Oncofertility case report: live birth 10 years after oocyte in vitro maturation and zygote cryopreservation.

PURPOSE: This study aims to report a case of urgent fertility preservation in an oncological patient with collection of immature oocytes in the absence of ovarian stimulation that, through in vitro maturation (IVM), followed by ICSI and cryopreservation of zygotes resulted, 10 years later, in the live birth of a healthy baby. METHODS: In September 2008, our clinic performed IVM in a 32-year-old woman diagnosed with a ductal invasive carcinoma with positive estradiol receptors, negative progesterone receptors and positive human epidermal growth factor receptor 2. The retrieval of immature oocytes was performed in the absence of ovarian stimulation after a simple mastectomy and prior to any chemotherapy treatment. The compact cumulus-oocyte complexes (COCs) collected were placed in Lag medium for 2 h, followed by incubation in IVM medium, supplemented with heat inactivated patient serum, recombinant FSH, and recombinant LH. After 30 h in culture, cumulus cells were removed, the metaphase II oocytes were microinjected, and the zygotes obtained were cryopreserved. In 2017, the zygotes were thawed and cultured until day 3. One embryo was transferred and the other cryopreserved. RESULTS: Four compact COCs were collected and subjected to IVM. Two oocytes reached metaphase II and were microinjected. Two zygotes were obtained and were cryopreserved at the two pronuclear stage. Approximately 9 years later, the two zygotes were thawed and cultured until day 3. An embryo with 10 cells was transferred and implanted, resulting in the birth of a healthy baby. CONCLUSIONS: In cases where urgency to start adjuvant therapy requires immediate oocyte collection, IVM may be the only option to obtain fully competent mature oocytes allowing for effective preservation of the reproductive potential.

Systemic treatment with resveratrol and/or curcumin reduces the progression of experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Periodontitis is a chronic inflammatory disease of periodontal tissues that leads to the destruction of bone and other connective tissues. Resveratrol and curcumin are plant-derived substances with biological properties that may have immunomodulatory properties. This study investigated the effect of continuous administration of resveratrol and curcumin and the association of resveratrol and curcumin on the progression of experimental periodontitis in rats. MATERIAL AND METHODS: Forty Wistar rats were assigned randomly to the following groups: group 1, experimental periodontitis + placebo (PL) (n = 10); group 2, experimental periodontitis + resveratrol (RSV) (n = 10); group 3, experimental periodontitis + curcumin (C) (n = 10); and group 4, experimental periodontitis + resveratrol + curcumin (COMBI) (n = 10). Periodontitis was induced in rats by tying a silk suture, as a ligature, around one of the first molars. Daily administration of the placebo solution, 10 mg/kg of resveratrol, 100 mg/kg of curcumin or 10 mg/kg of resveratrol plus 100 mg/kg of curcumin was carried out from day 0 to day 30. At the end of the relevant experimental periods, rats were killed and the specimens obtained were processed for morphometric analysis of bone loss. Gingival tissues surrounding the first molar were collected for quantification of interleukin (IL)-1ß, IL-4, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) using a Luminex/MAGPIX assay. RESULTS: Intergroup comparisons of the morphometric outcomes revealed higher bone-loss values in the PL group (p < 0.05) when compared with RSV, C and COMBI groups. There was no difference in bone-loss values among RSV, C and COMBI groups (p > 0.05). The immunoenzymatic assay of the gingival tissue showed a lower concentration of IL-1ß in the COMBI group in comparison with the PL group (p < 0.05). Higher values of IL-4 were demonstrated in groups RSV, C and COMBI in comparison with the PL group (p < 0.05). Only RSV caused a reduction in the levels of IFN-γ (p < 0.05). There was no difference in the concentration of TNF-α amongst the four groups (p > 0.05). CONCLUSION: Resveratrol and curcumin are capable of reducing alveolar bone loss in an animal model of periodontitis. This occurred when these agents were added singly or in combination with one another, but there did not appear to be either synergistic or additive effects.

Unraveling the variability and genetic structure of barker frog Physalaemus cuvieri (Leiuperinae) populations from different regions of Brazil.

The barker frog Physalaemus cuvieri is widely distributed in South America and is found in all regions of Brazil. Significant intraspecific morphological variation in this species has been reported. To determine the genetic structure of the natural Brazilian populations of P. cuvieri, 10 different populations geographically separated by 99.41 to 2936.75 km were evaluated using 10 polymorphic microsatellite loci. In addition, mitochondrial DNA data were analyzed to determine genetic distance between the populations. The genetic variation was found to be significant in most of the populations (HE ranged from 0.40 to 0.59, and allelic richness ranged from 2.07 to 3.54). An FST value of 0.27 indicated that high genetic structure was present among the P. cuvieri populations. STRUCTURE analyses grouped the 10 populations into nine clusters and indicated that only two of the populations were not genetically differentiated. The genetic distance calculated from the mitochondrial DNA data showed values <0.03 for seven of the populations.

Perinatal periodontal disease reduces social behavior in male offspring.

OBJECTIVE: Our objective was to verify whether prenatal maternal periodontitis is a risk factor for the development of central nervous system disorders in rats. METHODS: Periodontitis was induced by placing a ligature around the upper and lower first molars in 9 female Wistar rats (experimental group); 9 rats were left unligated (control group). The maternal general activity in an open field was observed on gestational day (GD) 0, GD 4, and GD 14, and the maternal performance was assessed on the second day after birth. The pups' play behavior was assessed on postnatal day 30. The relative level of reelin was measured in the frontal cortex by real-time PCR analysis. RESULTS: The results showed that, compared with the control group, (1) the general activity in female rats with periodontitis was decreased, (2) the maternal performance of these rats was not modified by periodontitis, (3) the play behavior of pups from dams with periodontitis was decreased, and (4) there were no differences in the frontal cortex reelin levels of pups from dams with periodontitis. CONCLUSIONS: We conclude that pre- and postnatal periodontitis induces maternal sickness behavior and reduces the pups' play behavior without interference with frontal cortex reelin expression.

Satellite DNA derived from 5S rDNA in Physalaemus cuvieri (Anura, Leiuperidae).

In the present study, we describe for the first time a family of 190-bp satellite DNA related to 5S rDNA in anurans and the existence of 2 forms of 5S rDNA, type I (201 bp) and type II (690 bp). The sequences were obtained from genomic DNA of Physalaemus cuvieri from Palmeiras, State of Bahia, Brazil. Analysis of the nucleotide sequence revealed that the satellite DNA obtained by digestion with EcoRI, called PcP190EcoRI, is 70% similar to the coding region of type I 5S rDNA and 66% similar to the coding region of type II 5S rDNA. Membrane hybridization and PCR amplification of the sequence showed that PcP190EcoRI is tandemly repeated. The satellite DNA as well as type I and type II 5S rDNA were localized in P. cuvieri chromosomes by fluorescent in situ hybridization. The PcP190EcoRI sequence was found in the centromeres of chromosomes 1-5 and in the pericentromeric region of chromosome 3. Type I 5S rDNA was detected in chromosome 3, coincident with the site of PcP190EcoRI. Type II 5S rDNA was located interstitially in the long arm of chromosome 5. None of these sequences co-localized with nucleolar organizer regions. Our data suggests that this satellite DNA originates from the 5S ribosomal multigene family, probably by gene duplication, nucleotide divergence and sequence dispersion in the genome.

Study of closely related species within the Physalaemus cuvieri group (Anura): contribution of microsatellite markers.

Various species of the Physalaemus cuvieri group of frogs are difficult to distinguish morphologically, making molecular analysis an attractive alternative for indentifying members of this group, which is considered to be at risk because of loss of habitat. The genetic structure of natural populations of P. ephippifer and P. albonotatus species was investigated and analyzed, together with that of five previously studied populations of P. cuvieri. Nine microsatellite loci were used in the analyses. The overall G(ST) value (0.46) revealed high genetic variation among the populations, as expected for different species. Bayesian analysis implemented by the STRUCTURE software clustered the seven populations into seven groups (K = 7). All the P. albonotatus and P. ephippifer specimens were grouped into a single cluster, both species showing clear differentiation from P. cuvieri. The different grouping based on these microsatellites of some P. cuvieri individuals from Porto Nacional and from Passo Fundo suggests that they could be a new species, indicating a necessity for taxonomic reevaluation. Despite the intrinsic difficulties in analyzing closely related species, the nine microsatellite loci were found to be adequate for distinguishing these three species of the P. cuvieri group and their populations.

Could implant position influence the peri-implant parameters in edentulous mandibles of diabetics rehabilitated with overdentures? A split-mouth randomized study.

This randomized controlled trial assessed the impact of crestal level position of implants installed in type 2 diabetes mellitus (T2DM) patients rehabilitated with overdentures. Twenty-two mandibular edentulous T2DM patients were submitted to implant placement for retention of an overdenture. By means of a split-mouth design, two implants were installed: one at supracrestal level (SL) and one at crestal level (CL). Clinical, immunoenzymatic and tomographic analyses were performed at prosthesis placement (baseline) and after 6, 12 and 24 months following implant loading. Increased peri-implant probing depths were detected in CL implants when compared with SL implants at all time-points (baseline P=0.047; 6 months P=0.014; 12 months P=0.027; 24 months P=0.036). Indeed, augmented clinical attachment levels were also detected in CL implants when compared with SL implants at all time-points (baseline P=003; 6 months P=0.045; 12 months P=0.029; 24 months P=0.026). CL implants demonstrated increased amounts of interleukin-6 (IL-6) at 6 months (P=0.043) and higher IL-17 (P=0.021), IL-21 (P=0.034) and tumour necrosis factor alpha (TNF-α) concentrations (P=0.030) at 24 months in comparison with SL implants. CL group revealed enhanced bone loss from baseline to 6 (P=0.032), 12 (P=0.043) and 24 months (P=0.028) when compared with SL. In conclusion, this study showed that implants placed supracrestally in T2DM patients rehabilitated with overdentures demonstrated lower bone loss and better clinical parameters with beneficial modulation of peri-implant immunoinflammatory biomarkers when compared with implants positioned at crestal level.

Lumbar Myelopathy Caused by Multiple Cartilaginous Exostoses in a Dog.

A 6-months-old male mixed-breed dog was admitted to the veterinary hospital with lameness in the right pelvic limb, proprioceptive ataxia, and suspected spinal cord injury in the lumbosacral segment. Upon palpation, firm nodules were noted on the bony surface of the thoracic and pelvic limbs, ribs, vertebrae, and tail. The radiographic study showed nodules of low radiopacity with well-defined limits and smooth contours, one of which was overlapping the fifth lumbar vertebra. Given the clinical presentation indicative of spinal cord injury, the animal was subjected to hemilaminectomy for nodular resection. Histopathological examination of the biopsy enabled the diagnosis of osteochondroma. The clinical follow up after surgery showed improvement in walking, despite persistent monoparesis in the right pelvic limb. The radiographic study in association with clinical and histopathological exams allowed the diagnosis and monitoring of the lesions.

Cytogenetic contributions for the study of the Amazonian Engystomops (Anura; Leiuperidae) assessed in the light of phylogenetic relationships.

Genetic divergence and speciation mechanisms of the Amazonian Engystomops frog have been inferred mainly from mtDNA sequences, microsatellite and male advertisement call. Although many aspects of this divergence remain unclear, cytogenetic analyses of Amazonian Engystomops populations are described and are compared to the relationships inferred from mitochondrial and nuclear DNA sequence data. High cytogenetic variation distinguished karyotypic patterns among the populations, even between populations which had no prezygotic isolation previously inferred from mating call analysis. Interestingly, the Puyo and Acre populations, which are in different clades, showed heteromorphic sex chromosomes (XX/XY), not identified in the other Ecuatorian populations analyzed. The analysis of a specimen collected in a site near Yasuní (Ecuador) which was cytogenetically related to the specimens from Puyo (Ecuador), was also phylogenetically closely related these specimens. In the rhodopsin nucleotide sequences, six polymorphic sites were identified and various specimens were heterozygous for them. All the data presented herein, in conjunction with those previously reported, corroborate the hypothesis that Engystomops petersi is a complex of distinct species. It also indicates that karyotypic evolution patterns may have played a substantial role in the Engystomops speciation and the occurrence of sporadic mating events between divergent evolutive lineages is discussed.

Engineering the surface of prostate tumor cells and hyaluronan/chitosan multilayer films to modulate cell-substrate adhesion properties.

This paper explores different film assembly conditions of the polyelectrolyte solutions of hyaluronan (HA) and chitosan (CHI), as well as both substrate and cell surface modifications, to investigate PC3 cells adhesion properties. UV-Visible, AFM-IR and Zeta potential techniques indicate that the solution ionic strength is a relevant parameter to modulate the free carboxylic groups of HA on the film surface. In addition, capacitive coupling measurements suggest that assembly conditions that favor surface charge mobility inhibit cell adhesion due to polymer rearrangements that support non-specific electrostatic interactions of positively charged CHI residues and the negatively charged cell moieties, rather than specific CD44-hyaluronan interactions. Moreover, the PC3 cells treatment with hyaluronidase and anti-CD44 antibody also highlighted the importance of CD44 binding site availability on the tumor cell adhesion properties. Finally, the conjugation of wheat germ agglutinin on the film surface proved to be a suitable strategy to boost the PC3 cell adhesion properties. Our results reveal the remarkable capacity of HA/CHI films to modulate cell-substrate properties, which pave the road for the development of surfaces suitable for several applications based on biosensing.

Subgingival Host-Microbial Interactions in Hyperglycemic Individuals.

Type 2 diabetes mellitus (T2DM) is an established risk factor for periodontitis, yet its contribution to creating host-bacterial disequilibrium in the subgingival crevice is poorly understood. The present investigation aimed to quantify the impact of hyperglycemia on host-bacterial interactions in established periodontitis and to map shifts in these dynamics following mechanical nonsurgical therapy. Seventeen T2DM and 17 non-T2DM subjects with generalized severe chronic periodontitis were recruited along with 20 periodontally healthy individuals. Subjects with periodontitis were treated with scaling and root planing (SRP). Samples of subgingival biofilm and gingival crevicular fluid were collected at baseline and at 1-, 3-, and 6 mo postoperatively. Correlations were generated between 13.7 million 16S ribosomal DNA sequences and 8 immune mediators. Intermicrobial and host-microbial interactions were modeled using differential network analysis. Periodontal health was characterized by a sparse interbacterial and highly connected cytokine-bacterial network, while both normoglycemics and T2DM subjects with periodontitis demonstrated robust congeneric and intergeneric hubs but significantly fewer cytokine-bacterial connections. Following SRP, the cytokine-bacterial edges demonstrated a 2-fold increase 1 mo postoperatively and a 10-fold increase at 6 mo in normoglycemics. In hyperglycemics, there was a doubling at 1 mo but no further changes thereafter. These shifts accompanied an increasingly sparse interbacterial network. In normoglycemics, the nodes anchored by interleukin (IL)-4, IL-6, and IL-10 demonstrated greatest rewiring, while in hyperglycemics, IL-1ß, IL-6, INF-γ, and IL-17 exhibited progressive rewiring. Thus, the present investigation points to a breakdown in host-bacterial mutualism in periodontitis, with interbacterial interactions rather than host-bacterial interactions primarily determining community assembly. Hyperglycemia further exacerbates this uncoupled mutualism. Our data also demonstrate that while nonsurgical therapy might not consistently alter microbial abundances or lower proinflammatory molecules, it "reboots" the interaction between the immunoinflammatory system and the newly colonizing microbiome, restoring a role for the immune system in determining bacterial colonization. However, this outcome is lower and delayed in hyperglycemics.

NOR dispersion, telomeric sequence detection in centromeric regions and meiotic multivalent configurations in species of the Aplastodiscus albofrenatus group (Anura, Hylidae).

In the present study, we examined the chromosome number and morphology, heterochromatin pattern, and location of the nucleolar organizer region (NOR) and telomeric sequences in Aplastodiscus albofrenatus, A. arildae, A. ehrhardti and A. eugenioi in order to cytogenetically characterize the Aplastodiscus albofrenatus group. The 4 species analyzed in this study had diploid numbers of 2n = 22 and very similar chromosome morphology. These species could be differentiated based on the distribution and amount of heterochromatin and the location of the nucleolar organizer region (NOR). Six of the 8 A. albofrenatus individuals had an NOR polymorphism previously unknown in anurans since only one of the homologs of pairs 1 and 7 was stained. In the other 2 specimens, the NOR occurred on both homologs of pair 7. In A. ehrhardti, pairs 6 and 10 were stained by the AgNOR technique, but only pair 6 was confirmed by in situ hybridization. The NOR was located on pair 10 in A. arildae and on pair 7 in A. eugenioi. In A. albofrenatus and A. arildae, multivalent rings involving NOR-containing chromosomes were observed during prophase I of meiosis. The telomeric probe identified the telomeres in all species and also centromeric regions in the chromosomes of A. albofrenatus and A. arildae, which were coincident with centromeric heterochromatin. The conserved chromosomal morphology seen mainly in the first 7 pairs among species of the A. albofrenatus group suggests that all of these species probably originated from a common ancestral karyotype and that chromosomal rearrangements resulted in karyotype differentiation, with changes in NOR location, as well as telomeric and heterochromatin dispersal.

Comparative analysis of the chromosomal positions of rDNA genes in species of the tripunctata radiation of Drosophila.

The location of rDNA on chromosomes has been found to be highly variable in different groups of insect families of various orders. Yet, in other insect families the rDNA position is relatively constant. This contrast so far has received limited attention. We investigated the rDNA position on mitotic chromosomes in 18 species of Drosophila, 16 of which are from the tripunctata radiation, subgenus Drosophila, through fluorescent in situ hybridization (FISH). All species showed fluorescent signals only on the sex chromosomes. On the X chromosome a single fluorescent mark, but with variable locations, was found. On the Y, we observed variation both in location and in number of fluorescent marks (from 1 to 5). This constancy of chromosome location, in contrast to the great variability found in other groups, is consistent with the work carried out in other species of Drosophila. This suggests that the presence of a nucleolus organizer region (NOR) on each sex chromosome is probably an ancestral condition in the genus. Moreover, this difference in the variation of rDNA position among groups points out an interesting evolution question, which deserves further study.

Comparison of the spermatozoan morphology of Isognomon bicolor and Isognomon alatus (Mollusca, Bivalvia, Isognomonidae).

In this study we used transmission and scanning electron microscopy to examine the spermatozoan structure of Isognomon bicolor and Isognomon alatus. The spermatozoa of both species were of the primitive or ect-aquasperm type. The acrosomal morphologies were essentially similar but the top of the acrosomal vesicle in I. bicolor sperm had a slightly flattened edge whereas the apex of the acrosomal vesicle of I. alatus sperm had a rounded outline. This difference suggested that acrosomal morphology could be an important character for taxonomic differentiation. In the present work, the results demonstrated that the gamete ultrastructure of the two distinct species I. alatus, from Panama, and I. bicolor, from the southeastern region of Brazil, were similar to the other studied species of the superfamily Pterioidea.

Cytogenetic comparison of tree frogs of the genus Aplastodiscus and the Hypsiboas faber group (Anura, Hylidae).

Four species of Aplastodiscus and two species of Hypsiboas were cytogenetically compared. Aplastodiscus perviridis, A. cochranae, H. albomarginaus, and H. faber had 2n = 24 chromosomes, while A. albosignatus and A. leucopygius had 2n = 20 and 2n = 18 chromosomes, respectively. Aplastodiscus perviridis and A. cochranae had identical karyotypes, as indicated by their chromosomal morphology, the location of the nucleolus organizer region (NOR) on chromosome pair 12, and the heterochromatin pattern. The NOR-bearing chromosomes of A. albosignatus and A. leucopygius (pair 9) were very similar in size and morphology (metacentric) when compared to A. perviridis and A. cochranae (pair 12) and to H. faber (pair 11); the NOR of these chromosomes also occurred in the same region, suggesting that these chromosomes are homologous. Although H. albomarginatus differs from the other species with regard to the location of its NOR on pair 2, this species had the same diploid number and a chromosomal morphology similar to that of A. perviridis and A. cochranae. Chromosomal differentiation among the species appears to have occurred by reduction in chromosome number.

Bilateral Primary Obstructive Giant Megaureter in an Adult Dog.

A rare case of bilateral, primary, obstructive, giant megaureter was found during necropsy examination of an 11-year-old female German shepherd dog. On ultrasound examination and at necropsy examination, both ureters were tortuous and extensively dilated with diameter ranging from 1.86 to 4.8 cm. Both vesicoureteral junctions were obstructed by uroliths. A diagnosis of giant megaureter was established using human parameters since these values are not recognized in animals. The classification of obstructive and primary megaureter was determined because the obstruction was due to uroliths at the vesicoureteral junctions.

The impact of implant abutment surface treatment with TiO2 on peri-implant levels of angiogenesis and bone-related markers: a randomized clinical trial.

The goal of this randomized, blinded, split-mouth controlled clinical trial was to assess the influence of abutment surface treatment on tissue healing. Fifteen patients received two implants distributed randomly to two groups: test (TiO2 abutment surface), control (standard abutment surface). Levels of epidermal growth factor (EGF), bone morphogenetic protein 9 (BMP-9), endothelin 1 (ET-1), fibroblast growth factor (FGF), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) were quantified in the peri-implant fluid after 3, 14, 30, and 60 days. Inter-group comparisons indicated higher levels of EGF, BMP-9, ET-1, FGF, and PlGF in the test group after 30days (P<0.05). PlGF levels were also higher in the test group after 60 days. In the test group, intra-group analysis revealed different levels of ET-1 and FGF between days 3 and 30, and days 3 and 60 (P<0.05); furthermore, VEGF levels were significantly higher on day 60 than on day 3 (P <0.05). In the control group, intra-group analysis demonstrated significantly different levels of ET-1, FGF, and PlGF between days 3 and 60 and of PlGF between days 14 and 60 (P<0.05). In conclusion, abutment surfaces treated with TiO2 influenced the levels of angiogenesis and bone-related markers.

Helmet retention system types as a risk factor to properly attach the chin strap.

OBJECTIVES: This study aimed to determine the factors associated with the incorrect use of a helmet retention system (loose or open) and how often this happens. METHODS: This was an observational transversal study conducted in Curitiba, Brazil. Trained observers positioned at traffic lights collected information about the helmet fixation mode, the helmet model (full-face, open-face, modular, half), and the helmet retention system model (micrometric, double-D, fast-release). Additional data including position on the motorcycle, gender, and function of the motorcycle (as a work vehicle) were collected. The observers, collection site, and periods were randomly selected by lots. RESULTS: From a total of 3,050 motorcyclists, 1,807 (59.2%) had their helmets fastened correctly, 907 (29.7%) had the retention system fastened loosely, and in 336 (11.0%), the retention system was completely open. Increased odds of incorrect use were observed for the fast-release and double-D buckles compared to the micrometrics buckles, with a fixed odds ratio (OR) of 4.62 (95% confidence interval [CI], 3.89-5.51) and 3.54 (95% CI, 2.46-5.09), respectively (P <.0001). Full-face helmets had a higher chance of incorrect use (P <.0001), and passengers had a higher incidence of incorrect use of the helmet than drivers (P <.0001). CONCLUSION: An important risk factor related to the incorrect use of the helmet was the type of retention system. The helmet model and being a passenger had a secondary influence on incorrect use of helmets.

The impact of different torques for the insertion of immediately loaded implants on the peri-implant levels of angiogenesis- and bone-related markers.

The aim of this split-mouth, randomized, double-blind, controlled clinical trial was to evaluate the influence of different insertion torque values for dental implants on bone- and angiogenesis-related marker profiles. Eighteen edentulous patients received dental implants and fixed complete-arch mandibular prostheses. The implants (n=36) were assigned randomly to two groups: reduced torque (n=18), with insertion torque <30Ncm; and conventional torque (n=18), with insertion torque ≥30Ncm. Levels of vascular endothelial growth factor (VEGF), placental growth factor, bone morphogenetic protein 9 (BMP-9), periostin, osteoprotegerin (OPG), and tartrate-resistant acid phosphatase (TRAP) in the peri-implant fluid were quantified at 7, 14, 30, and 120days after implant placement. Inter-group comparisons showed that VEGF and OPG levels were higher in the low-level torque group than in the conventional torque group on days 7 and 30, respectively (P<0.05). BMP-9 and periostin levels were higher in the conventional group than in the low-level torque group on day 120, and TRAP was up-regulated around implants inserted with conventional torque when compared to those inserted with lower-level torque at all time points evaluated (P<0.05). In conclusion, the use of different levels of torque for implantation of immediately loaded implants significantly influenced the levels of bone- and angiogenesis-related markers during early peri-implant repair.