Interleukin-1 receptor antagonist mediates toll-like receptor 3-induced inhibition of trophoblast adhesion to endometrial cells in vitro.

STUDY QUESTION: Is interleukin-1 receptor antagonist (IL-1RA) involved in the toll-like receptor 3 (TLR 3)-induced inhibition of trophoblast cells' adhesion to endometrial cells in vitro? SUMMARY ANSWER: IL-1RA mediates the TLR 3-induced inhibition of trophoblast cells' adhesion to endometrial cells in vitro. WHAT IS KNOWN ALREADY: It is well documented that endometrial TLR 3 activation leads to impairment of trophoblast binding to endometrial cells in vitro. IL-1RA is known as an anti-implantation factor, as its injection significantly reduced implantation rates in mice by an effect on endometrial receptivity. STUDY DESIGN, SIZE, DURATION: Poly I:C was used as a TLR3 specific ligand and endometrial cells were either treated or not with Poly I:C (treated versus control) in vitro. IL-1RA was applied to block IL-1 signal transduction. IL-1RA was knocked down by Accell Human IL1RN siRNA. Flagellin was used to stimulate TLR 5. SP600125 (JNK) was applied to inhibit the mitogen-activated protein kinases (MAPK) pathway. BAY11 -7082 was used to inhibit the nuclear factor-κB (NF-κB) pathway. The experiments were performed in three replicates on three separate days. PARTICIPANTS/MATERIALS, SETTING, METHODS: An in vitro assay was developed using RL95-2 (an endometrial cell line) and JAr (a trophoblast cell line) cells. Initially, the production of IL-1RA in RL95-2 cells in response to TLR 3 activation was measured. To determine whether the TLR 3-induced inhibition of trophoblast binding was mediated through IL-1RA: (i) we evaluated the effect of IL-1RA on the attachment of trophoblast cells to endometrial cells; (ii) we knocked down TLR3-induced IL-1RA gene expression by IL-1RA Small interfering RNA (siRNA) and evaluated trophoblast attachment to endometrial cells. Finally, to clarify through which pathway TLR 3-induced inhibition of trophoblast binding occurs: (i) activation of NF-κB and MAPK was detected by transfecting the cells with secreted placental alkaline phosphatase reporter plasmids bearing promoter sequences for each transcription factor; (ii) the inhibitors for NF-κB and MAPK were used to block signaling; (iii) it was then investigated whether addition of these inhibitors could restore the TLR 3-induced impairment of trophoblast attachment to the endometrial cells. MAIN RESULTS AND THE ROLE OF CHANCE: Our results showed that addition of polyinosinic:polycytidylic acid (Poly I:C) to RL95-2 cells significantly increased the production of IL-1RA (P < 0.05). Addition of human recombinant IL-1RA to RL95-2 cells remarkably decreased the adhesion rate of trophoblast cells to endometrial cells (P < 0.05). In addition, suppression of TLR3-induced IL-1RA gene expression in RL95-2 cells significantly restored trophoblast cells attachment to endometrial cells in the presence of Poly I:C (P < 0.05). Only TLR3 and not TLR5 induced MAPK activation (P < 0.05). TLR3 ligation did not affect NF-κB activation. Of NF-kB and MAPK inhibitors, only MAPK's inhibitor could achieve restoration of spheroid adhesion to endometrial cells (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study has been only done in vitro. Future in vivo studies will confirm our data. WIDER IMPLICATIONS OF THE FINDINGS: The findings of this study have a potential clinical application in introducing IL-1RA as one of the diagnostic infertility markers in the endometrium, which can affect the process of embryo adhesion at the time of implantation. Moreover, based on the novel data obtained in the current study, blocking and regulating the MAPK pathway by its inhibitors can be used as a new strategy to prevent and treat virus-induced infertility cases in ART techniques. STUDY FUNDING/COMPETING INTEREST: This study was partially funded by a Marie Curie IIF-253948 grant to I.C. and was partially funded by the author's institutions. The authors have no conflict of interest to declare.

Activation of Toll-like receptor 3 reduces actin polymerization and adhesion molecule expression in endometrial cells, a potential mechanism for viral-induced implantation failure.

STUDY QUESTION: Does activation of endometrial Toll-like receptor 3 (TLR 3) affect cell receptivity to trophoblast adhesion? SUMMARY ANSWER: TLR 3 activation in vitro reduces the attachment of trophoblast cells to endometrial cells by altering the cell cytoskeleton and reducing the expression of adhesion molecules in human endometrial cells. WHAT IS KNOWN ALREADY: It is well documented that the presence of an infection at the time of implantation can lead to implantation failure. The female reproductive tract recognizes invading micro-organisms through the innate pathogen recognition receptors such as the TLRs. STUDY DESIGN, SIZE, DURATION: Poly I:C was used as a TLR 3-specific ligand and endometrial cells were either treated or not with Poly I:C (treated versus control) in vitro. The experiments were performed in three replicates on three separate days. PARTICIPANTS/MATERIALS, SETTING, METHODS: An in vitro assay was developed using RL95-2 (a human endometrial cell line) and JAr (a human trophoblast cell line) cells. Initially, the percentage of attached JAr spheroids to RL95-2 was measured in response to TLR 3 activation. Next, actin polymerization in RL95-2 cells was assessed in response to TLR 2/6, 3 and 5 activation. Phalloidin was used to assess the mean fluorescence intensity of F-actin by flow cytometry or confocal microscopy. Secondly, the influence of TLR 2/6, 3 and 5 activation on the expression of cluster of differentiation 98 (CD98) and ß3 integrin was determined. To further understand through which pathways the TLR 3-induced alterations occur, inhibitors were applied for Toll/interleukin-1 receptor domain-containing adaptor inducing interferon-beta (TRIF), myeloid differentiation primary response 88 (MYD88), mitogen-activated protein kinases (MAPK) and nuclear factor pathways. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that stimulation of TLR 3 in endometrial cells with different concentrations of Poly I:C led to a reduction in the percentage of trophoblasts attached to the endometrial cells in a dose-dependent manner (P < 0.05). This decrease was consistent in the Poly I:C treated group regardless of the co-incubation time (P < 0.05). In addition, our results demonstrated that actin polymerization and CD98 expression significantly decreased only in response to TLR 3 activation (P < 0.05). Activation of endometrial cells with TLR 2/6, 3 and 5 significantly reduced ß3 integrin expression (P < 0.05). These alterations were shown to work via MYD88-MAPK pathways (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: This study has been performed in vitro. Future in vivo studies will be required in order to confirm our data. WIDER IMPLICATIONS OF THE FINDINGS: This is a novel discovery which extends our current knowledge concerning diagnosis and treatment of viral-induced infertility cases. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the COST Action FA1201 (GEMINI) by granting a Short Term Scientific Mission and the Instituto de Salud Carlos III by granting Grant PI11/01645. The authors have no conflict of interest to declare.

Severe rigid hip flexion-abduction contracture in cerebral palsy: a case report and review of the literature.

INTRODUCTION: severe, rigid hip abduction deformity in individuals with cerebral palsy (CP) is an exceptionally uncommon condition. This posture hinders the positioning in the wheelchair and the completion of basic activities of daily living (ADL). Addressing such severe deformities can be quite challenging. MATERIAL AND METHODS: a 14-year-old male, with spastic-dystonic quadriplegic CP, developed rigid and severe flexion-abduction contractures in both hips, characterized by 90 degrees of flexion and 100 degrees of abduction. These contractures severely impeded his ability to comfortably use a wheelchair and even pass through doorways. Performing basic ADLs became a significant challenge for both the patient and his caregivers. RESULTS: the treatment approach involved a two-stage surgical procedure, one for each hip, with a two-month interval between them. An extensive release of the fascia latae, gluteus maximus, external rotators, and hip flexors; in combination with a proximal femur osteotomy were performed. To maintain the corrections achieved, long-leg casts connected with two bars were employed, followed by orthotic support and physiotherapy. Following the procedure, lower limb adduction was achieved, and the patient and caregivers were highly satisfied, as ADLs and basic caregiving had been greatly facilitated. CONCLUSIONS: while the available literature on the management of severe rigid abduction hip contractures in non-ambulatory CP patients is limited, and treatment options are often complex, the present case underscores the effectiveness of a comprehensive approach involving soft tissue release and bone surgery. Achieving a more favorable wheelchair positioning and facilitating basic ADLs and care represents a significant success for patients and families.

INTRODUCCIÓN: la deformidad severa y rígida en abducción de cadera en individuos con parálisis cerebral (PC) es una condición infrecuente. Esta postura dificulta el posicionamiento en la silla de ruedas y la realización de actividades básicas de la vida diaria (AVD). El tratamiento de estas deformidades tan severas puede ser todo un reto. MATERIAL Y MÉTODOS: varón de 14 años, con PC tetrapléjica espástica-distónica, que desarrolló contracturas rígidas y severas de flexión-abducción en ambas caderas, caracterizadas por 90 grados de flexión y 100 grados de abducción. Estas contracturas impedían gravemente su capacidad para utilizar cómodamente una silla de ruedas e incluso pasar por las puertas. La realización de actividades básicas de la vida diaria se convirtió en un reto importante tanto para el paciente como para sus cuidadores. RESULTADOS: el tratamiento consistió en una intervención quirúrgica en dos fases, una para cada cadera, con un intervalo de dos meses entre ellas. Se realizó una amplia liberación de la fascia lata, el glúteo mayor, los rotadores externos y los flexores de la cadera; en combinación con una osteotomía proximal del fémur. Para mantener las correcciones conseguidas, se emplearon escayolas de pierna larga conectadas con dos barras, seguidas de soporte ortésico y fisioterapia. Tras la intervención, se consiguió la aducción de los miembros inferiores y el paciente y los cuidadores se mostraron muy satisfechos, ya que se habían facilitado en gran medida las AVD y los cuidados básicos. CONCLUSIONES: aunque la bibliografía disponible sobre el tratamiento de las contracturas rígidas graves de la cadera en abducción en pacientes no deambulantes con PC es limitada, y las opciones de tratamiento suelen ser complejas, el presente caso subraya la eficacia de un enfoque integral que incluye la liberación de los tejidos blandos y la cirugía ósea. Conseguir una posición más favorable en la silla de ruedas y facilitar las AVD básicas y los cuidados representa un éxito significativo para los pacientes y sus familias.

Analysis of the surgical activity in the Dermatology Department of Fuenlabrada University Hospital, Madrid, Spain, between 2005 and 2010: determination of the standard operating times.

OBJECTIVES: To analyze the characteristics of the dermatologic surgery lists and to compare different indicators of surgical productivity. MATERIALS AND METHODS: This was a retrospective, descriptive study of the routine surgical activity undertaken in the Dermatology Department of Fuenlabrada University Hospital, Madrid, Spain, between January 2005 and December 2010. Data about the procedures performed and about the surgical lists were analyzed and an analysis was made of operating room occupancy. Surgical productivity was analyzed using standard operating times (SOTs) and surgical scheduling adequacy values (SA1 and SA2). Variables were recorded as relative frequencies. Covariance and the Pearson linear correlation index were used for comparisons. RESULTS: The study period included a total of 11,481 surgical sessions, of which 71% were minor surgery and the remainder were major outpatient surgery. A mean of 9.7 operations were performed in each session and the overall operating room occupancy was 71.9%. Four SOT descriptors (minor surgery, multiple minor skin lesions, nail surgery, and malignant tumors with primary closure) were sufficient to classify 86.7% of the operations performed. The operating room down-time was 0.15 SOT (9minutes) in 91.3% of operations. The mean SA1 index was 1.20. Overall surgical productivity measured using the SA2 index was 96.46%. The Pearson correlation showed a statistically significant relationship between the increase in the number of patients per surgical session, the reduction in operating room occupancy, and the increase in SA2 operating room productivity. CONCLUSIONS: The application of SOT descriptors enables us to quantify the complexity of the operations included in the surgical waiting list and to obtain indicators for the evaluation of surgical activity, improving efficiency in surgical time management.

Analysis of operating room activities in the dermatology department at Hospital Universitario de Fuenlabrada (2005-2010).

OBJECTIVE: To analyze data corresponding to patients who underwent dermatological surgery in an operating room. MATERIAL AND METHODS: This was a descriptive, retrospective study of operating room activities in the dermatology department of Hospital Universitario de Fuenlabrada in Madrid between January 2005 and December 2010. We analyzed the relative frequency of a range of patient and procedure-related variables, as well as substitution and cancellation rates, the proportional risk of complications, and operating room efficiency. RESULTS: In the period analyzed, 11,516 patients underwent surgery: 9351 required minor surgery, 1998 major ambulatory surgery, and 167 surgery requiring hospitalization. Simple excision was the most common procedure (64.7%), and in the majority of cases (85%), the condition was benign. The mean number of patients treated per day was 9.7, and mean operating room efficiency was 71.9%. CONCLUSIONS: Accurate record-keeping is essential for analyzing operating room activities and comparing results with those from other centers. The analysis of patterns over time shows the effect of changes made on different indicators. In our case, a decrease in operating room efficiency was seen with an increase in the number of patients per day undergoing surgery.

Influence of TiO2 nanoparticles content as reinforce material to enhance the mechanical and corrosion resistance properties of Sn and Sn-Ag alloy for dental applications.

This study evaluated the influence of the TiO2 nanoparticles (NPs) on the mechanical and chemical performance of Sn and Sn-Ag alloys. The XRD (X-ray diffraction) and HR-TEM (high resolution-transmission electron microscopy) methods were used to characterize the NPs synthesized by the sol-gel microwave process. The chemical composition of the alloys was Sn, Sn+3TiO2 NPs, Sn-5Ag+1.5TiO2 NPs, Sn-10Ag, and Sn-10Ag+3TiO2 NPs, obtained from an experimental factorial design (EFD). A statistical model was used to determine the mechanical and chemical properties, showing the Vickers hardness response surface, tensile strength, wear, and corrosion resistance. The wear and corrosion tests for the various alloy compositions were performed using human artificial saliva solution. The results indicated that the Sn-10Ag+3TiO2 NPs exhibited the highest mechanical performance due to their increased hardness (380 HV), tensile strength (370 N), and wear resistance (0.34 × 10-3 mm3 Nm-1); in all the cases, the inclusion of TiO2 NPs enhanced the corrosion resistance of the alloys. According to the American Dental Association (ADA), Sn-10Ag+3TiO2 NPs alloy could be classified as a possible type IV restorative material.

Soil seed bank recovery occurs more rapidly than expected in semi-arid Mediterranean gypsum vegetation.

BACKGROUND AND AIMS: Seed banks are critical in arid ecosystems and ensure the persistence of species. Despite the importance of seed banks, knowledge about their formation and the extent to which a seed bank can recover after severe perturbation remains scarce. If undisturbed, soil seed banks reflect a long vegetation history; therefore, we would expect that new soil seed banks and those of undisturbed soils require long periods to become similar with respect to both density and composition. In contrast, if soil seed banks are only a short- to mid-term reservoir in which long-term accumulation constitutes only a tiny fraction, they will recover rapidly from the vegetation. To shed light on this question, we evaluated seed bank formation in a semi-arid gypsum community. METHODS: Soils from 300 plots were replaced with sterilized soil in an undisturbed semi-arid Mediterranean community. Seasonal changes in seed bank density and composition were monitored for 3 years by comparing paired sterilized and control soil samples at each plot. KEY RESULTS: Differences in seed bank density between sterilized and control soil disappeared after 18 months. The composition of sterilized seed banks was correlated with that of the control plots from the first sampling date, and both were highly correlated with vegetation. Nearly 24 % of the seed bank density could be attributed to secondary dispersal. Most seeds died before emergence (66·41-71·33 %), whereas the rest either emerged (14·08-15·48 %) or persisted in the soil (14·59-18·11 %). CONCLUSIONS: Seed banks can recover very rapidly even under the limiting and stressful conditions of semi-arid environments. This recovery is based mainly on the seed rain at small scales together with secondary dispersal from intact seed banks in the vicinity. These results emphasize the relevance of processes occurring on short spatial scales in determining community structure.

The effect of glycerol concentrations on the post-thaw in vitro characteristics of cryopreserved sex-sorted boar spermatozoa.

The objective of this study was to optimize protocols for the cryopreservation of sex-sorted boar spermatozoa. In the experiment 1, we evaluated the effects of a standard boar sperm cryopreservation procedure (3% final glycerol concentration) on the in vitro characteristics of sex-sorted sperm frozen at low sperm concentrations (20 × 10(6) sperm/ml; S20 group). Non-sorted spermatozoa frozen at 1000 × 10(6) (C1000 group) and 20 × 10(6) (C20 group) sperm/ml were used as the freezing control groups. In experiment 2, the effects of different final glycerol concentrations (0.16%, 0.5%, 1.0%, 2.0% and 3.0%) on post-thaw quality of the S20 and C20 groups were evaluated. In both experiments, the samples were evaluated prior to freezing (5°C) and at 30, 90 and 150 min after thawing. Experiment 1 indicated that freezing sperm at low concentrations decreased (p < 0.05) the total motility (TM) and progressive motility (PM) at 90 and 150 min after thawing regardless of whether the sperm were sorted or not. However, the sperm membrane integrity was not affected at any evaluation step. Inexperiment 2, significant effects on the TM and PM because of increased glycerol concentrations in the S20 and C20 groups were observed only at 90 and 150 min after thawing. The samples frozen in 3% glycerol showed lower (p < 0.05) TM and PM values when compared to those frozen in the presence of 0.5% and 1% glycerol. In both experiments, non-sorted control samples displayed higher percentages of spermatozoa with damaged DNA than sorted spermatozoa. In conclusion, the optimization of cryopreservation conditions by decreasing the glycerol concentrations can improve post-thaw motility of sex-sorted spermatozoa frozen at low concentrations.

Seminal plasma proteins as modulators of the sperm function and their application in sperm biotechnologies.

Seminal plasma (SP) is known to play an important role in mammalian fertilization. However, the variability found in its composition among species, males and even fractions of the same ejaculate has made difficult to completely understand its effect in sperm function. Proteins are one of the major SP components that modulate sperm functionality. During the last years, intensive work has been performed to characterize the role of these proteins. They have been found to influence sperm capacitation, formation of the oviductal sperm reservoir and sperm-oocyte interaction. Sperm biotechnologies, such as sperm cryopreservation and flow cytometric sex-sorting, that involve a substantial dilution of the SP are detrimental to sperm quality. Attempts to improve the outcome of these biotechnologies include the restoration of SP, which has produced contradictory results. To overcome this variability, different research groups have proposed the application of isolated SP proteins. Herein, we will review the current knowledge in the role of the major SP proteins as modulators of sperm functionality. Furthermore, we will discuss the possible applications of the SP proteins in sperm cryopreservation and flow cytometric sex-sorting.

Unmanned aerial vehicles (UAVs) as a tool for hazard assessment: The 2021 eruption of Cumbre Vieja volcano, La Palma Island (Spain).

Monitoring for assessment of natural disasters, such as volcanic eruptions, presents a methodological challenge for the scientific community. Here, we present Unmanned Aerial Vehicles (UAVs) as a feasible, precise, rapid and safe tool for real time monitoring of the impacts of a volcanic event during the Cumbre Vieja eruption on La Palma Island, Spain (2021). UAV surveys with optical RGB (Red-Green-Blue), thermal and multispectral sensors, and a water sampling device, were carried out in different areas affected by the lava flow, including the upper volcanic edifice and the lava delta formed on the coastal fringe of the island. Our results have provided useful information for the monitoring of the advance of the lava flow and its environmental consequences during the volcanic emergency. Our data shows how La Palma island's growth, with the formation of a new lava delta of 28 ha and a total volume of lava injected into the sea of 5,138,852 m3. Moreover, our Digital Elevation Model (DEM) simulated, with a 70 % accuracy, the probabilistic simulation of the possible path followed by the lava flow in the vicinity of the fissure from which the magma emanates. In addition, significant changes of seawater physical-chemical parameters were registered in coastal surface waters by the in situ seawater samples collected with the automatic water sampling device of our UAV. The first meters of the water column, due to the instant evaporation of the seawater in contact with the hot lava, produce an increase of temperature and salinity of up to 4-5 °C and up to 5 units, respectively.

Intramuscular psoas lengthening at the pelvic brim plus proximal lengthening of the rectus femoris as a treatment for fixed knee flexion deformity in cerebral palsy. / Tenotomía intrapélvica del psoas y alargamiento proximal del recto anterior como tratamiento del flexo fijo de rodilla en parálisis cerebral infantil.

Although the correction of knee flexion by lengthening the hamstring musculature is traditionally contemplated in cerebral palsy, literature suggests that treatment of hip flexion also improves knee extension. The aim of the study was to first show the efficacy of the sequence of intrapelvic tenotomy of the psoas followed by intramuscular lengthening of the proximal rectus anterior and, later, that of both surgical soft tissue surgeries separately. For this, a prospective study was carried out in 10 patients with a mean age of 14 years, which presented 16 fixed knee flexes with a mean of deformity of 22°. The data was analyzed through means of an ANOVA of repeated measures and to determine the effect separately of each one of the techniques, the improvements obtained with respect to the previous level were contrasted. The mean extension achieved was 12°, 7° corresponding to the intrapelvic tenotomy of the psoas and, on the remaining knee flexion, a correction of 5° after intramuscular lengthening of the anterior rectus at the proximal level. Both the sequence of proposed gestures and those that form separately, showed statistically significant differences (P <0.001) in the correction of the deformity. In conclusion, the proposed techniques applied sequentially or separately are effective in reducing knee flexion in predominantly spastic cerebral palsy, facilitating or even being able to avoid the treatment that is directly needed.

Incidence of venous thromboembolism in patients with colorectal cancer according to oncogenic status.

BACKGROUND: There are conflicting data regarding the role of KRAS mutation on the risk of venous thromboembolism (VTE) in colorectal cancer (CRC) patients. Moreover, the role of other biomarkers such as NRAS or BRAF has not been studied. PURPOSE: To analyze the incidence of VTE in a cohort of patients with CRC based on KRAS, NRAS, and BRAF status. METHODS: We performed a retrospective review of patients with unresectable locally advanced and metastatic CRC (mCRC) and known KRAS/NRAS/BRAF status, attended in the Medical Oncology Department of the Hospital General Universitario Gregorio Marañón (Madrid, Spain). The primary outcome was VTE defined as any venous thromboembolic event that occurred either 6 months before or at any time after the diagnosis of CRC. The biomarker status (KRAS, NRAS, and BRAF) and other predictors of thrombosis were collected. RESULTS: One hundred and ninety-four patients were identified and included in the analysis. Forty-one patients (21.1%) experienced VTE. The incidence was 19.1% in RAS-mutated patients, 28.6% in BRAF-mutated patients and 21% in triple wild-type patients (p = NS). In multivariate analysis, ECOG ≥ 2 was the only independent predictor of VTE (OR 8.73; CI 95% 1.32-57.82; p = 0.025). CONCLUSIONS: In our study, biomarkers have not been associated with an increased risk of VTE in CRC patients. A high incidence of VTE in BRAF-mutated patients has been observed and should be explored in further studies.

[Evaluation of function and limiting factors of gait disorder treatment in cerebral palsy: development of the Walking Abilities Levels Classification System]. / Evaluación funcional y de factores limitantes del tratamiento de los trastornos de la marcha en la parálisis cerebral infantil: desarrollo del sistema de clasificación de niveles de deambulación funcional.

INTRODUCTION: The Gross Motor Function Classification System has allowed us to stratificate cerebral palsy patients, according to their walking abilities. The lack of sensitivity about detecting changes and the absence of a global patient evaluation, justify the search of new pre-operative evaluation tools. AIMS: To present the Walking Abilities Levels Classification System (WALCS) and to show the first inter-observer agreement study that has been carried out. This system uses first a different pattern for ordering gait functional skills, and after that, evaluates the reversibility of the contextual factors that may limit the result of a gait disorder treatment. SUBJECTS AND METHODS: A new evaluation frame was built by an interdisciplinary team with an average professional experience of more than 15 years, initially focused as part of the pre-surgical patient evaluation. An inter-observer agreement study was held to gain the first insight of it. 14 participants studied the medical reports and gait lab video images of 10 cases. RESULTS: The kappa index was 0.76 for the walking ability level, 0.79 for the biological type, 0.69 psychological type and 0.64 social type of limiting factors. CONCLUSIONS: The WALCS offers a new evaluation frame gathering patient walking skills and limiting factors treatment. The initial inter-observer agreement rate endorsed more intra- and inter-studies in order to achieve a more robust validation.

TITLE: Evaluación funcional y de factores limitantes del tratamiento de los trastornos de la marcha en la parálisis cerebral infantil: desarrollo del sistema de clasificación de niveles de deambulación funcional.Introducción. El Gross Motor Function Classification System ha permitido estratificar, según su habilidad para caminar, a los pacientes que padecen parálisis cerebral infantil. La falta de sensibilidad en la detección de cambios y la ausencia de una evaluación del paciente en el contexto en el que se encuentra justifican la búsqueda de alternativas de evaluación pretratamiento. Objetivos. Presentar y mostrar la concordancia interobservador inicial del sistema de clasificación de niveles de deambulación funcional. Con él se evalúa la destreza para caminar y la necesidad de asistencia para realizar transferencias desde la silla de ruedas, y, posteriormente se analiza el escenario que la salud y el entorno del paciente ofrecen como condicionantes en la corrección de la marcha o la bipedestación asistida. Sujetos y métodos. Se describe un nuevo marco de evaluación, elaborado por un grupo interdisciplinar con más de 15 años de experiencia media, enfocado inicialmente a la toma de decisiones antes de un tratamiento quirúrgico. Como control interno, 14 participantes evaluaron la historia clínica y los vídeos de marcha de 10 casos. Resultados. Se alcanzó un índice kappa de acuerdo de 0,76 en niveles funcionales y de 0,79 en el tipo de escenario biológico, de 0,69 en el psicológico y de 0,64 en el social. Conclusiones. El sistema de clasificación de niveles de deambulación funcional ofrece un marco para la evaluación conjunta de la deambulación y de los factores limitantes en la eficacia de un tratamiento. La concordancia interobservador avala iniciar su validación.

Evaluation of the Serratia marcescens nuclease (NucA) as a transgenic cell ablation system in porcine.

The efficiency of the Serratia marcescens nuclease encoded by the NucA gene, with or without a nuclear localization signal (NLS), and the commonly used diphtheria toxin A (DTA) were compared for their ability to ablate cells in culture. Constructs containing the test genes driven by the beta-actin promoter coupled with enhancer elements from the cytomegalovirus promoter and rabbit beta-globin gene (pCAG) and the blasticidin resistance gene driven by the phosphoglycerate kinase (PGK) promoter were generated and electroporated into porcine fetal fibroblasts. Three independent replicates were completed. Following blasticidin selection, the number of surviving colonies was counted to assess the efficiency of the toxic gene. Both NucA and DTA proved to be effective in killing porcine fibroblasts compared to controls. However, the efficiency of cell ablation was significantly higher with DTA than with NucA or NucANLS (p < 0.05). Gene expression analysis of surviving colonies indicated that survival is related to low or absent expression of the toxic genes. These results indicate that the NucA gene, while capable of mammalian cell ablation, is less efficient than DTA.

In vitro maturation of porcine oocytes with retinoids improves embryonic development.

In the present study, the effects of retinoid metabolite administration during in vitro maturation (IVM) on oocyte maturation, parameters of in vitro fertilisation (IVF) and embryo development were examined. Varying concentrations of 9-cis retinoic acid (RA; 0, 5, 50 and 500 nm; Experiment 1) and all-trans retinol (ROH; 0, 125, 1250 and 12 500 nm; Experiment 2) were included in the maturation medium. Cumulus-oocyte complexes were matured in vitro and inseminated with frozen-thawed spermatozoa. Presumptive zygotes were cultured for 16 h to assess IVF parameters or for 7 days to assess embryo development and quality. In Experiment 1, the oocyte maturation rate to metaphase II was significantly decreased (P < 0.001), with values below 5%, in the presence of the highest concentration of RA (500 nm). However, 5 and 50 nm RA had no effect compared with control. Treatment with 5 nm RA improved the blastocyst development rate (P < 0.001). In Experiment 2, the oocyte maturation rate did not differ between 125 and 1250 nm ROH treatment groups and control. However, treatment with 12 500 nm ROH was deleterious because no matured oocytes were observed following the treatment. The penetration rate was lower in the group treated with 1250 nm ROH compared with the 125 nm ROH-treated and control groups, but the blastocyst formation rate did not differ among the three groups. In conclusion, 5 nm RA in the IVM medium significantly increased the blastocyst formation rate, suggesting that RA may play an important role during IVM.

Major proteins of boar seminal plasma as a tool for biotechnological preservation of spermatozoa.

Boar seminal plasma is a complex mixture of secretions from the testes, epididymides, and the male accessory reproductive organs which bathe the spermatozoa at ejaculation. The seminal plasma contains factors, mostly proteins, which influence the spermatozoa, the female genital tract, and the ovum. In boars, most of the proteins belong to the spermadhesin family and bind to the sperm surface. Spermadhesins are multifunctional proteins with a wide range of ligand-binding abilities to heparin, phospholipids, protease inhibitors and carbohydrates; the family can be roughly divided into heparin-binding (AQN-1, AQN-3, AWN) and non-heparin-binding spermadhesins (PSP-I/PSP-II heterodimer). These proteins have various effects promoting or inhibiting sperm functions including motility, oviduct binding, zona binding/penetration, and ultimately fertilization. The complexity of the environmental signals that influence these actions have implications for the uses of these proteins in vivo and in vitro, and may lead to uses in improving sperm storage.

In vitro fertilization (IVF) in straws and a short gamete coincubation time improves the efficiency of porcine IVF.

The present study was designed to evaluate three different in vitro fertilization (IVF) systems: a straw-IVF system with 10 min of coincubation, a straw-IVF system with 6-h coincubation and the microdrop-IVF system with 6-h coincubation (the traditional IVF system used routinely in most of IVF laboratories) in an attempt to reduce polyspermic penetration (Experiment 1). When the straw-IVF system was tested in combination with two coincubation times, the use of 10 min of coincubation significantly increased (p < 0.001) the penetration rate and the efficiency of fertilization (67.7 +/- 6.4% vs 31.9 +/- 6.5% and 41.5 +/- 2.5% vs 17.6 +/- 2.5% for 10 min and 6 h, respectively), while there were no significant differences in the incidence of monospermy between both systems (64.3 +/- 5.1% and 67.7 +/- 3.4%, for 10 min and 6 h, respectively). The penetration rate in the 6-h microdrop-IVF system was higher (93.8 +/- 3.6%; p < 0.001) compared with the 10-min straw-IVF system (67.7 +/- 6.4%), however, monospermy was severely reduced (25.0 +/- 4.3% vs 67.7 +/- 3.4%, for the 6-h microdrop-IVF system and 10-min straw-IVF system, respectively). The efficiency of the IVF showed similar values between microdrop and 6-h straw-IVF systems, but efficiency was significantly improved (p < 0.05) when the 10-min straw-IVF system was used. Experiment 2 was designed to compare porcine in vitro embryo production in two IVF systems, the 6-h microdrop-IVF system (1000 sperm per oocyte) and 10-min straw-IVF system (30 000 sperm per oocyte). The blastocyst formation rates tended (p = 0.06) to be higher when the 10-min straw-IVF system was used compared with the 6-h microdrop-IVF system. In addition, the number of total cells per blastocyst increased significantly (p < 0.05) in the 10-min straw-IVF system. These results showed that the 10-min straw-IVF system is an effective way to decrease polyspermic penetration, and improve the efficiency of fertilization and the quality of blastocysts in terms of cell number per embryo.

Improving the efficiency of insemination with sex-sorted spermatozoa.

The sorting of X- and Y-chromosome-bearing spermatozoa by flow cytometry is nowadays one of the most apt assisted-reproduction technologies in livestock production. Potential economic and biological benefits, as well as those related to easier management of herds, have been reported arising out of the application of this technique, especially in cattle. Yet, the sex-sorting procedure induces damage to spermatozoa, affecting their function and fertilizing ability. Different species present varying degrees of susceptibility to damage from the sorting process and each has its own requirements for sex-sorted insemination procedures. Thus, several new protocols and strategies have been designed for the handling of sorted spermatozoa, with the main objective of optimizing their fertilizing ability and the consequent application of flow-cytometric sex-sorting technology. This article reviews current advances in this technology, pointing out the components to be improved before this technology may be widely applied in different domestic species.

Low-dose insemination in pigs: problems and possibilities.

Low-dose AI procedures are required by the pig industry to efficiently utilize emerging sperm technologies, such as cryopreservation and sex-sorting. Currently, several different procedures for inseminating with a low or very low number of spermatozoa have been described. Deep intrauterine insemination allows the deposition of the spermatozoa in the depth of the uterine horn, allowing a significant reduction in the number of spermatozoa inseminated with maintenance of optimal reproductive performance. Intra-oviductal laparoscopic insemination has been recently applied in pigs. This technique has proved to be applicable with diluted and sex-sorted spermatozoa. This review discusses several problems encountered during the development of deep intrauterine insemination and intra-oviductal laparoscopic insemination of pigs and provides potential solutions for the practical application of both the technologies.

Vitrification of in vitro cultured porcine two-to-four cell embryos.

The objective of this experiment was to evaluate the effect of a 5-day period of in vitro culture of two-to-four cell porcine embryos up to the blastocyst stage on their ability to survive vitrification and warming. In order to increase the cooling rate, superfine open pulled straws and Vit-Master((R)) technology were used for vitrification. Two-to-four cell embryos were collected from weaned sows (n=11) on day 2 (D0=onset of estrus). Some embryos (N=63) were vitrified within 3h after collection, warmed and cultured for 120h (Group V2). Additionally, 81 two-to-four cell embryos were cultured for 96h in order to obtain blastocysts; these were then vitrified, warmed and cultured for 24h (Group VB; N=65). The remaining two-to-four cell embryos were used as controls and thus not vitrified (control embryos; N=70) but were cultured in vitro for 120h. The V2, VB and control embryos were evaluated for their developmental progression and morphology during culture. All embryos (V2, VB and controls) were fixed on the same day of development in order to assess the total number of blastomeres. The survival and blastocyst formation rates obtained from V2 embryos were very poor (9.6+/-0.7% and 3.2+/-0.5%, respectively). The survival and hatching rates of VB embryos (75.0+/-0.69% and 33.6+/-0.13%) were lower (p<0.001) than those obtained with control embryos (89.1+/-0.8% and 47.5+/-0.12%). Hatched VB embryos had a lower (p<0.01) total cell number than hatched control embryos (70.3+/-4.5 versus 90.6+/-3.2, respectively). There was no difference between expanded VB and control blastocysts. In conclusion, blastocysts derived from in vitro culture of two-to-four cell pig embryos could be successfully vitrified using SOPS straws and Vit-Master.