation at 37 °C promoted a decrease in these parameters. The ROS levels were increased at both incubation conditions. The progressive motility was decreased in all experimental groups and the decrease was more pronounced under incubation at RT. No increase in phosphatidylserine externalization was observed. In conclusion, prior to use in assisted reproduction procedures, devitrified spermatozoa at RT conserve a better viability and ΔΨM than at 37 °C.

Clinical predictors of maxillary canine impaction: a novel approach using multivariate analysis.

Background: Ectopic eruption and/or impaction of maxillary permanent canines is a frequent problem in clinical dentistry. Previous studies aimed to identify potential associated factors and predictors for impacted maxillary canines have only used conventional univariate statistics, which does not allow the analysis of the interaction between and within variables. Multivariate data analysis (MVDA) is a better and more powerful tool for the integration and interpretation of complex datasets. Aim: The aim of this study was to validate previously explored predictors of permanent maxillary canine impaction using MVDA. Subjects and methods: This cohort study included all the patients referred during 2011 to Mölndal Hospital, Sweden for surgical exposure of impacted canines (N = 45). Age- and gender-matched orthodontic patients (N = 45) with normally erupting canines comprised the control group. The age range for both groups was 11-17 years. The positions of the canine teeth (orthopantograms), the skeletal variables (profile radiographs), and dentoalveolar traits (casts) were evaluated as potential predictive factors for impaction. Results: None of the parameters evaluated with either profile radiography or casts were positively correlated with impacted maxillary canines, with the exception of the location of the already impacted canines, as identified by orthopantogram. Conclusion: No correlation between clinical variables and impaction was found using MVDA. Therefore, these variables could not be used as predictors of canine impaction. Other types of parameters, such as inheritance and molecular factors that regulate the biological mechanisms of the eruption process, need to be further investigated.