Albert Salas-Huetos

New insights into the expression profile and function of micro-ribonucleic acid in human spermatozoa

OBJECTIVE To characterize the microRNA (miRNA) expression profile in spermatozoa from human fertile individuals and their implications in human fertility. DESIGN The expression levels of 736 miRNAs were evaluated using TaqMan arrays. Ontologic analyses were performed to determine the presence of enriched biological processes among their targets. SETTING University research and clinical institutes. PATIENT(S) Ten individuals with normal seminogram, standard karyotype, and proven fertility. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Expression levels of 736 miRNAs, presence of enriched metabolic routes among their targets, homogeneity of the population, influence of demographic features in the results, presence of miRNA stable pairs, and best miRNA normalizing candidates. RESULT(S) A total of 221 miRNAs were consistently present in all individuals, 452 were only detected in some individuals, and 63 did not appear in any sample. The ontologic analysis of the 2,356 potential targets of the ubiquitous miRNAs showed an enrichment of processes related to cell differentiation, development, morphogenesis, and embryogenesis. None of the miRNAs were significantly correlated with age, semen volume, sperm concentration, motility, or morphology. Correlations between samples were statistically significant, indicating a high homogeneity of the population. A set of 48 miRNA pairs displayed a stable expression, a particular behavior that is discussed in relationship to their usefulness as fertility biomarkers. Hsa-miR-532-5p, hsa-miR-374b-5p, and hsa-miR-564 seemed to be the best normalizing miRNA candidates. CONCLUSION(S) Human sperm contain a stable population of miRNAs potentially related to embryogenesis and spermatogenesis.

Dietary patterns, foods and nutrients in male fertility parameters and fecundability: a systematic review of observational studies

BACKGROUND Infertility is a global public health issue, affecting 15% of all couples of reproductive age. Male factors, including decreased semen quality, are responsible for ~25% of these cases. The dietary pattern, the components of the diet and nutrients have been studied as possible determinants of sperm function and/or fertility. OBJECTIVE AND RATIONALE Previous systematic reviews have been made of the few heterogeneous low-quality randomized clinical trials (RCTs) conducted in small samples of participants and investigating the effect of specific nutrients and nutritional supplements on male infertility. However, as yet there has been no systematic review of observational studies. SEARCH METHODS A comprehensive systematic review was made of the published literature, from the earliest available online indexing year to November 2016, in accordance with the guidelines of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses. We have included cross-sectional, case-control and prospective and retrospective studies in which fertile/infertile men were well defined (men with sperm disorders, sperm DNA damage, varicocele or idiopathic infertility). The primary outcomes were semen quality or fecundability. With the data extracted, we evaluated and scored the quality of the studies selected. We excluded RCTs, animal studies, review articles and low-quality studies. OUTCOMES A total of 1944 articles were identified, of which 35 were selected for qualitative analysis. Generally, the results indicated that healthy diets rich in some nutrients such as omega-3 fatty acids, some antioxidants (vitamin E, vitamin C, β-carotene, selenium, zinc, cryptoxanthin and lycopene), other vitamins (vitamin D and folate) and low in saturated fatty acids and trans-fatty acids were inversely associated with low semen quality parameters. Fish, shellfish and seafood, poultry, cereals, vegetables and fruits, low-fat dairy and skimmed milk were positively associated with several sperm quality parameters. However, diets rich in processed meat, soy foods, potatoes, full-fat dairy and total dairy products, cheese, coffee, alcohol, sugar-sweetened beverages and sweets have been detrimentally associated with the quality of semen in some studies. As far as fecundability is concerned, a high intake of alcohol, caffeine and red meat and processed meat by males has a negative influence on the chance of pregnancy or fertilization rates in their partners. WIDER IMPLICATIONS Male adherence to a healthy diet could improve semen quality and fecundability rates. Since observational studies may prove associations but not causation, the associations summarized in the present review need to be confirmed with large prospective cohort studies and especially with well-designed RCTs.

Cultural and historical aspects of Mediterranean nuts with emphasis on their attributed healthy and nutritional properties

BACKGROUND AND AIMS Nuts have been part of the human diet since prehistoric times. The aim of the present article is to describe the most important historical and cultural aspects of nut consumption throughout history. DATA SYNTHESIS We discuss the following historical aspects of nuts originating in the Mediterranean: prehistory, the Egyptian civilization, their spread through the Mediterranean region by the Greek, Phoenician and Roman civilizations, and their reintroduction into Europe by means of the Al-Andalus culture. Particular emphasis is placed on the healthy and nutritional attributes that nuts have had throughout history. We also consider the role of the first globalization of food--the exchange of nuts between continents--and discuss the symbolism that nuts have had for humans throughout history in the context of cultural aspects of the Mediterranean region. CONCLUSIONS Nuts and fruits are probably the earliest foods consumed by humans and are considered to be important because of their nutritional properties. Nuts have also been used in the past by different civilizations as drugs to prevent or treat several diseases.

Spermatozoa from patients with seminal alterations exhibit a differential micro-ribonucleic acid profile

OBJECTIVE To compare the microRNA (miRNA) expression profile in spermatozoa from three infertile populations vs. a group of fertile men. DESIGN Evaluation of the expression level of 736 miRNAs in human spermatozoa using TaqMan quantitative reverse transcription-polymerase chain reaction. SETTING University research facility. PATIENT(S) Semen samples with a single seminal alteration were collected from infertile individuals: asthenozoospermic (n = 10), teratozoospermic (n = 10), and oligozoospermic (n = 10). INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) Correlation of the expression level of each miRNA with seminal parameters, age, and chromosome instability; clustering of the individuals according to their miRNA expression profiles and influence of the seminogram, age, chromosome instability, and assisted reproductive technology outcome in the clustering; analysis of the differentially expressed miRNAs (DE-miRNAs) in each infertile population; genome annotation of these DE-miRNAs; and ontological analysis of their predicted targets. RESULT(S) The hsa-miR-34b-3p correlated with age, the hsa-miR-629-3p with sperm motility, and the hsa-miR-335-5p, hsa-miR-885-5p, and hsa-miR-152-3p with sperm concentration. The individuals clustered into two groups, and only the seminogram was differentially distributed. We identified 32 DE-miRNAs in the asthenozoospermic group, 19 in the teratozoospermic group, and 18 in the oligozoospermic group. The up-regulated miRNAs presented an enriched localization in introns, affecting relevant genes for spermatogenesis. The predicted targets of the DE-miRNAs contained critical genes associated to infertility, and their ontological analysis revealed significantly associated functions related to the seminal alterations of each group. CONCLUSION(S) Spermatozoa from patients with seminal alterations exhibit a differential miRNA profile. This provides new evidence that miRNAs have an essential role in spermatogenesis, contributing to the mechanisms involved in human fertility.

Mediterranean nuts: origins, ancient medicinal benefits and symbolism

OBJECTIVE To consider historical aspects of nuts in relation to origin and distribution, attributed medicinal benefits, symbolism, legends and superstitions. DESIGN Review of historical aspects of nuts. SETTING Mediterranean region. SUBJECTS The varieties reviewed include almonds, walnuts, hazelnuts, pine nuts and pistachios. RESULTS AND CONCLUSIONS Like other foods, nuts have a wide variety of cultural connections to the areas where they grow and to the people who live there or eat them. History, symbolism and legends reveal the ancient tradition of nuts and how they are related to the lives of our ancestors. Archaeological excavations in eastern Turkey have uncovered the existence of a non-migratory society whose economy centred on harvesting nuts. This shows that nuts have been a staple in the human diet since the beginnings of history. Moreover, since ancient times nuts have been used for their medicinal properties. They also play a role in many old legends and traditions.

Spermatozoa from infertile patients exhibit differences of DNA methylation associated with spermatogenesis-related processes: an array-based analysis

The influence of aberrant sperm DNA methylation on the reproductive capacity of couples has been postulated as a cause of infertility. This study compared the DNA methylation of spermatozoa of 19 fertile donors and 42 infertile patients using the Illumina 450K array. Clustering analysis of methylation data arranged fertile and infertile patients into two groups. Bivariate clustering analysis identified a differential distribution of samples according to the characteristics of seminogram and age, suggesting a possible link between these parameters and specific methylation profiles. The study identified 696 differentially methylated cytosine-guanine dinucleotides (CpG) associated with 501 genes between fertile donors and infertile patients. Ontological enrichment analysis revealed 13 processes related to spermatogenesis. Data filtering identified a set of 17 differentially methylated genes, some of which had functions relating to spermatogenesis. A significant association was identified between RPS6KA2 hypermethylation and advanced age (P = 0.016); APCS hypermethylation and oligozoospermia (P = 0.041); JAM3/NCAPD3 hypermethylation and numerical chromosome sperm anomalies (P = 0.048); and ANK2 hypermethylation and lower pregnancy rate (P = 0.040). This description of a set of differentially methylated genes provides a framework for further investigation into the influence of such variation in male fertility in larger patient cohorts.

MicroRNAs and Drinking: Association between the Pre-miR-27a rs895819 Polymorphism and Alcohol Consumption in a Mediterranean Population

Recently, microRNAs (miRNA) have been proposed as regulators in the different processes involved in alcohol intake, and differences have been found in the miRNA expression profile in alcoholics. However, no study has focused on analyzing polymorphisms in genes encoding miRNAs and daily alcohol consumption at the population level. Our aim was to investigate the association between a functional polymorphism in the pre-miR-27a (rs895819 A>G) gene and alcohol consumption in an elderly population. We undertook a cross-sectional study of PREvención con DIeta MEDiterránea (PREDIMED)-Valencia participants (n = 1007, including men and women aged 67 ± 7 years) and measured their alcohol consumption (total and alcoholic beverages) through a validated questionnaire. We found a strong association between the pre-miR-27a polymorphism and total alcohol intake, this being higher in GG subjects (5.2 ± 0.4 in AA, 5.9 ± 0.5 in AG and 9.1 ± 1.8 g/day in GG; padjusted = 0.019). We also found a statistically-significant association of the pre-miR-27a polymorphism with the risk of having a high alcohol intake (>2 drinks/day in men and >1 in women): 5.9% in AA versus 17.5% in GG; padjusted < 0.001. In the sensitivity analysis, this association was homogeneous for sex, obesity and Mediterranean diet adherence. In conclusion, we report for the first time a significant association between a miRNA polymorphism (rs895819) and daily alcohol consumption.

What the human sperm methylome tells us.

AIM To characterize the sperm methylome in semen samples from 19 donors with proven fertility. MATERIALS & METHODS Bisulfite-converted sperm DNA was hybridized on the HumanMethylation450 Infinium BeadChip platform. CpG fluorescence intensities were extracted and converted to β-values. RESULTS The sperm methylome is highly homogeneous and hypomethylated. Genes with hypomethylated promoters are ontologically associated to biological functions related to spermatogenesis and embryogenesis. Sex chromosomes are the most hypomethylated chromosomes, supporting data that indicated their essential role in spermatogenesis. A total of 94 genes are resistant to demethylation, being strong candidates for transgenerational inheritance. CONCLUSION Spermatozoa carry a homogeneous methylation profile that is a footprint of past events (spermatogenesis), is designed to facilitate future events (embryogenesis) and has a possible influence in the adult life (transgenerational effects).

Análisis de la expresión de 4 micro-ARN en espermatozoides y su implicación en la fertilidad masculina

Introduccion: Los micro-ARN (mi-ARN) son moleculas de 22�24 nucleotidos implicadas en la regulacion de la expresion genica de numerosos procesos. Recientemente, se han identificado perfiles alterados de expresion de mi-ARN en diferentes casos de infertilidad idiopatica, sugiriendo un papel relevante de estas moleculas en la regulacion de la fertilidad. Objetivo: Caracterizar los patrones de expresion de 4 mi-ARN en acido ribonucleico espermatico procedente de individuos fertiles e infertiles. Material y metodos: Se obtuvo la fraccion espermatica de 4 muestras de semen de individuos fertiles y 4 muestras de individuos que consultaban por infertilidad. Se aislo el acido ribonucleico utilizando el metodo TRIzol® y seguidamente se efectuo un tratamiento con DNasa. La ausencia de acido desoxirribonucleico en las muestras extraidas se confirmo mediante una reaccion en cadena de la polimerasa para el gen de la protamina 1. Seguidamente, con cebadores especificos para los mi-ARN hsa-miR-23a, hsa-miR-744, hsa-let-7f y hsa-miR-1, y el normalizador Mamm-U6, se realizaron reacciones en cadena de la polimerasa a tiempo real mediante tecnologia TaqMan. La cuantificacion de las diferencias de expresion se realizo mediante el calculo del estadistico Fold Change y la utilizacion de un intervalo de confianza del 95%. Resultados: Para cada individuo, se obtuvo una media de 2,6 × 10-5 ng de acido ribonucleico/espermatozoide con una pureza de 1,72 (± 0,05) y sin trazas de acido desoxirribonucleico. El analisis TaqMan revelo la presencia de hsa-miR-23a, hsa-miR-744 y hsa-let-7f en los espermatozoides de todos los individuos estudiados. La expresion de hsa-let-7f mostro una reduccion significativa en 3 de los 4 individuos problema respecto a los individuos control. Conclusion: Los espermatozoides humanos contienen los mi-ARN hsa-miR-23a, hsa-miR-744 y hsa-let-7f. Este ultimo muestra una reduccion significativa de expresion en 3 de los 4 individuos infertiles analizados sugiriendo la implicacion de alteraciones en la expresion de mi-ARN como causa subyacente de algunos casos de infertilidad masculina.

Changes in circulating miRNAs in healthy overweight and obese subjects: Effect of diet composition and weight loss

BACKGROUND MicroRNAs (miRNAs) are small non-coding RNA molecules that can play an important role in several chronic metabolic conditions, including obesity. However, to date little is known about how they are regulated. Weight loss induced by surgical procedures has been effective at modulating specific circulating miRNAs, but the effect of energy-restricted diets with different macronutrient compositions on circulating miRNAs is not well understood. The objective of the present analysis was to explore the effect of three energy-restricted diets of different macronutrient composition and carbohydrate quality on plasma miRNA levels. METHODS The GLYNDIET study is a 6-month, parallel, randomized clinical trial conducted on overweight and obese subjects who were randomized to one of three different dietary intervention groups: i) a moderate-carbohydrate and low glycemic index diet (LGI), ii) a moderate-carbohydrate and high glycemic index diet (HGI), and iii) a low-fat and high glycemic index diet (LF). We assessed the genome-wide circulating miRNA profile in a subsample of eight randomly selected participants. A total of 8 miRNAs (miR-411, miR-432, miR-99b, miR-340, miR-423, miR-361, let-7c) were differently quantified according to diet intervention, and were therefore longitudinally validated in 103 participants before and after the energy-restricted diets. RESULTS Circulating miR-361 levels were lower in the LGI group than in the HGI group, even after adjusting for differences in weight loss. The intra-group analyses demonstrated a significant down-regulation of all miRNAs screened in our study subjects after the LGI intervention. Similarly, miR-139 and miR-340 were down-regulated after the HGI intervention, while miR-139, miR-432 and miR-423 were down-regulated after the low-fat diet. Changes in circulating miR-139 and let-7c were significantly associated with changes in lipid profile and insulin resistance. CONCLUSION An energy-restricted low-glycemic index diet down-regulates circulating miRNA-361 more than an energy-restricted high-glycemic index, regardless of the magnitude of the weight loss.