Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes.

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

[Cognitive performance of preschoolers with Congenital Hypothyroidism enrolled in a follow-up program]. / Desempeño cognitivo en preescolares con Hipotiroidismo Congénito incorporados en un programa de seguimiento.

The age at treatment initiation is decisive for limiting the neurological sequelae of Congenital Hypothyroidism (CH). Incorporating children into follow-up programs could be very helpful. OBJECTIVE: To evaluate the cognitive performance of preschool children with CH incorporated into a follow- up program. PATIENTS AND METHOD: Prospective study of 93 patients with a confirmed diagnosis of CH. Intelligence quotient (IQ) was assessed using the Wechsler Preschool and Primary Intelligence Scale (WPPSI) at 4 and 5 years, and the WISC-R at 6 years of age. Full-Scale IQ (FSIQ), Verbal IQ (VIQ), and Performance IQ (PIQ) scores were analyzed. RESULTS: The study sample was 80 children. The average age at starting hormonal treatment was 42 ± 18 days; treatment started early in 25 patients (24 ± 6 days) and late in 55 patients (50 ± 16 days). The mean initial dose of Levothyroxine was 13.5 ± 1.5µg/kg/day. Children with athyrosis and late initiation of treatment had lower scores on the VIQ (85 ± 14), the PIQ (89 ± 12), and the FSIQ (86 ± 13) scales at 4 years of age, in comparison with patients with early initiation of treatment. These patients scored within the cut-off point for the normal IQ classification (90-109 points). IQ comparison at 6 years of age revealed differences up to 14 points in the PIQ and 11 points in the FSIQ between children with athyrosis and early initiation of treatment, with and without regular attendance to the follow-up program. DISCUSSION: These results support the importance of early initiation of treatment and the incorporation of children in follow-up programs and early stimulation. The etiology of hypothyroidism and the age at initiation of treatment were the most significant factors that affected cognitive performance.

Influence of soil chemical composition on U, 226Ra and 210Pb uptake in leaves and fruits of Quercus ilex L.

To determine their transfer factors, activity concentrations of natural radionuclides were measured in the leaves and acorns of holm oak (Quercus ilex L.) trees collected from seven locations with different soil properties and radionuclide activity concentrations. The chemical and mineralogical compositions of the soils were also analysed to investigate the effect these had on radionuclide absorption by the trees. Soil chemistry showed significant effects on radionuclide incorporation into Quercus ilex L. tissues. A significant relationship was established between activity concentrations and soil content of Ca and P with 238U and 226Ra in the leaves and acorns of Quercus ilex L. Differentiated transfer was found for 40K, which showed greater transfer to the leaves than the other radionuclides. The activity concentration of U and 226Ra was higher in the fruits than in the leaves, with the opposite effect being observed for 40K. The risk of U and 226Ra transfer into the food chain through acorn consumption by livestock is predicted to increase in soils poor in Ca and rich in P.

Immune dysregulation and pathophysiology of alcohol consumption and alcoholic liver disease.

Alcoholic liver disease (ALD) is a clinical-pathologic entity caused by the chronic excessive consumption of alcohol. The disease includes a broad spectrum of anomalies at the cellular and tissual level that can cause acute-on-chronic (alcoholic hepatitis) or chronic (fibrosis, cirrhosis, hepatocellular cancer) injury, having a great impact on morbidity and mortality worldwide. Alcohol is metabolized mainly in the liver. During alcohol metabolism, toxic metabolites, such as acetaldehyde and oxygen reactive species, are produced. At the intestinal level, alcohol consumption can cause dysbiosis and alter intestinal permeability, promoting the translocation of bacterial products and causing the production of inflammatory cytokines in the liver, perpetuating local inflammation during the progression of ALD. Different study groups have reported systemic inflammatory response disturbances, but reports containing a compendium of the cytokines and cells involved in the pathophysiology of the disease, from the early stages, are difficult to find. In the present review article, the role of the inflammatory mediators involved in ALD progression are described, from risky patterns of alcohol consumption to advanced stages of the disease, with the aim of understanding the involvement of immune dysregulation in the pathophysiology of ALD.

An optimized MNK1b aptamer, apMNKQ2, and its potential use as a therapeutic agent in breast cancer.

Breast cancer is the most commonly diagnosed and leading cause of cancer death among women worldwide. Mitogen-activated protein kinase-interacting kinases (MNKs) promote the expression of several oncogenic proteins and are overexpressed in several types of cancer. In human cells, there are four isoforms of MNKs. The truncated isoform MNK1b, first described in our laboratory, has a higher basal activity and is constitutively active. Aptamers are emerging in recent years as potential therapeutic agents that show significant advantages over drugs of other nature. We have previously obtained and characterized a highly specific aptamer against MNK1b, named apMNK2F, with a dissociation constant in the nanomolar range, which produces significant inhibition of proliferation, migration, and colony formation in breast cancer cells. Furthermore, its sequence analysis predicted two G-quadruplex structures. In this work, we show the optimization process of the aptamer to reduce its size, improving its stability. The obtained aptamer, named apMNKQ2, is able to inhibit proliferation, colony formation, migration, and invasion in breast cancer cells. In murine models of breast cancer, apMNKQ2 has demonstrated its efficacy in reducing tumor volume and the number of metastases. In conclusion, apMNKQ2 could be used as an anti-tumor drug in the future.

Improvements in the radiochemical method for separating 226Ra in solid samples through coprecipitation with BaSO4.

The aim of this work was to improve the commonly used method for 226Ra determination in water and to establish its application in solid samples. This method is based on the coprecipitation of Ra with BaSO4 and gross alpha counting of the precipitate. An exhaustive study of the coprecipitation behaviour of the most abundant cations present in solid samples was performed to avoid incorrect radiochemical yields. As a result, it was considered necessary to introduce two new purification steps into the conventional method. Likewise, two nuclides, 241Am and 226Ra, were compared to obtain the mass efficiency curve given their different behaviour in the coprecipitation process. While Ra behaves similarly to Ba, Am coprecipitates, forming mixed crystals that may behave differently in the self-absorption process. The influence of the cations on the chemical yield with no precipitate purification was: Sr2+≫Fe3+>Mg2+≈Ca2+>K+≈Na+. The method was successfully applied to soil, sediment, and plant ash samples.

Selective expression of the neurexin substrate for presenilin in the adult forebrain causes deficits in associative memory and presynaptic plasticity.

Presenilins (PS) form the active subunit of the gamma-secretase complex, which mediates the proteolytic clearance of a broad variety of type-I plasma membrane proteins. Loss-of-function mutations in PSEN1/2 genes are the leading cause of familial Alzheimer's disease (fAD). However, the PS/gamma-secretase substrates relevant for the neuronal deficits associated with a loss of PS function are not completely known. The members of the neurexin (Nrxn) family of presynaptic plasma membrane proteins are candidates to mediate aspects of the synaptic and memory deficits associated with a loss of PS function. Previous work has shown that fAD-linked PS mutants or inactivation of PS by genetic and pharmacological approaches failed to clear Nrxn C-terminal fragments (NrxnCTF), leading to its abnormal accumulation at presynaptic terminals. Here, we generated transgenic mice that selectively recreate the presynaptic accumulation of NrxnCTF in adult forebrain neurons, leaving unaltered the function of PS/gamma-secretase complex towards other substrates. Behavioral characterization identified selective impairments in NrxnCTF mice, including decreased fear-conditioning memory. Electrophysiological recordings in medial prefrontal cortex-basolateral amygdala (mPFC-BLA) of behaving mice showed normal synaptic transmission and uncovered specific defects in synaptic facilitation. These data functionally link the accumulation of NrxnCTF with defects in associative memory and short-term synaptic plasticity, pointing at impaired clearance of NrxnCTF as a new mediator in AD.

Overview of virus and cancer relationships. Position paper.

The role of certain viruses in the etiology of some tumors is today indisputable, but there is a lack, however, of annoverview of the relationship between viruses and cancer with amultidisciplinary approach. For this reason, the Health Sciences Foundation has convened a group of professionals from different areas of knowledge to discuss the relationship between viruses and cancer, and the present document is the result of these deliberations. Although viruses cause only 10-15% of cancers, advances in oncology research are largely due to the work done during the last century on tumor viruses. The clearest cancer-inducing viruses are: HPV, HBV, HCV, EBV and, depending on the geographical area, HHV-8, HTLV-1 and HIV. HPVs, for example, are considered to be the causative agents of cervical carcinomas and, more recently, of a proportion of other cancers. Among the Herpes viruses, the association with the development of neoplasms is well established for EBV and HHV-8. Viruses can also be therapeutic agents in certain neoplasms and, thus, some oncolytic viruses with selective tropism for tumor cells have been approved for clinical use in humans. It is estimated that the prophylaxis or treatment of viral infections could prevent at least 1.5 million cancer deaths per year.

Undetectable viral RNA in oocytes from SARS-CoV-2 positive women.

A central concern for the safe provision of ART during the current coronavirus disease 2019 (COVID-19) pandemic is the possibility of vertical transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection through gametes and preimplantation embryos. Unfortunately, data on SARS-CoV-2 viral presence in oocytes of infected individuals are not available to date. We describe the case of two women who underwent controlled ovarian stimulation and tested positive to SARS-CoV-2 infection by PCR on the day of oocyte collection. The viral RNA for gene N was undetectable in all the oocytes analyzed from the two women.

Efficiency and efficacy of vitrification in 35 654 sibling oocytes from donation cycles.

STUDY QUESTION: Is oocyte vitrification/warming as efficient and effective as using fresh oocytes in donation cycles? SUMMARY ANSWER: IVF with vitrified donor oocytes is less efficient than using fresh oocytes, but its efficacy remains comparable to that of fresh cycles. WHAT IS KNOWN ALREADY: Oocyte vitrification is used to preserve the reproductive potential of oocytes. A small number of randomized controlled trials carried out by experienced groups have shown that this technique provides fertilization, pregnancy, implantation and ongoing pregnancy rates comparable to those of fresh oocytes. However, large registry-based analyses have consistently reported lower live birth rates (LBRs) in cycles using vitrified oocytes. It is not clear whether this decrease may be due to the effect of vitrification per se on the oocytes or to the lower efficiency of the technique, as some of the oocytes do not survive after warming. STUDY DESIGN, SIZE, DURATION: Retrospective cohort analysis of 1844 cycles of oocyte donation (37 520 oocytes), each donor in the study provided enough oocytes for at least one reception cycle with fresh oocytes (2561 cycles) and one reception cycle with vitrified oocytes (2471 cycles) from the same ovarian stimulation (sibling oocytes). Overall, 35 654 oocytes were considered in the analysis. All embryo transfers (n = 5032) were carried out between 2011 and 2017. PARTICIPANTS/MATERIALS, SETTING, METHODS: Differences in reproductive outcomes after the first embryo transfer were evaluated using Pearson's Chi-squared test and regression analysis adjusted for recipient's age, BMI, sperm origin and state, day of embryo transfer, morphological score and number of transferred embryos. We performed two additional sub-analyses, to test whether the efficiency and/or effectiveness of vitrification/warming impacts reproductive results. One analysis included paired cycles where the same number of fresh and vitrified oocytes were available for ICSI (SAME sub-analysis), while the second analysis included those cycles with a 100% survival rate post-warming (SAME100 sub-analysis). MAIN RESULTS AND THE ROLE OF CHANCE: Baseline and cycle characteristics of participants were comparable between groups. Overall, fertilization rates and embryo morphological scores were significantly lower (P < 0.001) when using vitrified oocytes; moreover, vitrified oocytes also resulted in lower reproductive outcomes than sibling fresh oocytes using both unadjusted and adjusted analyses: ongoing pregnancy (32.1% versus 37.5%; P < 0.001; OR 0.88, 95% CI 0.77, 1.00) and live birth (32.1% versus 31.9%; P = 0.92; OR 1.16, 95% CI 0.90, 1.49). However, when the efficiency of warming was taken into account, reproductive outcomes in recipients became comparable: ongoing pregnancy (33.5% versus 34.1%; P = 0.82; OR 1.11, 95% CI 0.87, 1.43) and LBR (32.1% versus 32%; P = 0.97; OR 1.15, 95% CI 0.89, 1.48). Moreover, after selecting only cycles that, in addition to having the same number of oocytes available for ICSI, also had 100% post-warming survival rate in the vitrified group, reproductive outcomes were also comparable between fresh and vitrified oocytes: ongoing pregnancy (34.8% versus 32.4%; P = 0.42; OR 1.32, 95% CI 0.98, 1.77) and live birth (32.9% versus 31.0%; P = 0.52; OR 1.27, 95% CI 0.95, 1.71), indicating that reproductive outcomes of these cycles are affected by the efficiency of the vitrification/warming technique performed rather than the oocyte damage due to the fast cooling process to which oocytes are subjected. LIMITATIONS, REASONS FOR CAUTION: An open vitrification system was used for all cases, and oocyte vitrification/warming was performed by experienced embryologists with consistently high survival rates; caution must be exerted when extrapolating our results to data obtained using other open vitrification systems, closed vitrification systems or to IVF units with survival rates <90%. WIDER IMPLICATIONS OF THE FINDINGS: This is the largest cohort study comparing reproductive outcomes of vitrified and fresh sibling donor oocytes to date. We found that, when the number of oocytes available after warming is equal to the number of fresh oocytes, reproductive results including live birth are comparable. Consequently, the efficiency of vitrification must be taken into account to achieve the same reproductive outcomes as with fresh oocytes. We recommend implementing strict indicators of vitrification/warming efficiency in clinics and refining vitrification/warming protocols to maximize survival. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funding of Clínica EUGIN and by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia (GENCAT 2015 DI 048). The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.

Echinococcus granulosus sensu stricto, Echinococcus ortleppi; and E. intermedius (G7) are present in Bolivia.

Cystic echinococcosis (CE) is a zoonotic disease caused by a complex of species known as Echinococcus granulosus sensu lato. CE is endemic in Argentina, Chile, Peru, Uruguay and the South part of Brazil. In contrast, little is known regarding the presence of CE in Bolivia. In this study, 35 cysts isolated from livestock (mostly from the Department of La Paz) and 3 from humans (La Paz, Oruro and Potosi) were genetically characterized analysing the sequence of the cox1 gene (1609 bp). In total, 30 cysts (from La Paz, Cochabamba and Beni) were characterized as E. granulosus sensu stricto (3 fertile and 4 non-fertile cysts from sheep, 8 fertile and 12 non-fertile cysts from cattle and 3 fertile cysts from humans). A detailed analysis of the cox1 haplotypes of E. granulosus s.s. is included. Echinococcus ortleppi (G5) was found in 5 fertile cysts from cattle (from La Paz and Cochabamba). Echinococcus intermedius (G7) was identified in 3 fertile cysts from pigs (from Santa Cruz). Additionally, E. granulosus s.s. was detected in 4 dog faecal samples, while E. ortleppi was present in other two dog faecal samples. The implications of these preliminary results in the future implementation of control measures are discussed.

Altered cytoplasmic maturation in rescued in vitro matured oocytes.

STUDY QUESTION: Do culture conditions affect cytoplasmic maturation in denuded immature non-GV oocytes? SUMMARY ANSWER: The maturation rate of denuded non-GV oocytes is not affected by culture media, but in vitro maturation seems to alter the mitochondrial membrane potential, endoplasmic reticulum (ER) and actin cytoskeleton compared with in vivo maturation. WHAT IS KNOWN ALREADY: In vitro maturation of denuded immature non-GV oocytes benefits cycles with poor in vivo MII oocyte collection, but maturation levels of non-GV oocytes are only scored by polar body extrusion. Since oocyte maturation involves nuclear as well as cytoplasmic maturation for full meiotic competence, further knowledge is needed about cytoplasmic maturation in in vitro culture. STUDY DESIGN, SIZE, DURATION: This basic research study was carried out between January 2017 and September 2018. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 339 denuded immature non-GV oocytes were cultured in SAGE 1-Step (177) or G-2 PLUS (162) for 6-8 h after retrieval, and 72 in vivo matured MII oocytes were used as controls. Cultured immature non-GV oocytes were scored for polar body extrusion and analysed for mitochondrial membrane potential (ΔΨm), ER clusters, cortical granules number and distribution, spindle morphology and actin cytoskeleton organization. The obtained parameter values were compared to in vivo matured MII oocyte parameter values. MAIN RESULTS AND THE ROLE OF CHANCE: The maturation rates of oocytes cultured in G-2 PLUS and SAGE 1-Step were similar (65% vs 64.2%; P = 0.91). The differences observed in cortical granule density were not statistically significant. Also spindle morphometric parameters were mostly similar between in vitro and in vivo matured MII oocytes. However, the number of ER clusters, the ΔΨm and the cortical actin thickness showed significant differences between in vivo MII oocytes and denuded immature non-GV oocytes cultured in vitro until meiosis completion. LIMITATIONS, REASONS FOR CAUTION: Frozen-thawed oocytes together with fresh oocytes were used as controls. Due to technical limitations (fixation method and fluorochrome overlap), only one or two parameters could be studied per oocyte. Thus, a global view of the maturation status for each individual oocyte could not be obtained. WIDER IMPLICATIONS OF THE FINDINGS: Characterization of in vitro matured oocytes at the cellular level will help us to understand the differences observed in the clinical outcomes reported with rescue IVM compared to in vivo MII oocytes and to improve the culture methods applied. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by intramural funding of Clinica Eugin and by the Torres Quevedo Program to A.F.-V. from the Spanish Ministry of Economy and Competitiveness. No competing interests are declared.

Impact of fertilization by natural manure on the microbial quality of soil: Molecular approach.

The quality of soil is strongly bound by several interactions between chemical and biological components, including microbial composition, which are a key importance for soil performance. Cultural activities have a huge induction on soil health, through both modification of physicochemical proprieties and changing on soil microbial communities. This usually affects the safety of soil, and then the crop production and water. In the present work, the information on bacterial community composition was determined from a set of 6 soils collected from 2 farms in agricultural land of Marrakech (Morocco), one of which used poultry manure (PM) and the other cow manure (CM) as fertilizers. To profile this structure of the bacterial community Denaturing Gradient Gel Electrophoresis (DGGE) of 16S rDNA fragments has been used. These amendments resulted in the appearance of several novel bands and different relative intensities of bands between the control station and other sites studied. The stations most affected are those receiving a supply of manure rather high, which results in an organic and bacterial load in the soil. The results showed a bacterial diversity very important indicating a fecal contamination like Bacteroides, Pseudomonas, Staphylococcus,… etc. Bacteria pertain to the phylum Firmicutes and Bacteroidetes were noted to be the dominant ribotype in amended soil. Moreover, this work demonstrates also the existence of pathogens strains in soil amended by poultry manure (PM) belonging to the Clostridiales order and Pseudomonadales. The pathogenic bacteria detected posing a hazard of human contagion when they are used for soil practice.

The transcriptome of human oocytes is related to age and ovarian reserve.

STUDY QUESTION: How does the human oocyte transcriptome change with age and ovarian reserve? SUMMARY ANSWER: Specific sets of human oocyte messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) are affected independently by age and ovarian reserve. WHAT IS KNOWN ALREADY: Although it is well established that the ovarian reserve diminishes with increasing age, and that a woman's age is correlated with lower oocyte quality, the interplay of a diminished reserve and age on oocyte developmental competence is not clear. After maturation, oocytes are mostly transcriptionally quiescent, and developmental competence prior to embryonic genome activationrelies on maternal RNA and proteins. STUDY DESIGN, SIZE, DURATION: A total of 36 vitrified/warmed MII oocytes from 30 women undergoing oocyte donation were included in this study, processed and analyzed individually. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA from each oocyte was independently isolated, amplified, labeled, and hybridized on HTA 2.0 arrays (Affymetrix). Data were analyzed using TAC software, in four groups, each including nine oocytes, according to the woman's age and antral follicular count (AFC) (mean ± SD): Young with High AFC (YH; age 21 ± 1 years and 24 ± 3 follicles); Old with High AFC (OH; age 32 ± 2 years and 29 ± 7 follicles); Young with Low AFC (YL; age 24 ± 2 years and 8 ± 2 follicles); Old with Low AFC (OL; age 34 ± 1 years and 7 ± 1 follicles). qPCR was performed to validate arrays. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a set of 30 differentially expressed mRNAs when comparing oocytes from women with different ages and AFC. In addition, 168 non-coding RNAs (ncRNAs) were differentially expressed in relation to age and/or AFC. Few mRNAs have been identified as differentially expressed transcripts, and among ncRNAs, a set of Piwi-interacting RNAs clusters (piRNAs-c) and precursor microRNAs (pre-miRNAs) were identified as increased in high AFC and old groups, respectively. Our results indicate that age and ovarian reserve are associated with specific ncRNA profiles, suggesting that oocyte quality might be mediated by ncRNA pathways. LARGE SCALE DATA: Data can be found via GEO accession number GSE87201. LIMITATIONS, REASONS FOR CAUTION: The oldest woman included in the study was 35 years old, thus our results cannot readily be extrapolated to women older than 35 or infertile women. WIDER IMPLICATIONS OF THE FINDINGS: We show, for the first time, that several non-coding RNAs, usually regulating DNA transcription, are differentially expressed in relation to age and/or ovarian reserve. Interestingly, the mRNA transcriptome of in vivo matured oocytes remains remarkably stable across ages and ovarian reserve, suggesting the possibility that changes in the non-coding transcriptome might regulate some post-transcriptional/translational mechanisms which might, in turn, affect oocyte developmental competence. STUDY FUNDING AND COMPETING INTEREST(S): This work was supported by intramural funding of Clinica EUGIN and by the Secretary for Universities and Research of the Ministry of Economy and Knowledge of the Government of Catalonia. J.H. and A.S. are employees of Affymetrix, otherwise there are no competing interests.

WBP2NL/PAWP mRNA and protein expression in sperm cells are not related to semen parameters, fertilization rate, or reproductive outcome.

PURPOSE: WBP2NL/PAWP, a protein found in the post-acrosomal region of mammalian spermatozoa, has been proposed as a sperm-borne oocyte-activating factor (SOAF) contributing to Ca2+ release within the oocyte and subsequent fertilization and embryo development. However, its relevance as either a diagnostic or a prognostic marker of fertilization failure has been questioned in the recent literature. We analyzed WBP2NL/PAWP gene and protein expression level and localization in patients without previous intracytoplasmic sperm injection (ICSI) cycles in order to assess its association with both sperm characteristics and ability to fertilize. METHODS: Raw frozen-thawed semen samples from 33 couples referred for oocyte donation were included in the study during 2015. Relative protein expression versus α-tubulin (western blot, WB), proportion of post-acrosomal WBP2NL/PAWP-positive spermatozoa over the total number of sperm cells (immunofluorescence), and WBP2NL/PAWP gene expression (RT-qPCR) were analyzed and correlated with semen analysis parameters (number, motility, and morphology) and with reproductive outcomes. RESULTS: WBP2NL/PAWP protein was expressed in all samples with high variability: relative protein expression (1.77 ± 0.8, range [0.4-3.7]), proportion of positive cells (49.6% ± 16.1, range [22-89]), and relative gene expression (7.3 ± 8.2). No significant correlation (R 2 < 0.1) was found between gene and protein expression, neither between WBP2NL/PAWP gene or protein expression, and fertilization rate or other reproductive outcomes (i.e., pregnancy). In contrast, we found significant correlation between sperm morphology and WBP2NL/PAWP semiquantitative analysis in WB (r = -0.42, p < 0.05) and for sperm motility and WBP2NL/PAWP expression in IF (r = 0.52, p < 0.05). CONCLUSION: Taken into account that WBP2NL/PAWP gene and protein levels and distribution did not correlate with fertilization rates, this study questions the interest of WBP2NL/PAWP protein and gene expression analysis in sperm cells as a prognostic factor for the outcome of ICSI cycles. Larger studies focusing on WBP2NL/PAWP protein and gene expression are needed in order to evaluate the role of WBP2NL/PAWP as a prognostic factor for ART.

Effect of ribonucleic acid (RNA) isolation methods on putative reference genes messenger RNA abundance in human spermatozoa.

Although the male gamete participates in a significant proportion of infertility cases, there are currently no proven molecular markers of sperm quality. The search for significant gene expression markers is partially hindered by the lack of a recognized set of reference genes (RGs) to normalize reverse transcription quantitative PCR (RT-qPCR) data across studies. The aim of this study is to define a set of RGs in assisted reproduction patients undergoing different sample collection and RNA isolation methods. Twenty-two normozoospermic men were included in the study. From each man, semen was either cryopreserved by slow freezing or analyzed fresh, and, for each, RNA was extracted with either phenol-free or phenol-based methods. In two cases, both methods were used to isolate RNA. Twenty putative RGs were analyzed and their mRNA abundance across samples was estimated by RT-qPCR. To determine the genes whose steady-state mRNA abundance remains unchanged, three different algorithms (geNorm, BestKeeper and NormFinder) were applied to the qPCR data. We found that RGs such as GAPDH or ACTB, useful in other biological contexts, cannot be used as reference for human spermatozoa. It is possible to compare gene expression from fresh and cryopreserved sperm samples using the same isolation method, while the mRNA abundance of expressed genes becomes different depending on the RNA isolation technique employed. In our conditions, the most appropriate RGs for RT-qPCR analysis were RPLP1, RPL13A, and RPLP2. Published discrepancies in gene expression studies in human spermatozoa may be due in part to inappropriate RGs selection, suggesting a possible different interpretation of PCR data in several reports, which were normalized using unstable RGs.

Reducción amigdalar por radiofrecuencia en pediatría / No disponible

Introducción: Se ha realizado un estudio preliminar donde se evalúa la radiofrecuencia como nueva técnica disponible para tratar casos seleccionados de hipertrofia amigdalar. Objetivos: El objetivo principal es determinar los beneficios y seguridad de la reducción amigdalar por radiofrecuencia mediante punción en pacientes pediátricos. Como objetivos secundarios se expondrán las implicaciones quirúrgicas y anestésicas de la técnica. Material y métodos: Estudio retrospectivo y descriptivo de todos los pacientes pediátricos intervenidos de hipertrofia amigdalar mediante radiofrecuencia por punción durante el periodo junio2010-febrero 2011 en nuestra Unidad. Los pacientes seleccionados se incluyeron dentro de un protocolo de actuación. Se ha estudiado: edad, sexo, sintomatología previa, dispositivo mantenimiento vía aérea, tiempo quirúrgico, asociación de adenoidectomía,complicaciones, estancia hospitalaria, dolor postoperatorio, recuperación actividad normal y resultado final. Resultados: Durante el periodo señalado 15 pacientes han sido sometidos a dicha técnica, nueve niñas (60%) y seis niños(40%), con edad media de 3,9 años. Los síntomas de indicación quirúrgica más frecuentes fueron mala respiración, ronquido y alteraciones de deglución. En todos los casos se utilizó mascarilla laríngea reforzada como único dispositivo de mantenimiento de la vía aérea. Se asoció adenoidectomía en el 80% de los pacientes. El tiempo medio de la intervención fue de 14 minutos. No se constataron complicaciones intraoperatorias ni postoperatorias. Como tratamiento del dolor postoperatorio inmediato se emplearon antiinflamatorios no esteroideos y paracetamol y tras el alta hospitalaria sólo requirieron paracetamol. La (..) (AU)

Introduction: We performed a preliminary study where puncture radiofrequency was evaluated as a new technique, available to treat selected cases of tonsillar hypertrophy. Objectives: The main objective is to determine the benefits and safety of puncture radiofrequency tonsil reduction in pediatric patients. Secondary we evaluated the implications of surgical and anesthetic techniques. Material and methods: Retrospective and descriptive study of all pediatric patients who underwent radiofrequency tonsil hypertrophypuncture during June 2010-February 2011 in our unit. The selected patients were included in a protocol including: age, sex, previous symptoms, airway maintenance device, surgical time, adenoidectomy association, complications, hospital stay, postoperative pain, recovery and outcome. Results: During the reported period 15 children have undergone this technique, nine girls (60%) and six boys (40%), with a mean age of 3.9 years. The most frequent symptoms for surgery were breathing difficulties, snoring and swallowing disorders. In allcases reinforced laryngeal mask was used as the sole device for airway maintenance. Adenoidectomy was associated in 80% of patients. The average time for surgery was 14 minutes. There were neither intraoperative nor postoperative complications. NSAIDs and acetaminophen were used as postoperative analgesia. At home, patients only used acetaminophen for pain control. Hospital stay was less than 24 hours, with 48-72 hours of recovery time. No patient came back to our emergency units. After two months, there was tonsillar reduction demonstrated in 14 of the 15 cases (93%).Conclusions: Puncture Radio frequency tonsils reduction, inpediatric selected cases can be a safe, effective and a low morbidity technique (AU)

Hematoma cervical compresivo postquirúrgico en un paciente con prótesis traqueal / No disponible

No disponible

Complete meiosis from human induced pluripotent stem cells.

Gamete failure-derived infertility affects millions of people worldwide; for many patients, gamete donation by unrelated donors is the only available treatment. Embryonic stem cells (ESCs) can differentiate in vitro into germ-like cells, but they are genetically unrelated to the patient. Using an in vitro protocol that aims at recapitulating development, we have achieved, for the first time, complete differentiation of human induced pluripotent stem cells (hiPSCs) to postmeiotic cells. Unlike previous reports using human ESCs, postmeiotic cells arose without the over-expression of germline related transcription factors. Moreover, we consistently obtained haploid cells from hiPSCs of different origin (keratinocytes and cord blood), produced with a different number of transcription factors, and of both genetic sexes, suggesting the independence of our approach from the epigenetic memory of the reprogrammed somatic cells. Our work brings us closer to the production of personalized human gametes in vitro.

Predicting the onset of major depression in primary care: international validation of a risk prediction algorithm from Spain.

BACKGROUND: The different incidence rates of, and risk factors for, depression in different countries argue for the need to have a specific risk algorithm for each country or a supranational risk algorithm. We aimed to develop and validate a predictD-Spain risk algorithm (PSRA) for the onset of major depression and to compare the performance of the PSRA with the predictD-Europe risk algorithm (PERA) in Spanish primary care. METHOD: A prospective cohort study with evaluations at baseline, 6 and 12 months. We measured 39 known risk factors and used multi-level logistic regression and inverse probability weighting to build the PSRA. In Spain (4574), Chile (2133) and another five European countries (5184), 11 891 non-depressed adult primary care attendees formed our at-risk population. The main outcome was DSM-IV major depression (CIDI). RESULTS: Six variables were patient characteristics or past events (sex, age, sex×age interaction, education, physical child abuse, and lifetime depression) and six were current status [Short Form 12 (SF-12) physical score, SF-12 mental score, dissatisfaction with unpaid work, number of serious problems in very close persons, dissatisfaction with living together at home, and taking medication for stress, anxiety or depression]. The C-index of the PSRA was 0.82 [95% confidence interval (CI) 0.79-0.84]. The Integrated Discrimination Improvement (IDI) was 0.0558 [standard error (s.e.)=0.0071, Zexp=7.88, p<0.0001] mainly due to the increase in sensitivity. Both the IDI and calibration plots showed that the PSRA functioned better than the PERA in Spain. CONCLUSIONS: The PSRA included new variables and afforded an improved performance over the PERA for predicting the onset of major depression in Spain. However, the PERA is still the best option in other European countries.