IgG4-related disease mimicking a liver abscess.

IgG4-related disease (IGRD) is a complex medical condition affecting multiple organs, including the liver. The condition is characterized by excessive production of IgG4 antibodies, leading to chronic inflammation and tissue damage. We present a case of a 37-year-old man with a history of chronic pancreatitis was diagnosed with a liver mass. Initial treatment included piperacillin and tazobactam, but the patient's condition worsened. An ultrasound-guided biopsy revealed increased IgG4 positive cells, leading to the diagnosis of an inflammatory pseudotumor associated with IGRD. The patient was treated with prednisone taper therapy, and the liver mass resolved after six months of corticoid treatment.

Chromosome positioning and male infertility: it comes with the territory.

The production of functional spermatozoa through spermatogenesis requires a spatially and temporally highly regulated gene expression pattern, which in case of alterations, leads to male infertility. Changes of gene expression by chromosome anomalies, gene variants, and epigenetic alterations have been described as the main genetic causes of male infertility. Recent molecular and cytogenetic approaches have revealed that higher order chromosome positioning is essential for basic genome functions, including gene expression. This review addresses this issue by exposing well-founded evidences which support that alterations on the chromosome topology in spermatogenetic cells leads to defective sperm function and could be considered as an additional genetic cause of male infertility.

Normalization matters: tracking the best strategy for sperm miRNA quantification.

STUDY QUESTION: What is the most reliable normalization strategy for sperm microRNA (miRNA) quantitative Reverse Transcription Polymerase Chain Reactions (qRT-PCR) using singleplex assays? SUMMARY ANSWER: The use of the average expression of hsa-miR-100-5p and hsa-miR-30a-5p as sperm miRNA qRT-PCR data normalizer is suggested as an optimal strategy. WHAT IS KNOWN ALREADY: Mean-centering methods are the most reliable normalization strategies for miRNA high-throughput expression analyses. Nevertheless, specific trustworthy reference controls must be established in singleplex sperm miRNA qRT-PCRs. STUDY DESIGN, SIZE DURATION: Cycle threshold (Ct) values from previously published sperm miRNA expression profiles were normalized using four approaches: (i) Mean-Centering Restricted (MCR) method (taken as the reference strategy); (ii) expression of the small nuclear RNA RNU6B; (iii) expression of four miRNAs selected by the Concordance Correlation Restricted (CCR) algorithm: hsa-miR-100-5p, hsa-miR-146b-5p, hsa-miR-92a-3p and hsa-miR-30a-5p; (iv) the combination of two of these miRNAs that achieved the highest proximity to MCR. PARTICIPANTS/MATERIALS, SETTING, METHODS: Expression profile data from 736 sperm miRNAs were taken from previously published studies performed in fertile donors (n = 10) and infertile patients (n = 38). For each tested normalizer molecule, expression ubiquity and uniformity across the different samples and populations were assessed as indispensable requirements for being considered as valid candidates. The reliability of the different normalizing strategies was compared to MCR based on the set of differentially expressed miRNAs (DE-miRNAs) detected between populations, the corresponding predicted targets and the associated enriched biological processes. MAIN RESULTS AND THE ROLE OF CHANCE: All tested normalizers were found to be ubiquitous and non-differentially expressed between populations. RNU6B was the least uniformly expressed candidate across samples. Data normalization through RNU6B led to dramatically misguided results when compared to MCR outputs, with a null prediction of target genes and enriched biological processes. Hsa-miR-146b-5p and hsa-miR-92a-3p were more uniformly expressed than RNU6B, but their results still showed scant proximity to the reference method. The highest resemblance to MCR was achieved by hsa-miR-100-5p and hsa-miR-30a-5p. Normalization against the combination of both miRNAs reached the best proximity rank regarding the detected DE-miRNAs (Area Under the Curve = 0.8). This combination also exhibited the best performance in terms of the target genes predicted (72.3% of True Positives) and their corresponding enriched biological processes (70.4% of True Positives). LARGE SCALE DATA: Not applicable. LIMITATIONS, REASONS FOR CAUTION: This study is focused on sperm miRNA qRT-PCR analysis. The use of the selected normalizers in other cell types or tissues would still require confirmation. WIDER IMPLICATIONS OF THE FINDINGS: The search for new fertility biomarkers based on sperm miRNA expression using high-throughput assays is one of the upcoming challenges in the field of reproductive genetics. In this context, validation of the results using singleplex assays would be mandatory. The normalizer strategy suggested in this study would provide a universal option in this area, allowing for normalization of the validated data without causing meaningful variations of the results. Instead, qRT-PCR data normalization by RNU6B should be discarded in sperm-miRNA expression studies. STUDY FUNDING/COMPETING INTERESTS: This work was supported by the 2014/SGR00524 project (Agència de Gestió d'Ajuts Universitaris i de Recerca, Generalitat de Catalunya, Spain) and UAB CF-180034 grant (Universitat Autònoma de Barcelona). Celia Corral-Vazquez is a recipient of a Personal Investigador en Formació grant UAB/PIF2015 (Universitat Autònoma de Barcelona). The authors report no conflict of interest.

Altered segregation pattern and numerical chromosome abnormalities interrelate in spermatozoa from Robertsonian translocation carriers.

The aim of this study was to assess whether there is a relationship between numerical chromosome abnormalities and certain segregation modes in spermatozoa from Robertsonian translocation carriers. A sequential fluorescence in-situ hybridization protocol based on two successive hybridization rounds was performed on sperm samples from one t(13;22) and ten t(13;14) carriers. Patient inclusion criteria included the presence of a positive interchromosomal effect (ICE). In the first round, numerical abnormalities for chromosomes 15/22, 18, 21, X and Y were analysed. In the second round, the segregation outcome of the rearranged chromosomes was evaluated in the numerically abnormal spermatozoa detected in the first round, as well as in randomly assessed spermatozoa. Aneuploid spermatozoa showed statistical differences in all segregation modes when compared with randomly assessed spermatozoa: alternate (50.7% versus 84.3%), adjacent (36.6% versus 14.6%) and 3:0 (10.2% versus 1%). Diploid/multiple disomic spermatozoa showed differences in alternate (3.7% versus 84.3%) and 3:0 (67.6% versus 1%). We concluded that in Robertsonian translocation carriers that exhibit ICE, numerically abnormal spermatozoa preferentially contain unbalanced segregation products. This might be explained by heterosynapsis acting as a rescue mechanism that would lead to aberrant recombination, which is a predisposing factor for non-disjunction events.

Unraveling the Intricacies of the Seminal Microbiome and Its Impact on Human Fertility.

Although the microbial communities from seminal fluid were an unexplored field some decades ago, their characteristics and potential roles are gradually coming to light. Therefore, a complex and specific microbiome population with commensal niches and fluctuating species has started to be revealed. In fact, certain clusters of bacteria have been associated with fertility and health, while the outgrowth of several species is potentially correlated with infertility indicators. This constitutes a compelling reason for outlining the external elements that may induce changes in the seminal microbiome composition, like lifestyle factors, gut microbiota, pathologies, prebiotics, and probiotics. In this review, we summarize the main findings about seminal microbiome, its origins and composition, its relationship with fertility, health, and influence factors, while reminding readers of the limitations and advantages introduced from technical variabilities during the experimental procedures.

Sequential FISH allows the determination of the segregation outcome and the presence of numerical anomalies in spermatozoa from a t(1;8;2)(q42;p21;p15) carrier.

PURPOSE: To find out the meiotic segregation behaviour of the t(1;8;2)(q42;p21;p15), to evaluate the occurrence of interchromosomal effects, and to determine whether there is an accumulation of unbalanced products in aneuploid/diploid gametes. METHODS: A sequential FISH protocol based on two successive hybridization rounds over the same spermatozoa was performed to determine the segregation outcome of the rearranged chromosomes. The presence of numerical abnormalities for 13, 18, 21, X and Y was also evaluated by sperm FISH. Those aneuploid/diploid gametes were subsequently relocalized and analyzed for their segregation content through additional hybridization rounds. RESULTS: The segregation pattern observed reported a very low production of normal/balanced gametes (11.7 %). Significant increased frequencies of diploidies and disomies for chromosomes X/Y and 18 were detected (p < 0.001). Aneuploid and diploid spermatozoa displayed significant increases of 5:1, 6:0 and other unexpected disjunction modes (p < 0.001). CONCLUSIONS: The strategy developed in this study is a reliable new approach to establish the full segregation pattern of complex chromosome rearrangements (CCR). Results corroborate the low number of normal/balanced spermatozoa produced by CCR carriers and support previous findings regarding an altered segregation pattern in gametes with numerical abnormalities. Altogether this confirms the importance of PGD as a tool to prevent the transmission of chromosomal abnormalities to the offspring in CCR patients.

The courtship choreography of homologous chromosomes: timing and mechanisms of DSB-independent pairing.

Meiosis involves deep changes in the spatial organisation and interactions of chromosomes enabling the two primary functions of this process: increasing genetic diversity and reducing ploidy level. These two functions are ensured by crucial events such as homologous chromosomal pairing, synapsis, recombination and segregation. In most sexually reproducing eukaryotes, homologous chromosome pairing depends on a set of mechanisms, some of them associated with the repair of DNA double-strand breaks (DSBs) induced at the onset of prophase I, and others that operate before DSBs formation. In this article, we will review various strategies utilised by model organisms for DSB-independent pairing. Specifically, we will focus on mechanisms such as chromosome clustering, nuclear and chromosome movements, as well as the involvement of specific proteins, non-coding RNA, and DNA sequences.

A transcriptomic insight into the human sperm microbiome through next-generation sequencing.

The purpose of this study is to provide novel information through Next Generation Sequencing (NGS) for the characterization of viral and bacterial RNA cargo of human sperm cells from healthy fertile donors. For this, RNA-seq raw data of poly(A) RNA from 12 sperm samples from fertile donors were aligned to microbiome databases using the GAIA software. Species of viruses and bacteria were quantified in Operational Taxonomic Units (OTU) and filtered by minimal expression level (>1% OTU in at least one sample). Mean expression values (and their standard deviation) of each species were estimated. A Hierarchical Cluster Analysis (HCA) and a Principal Component Analysis (PCA) were performed to detect common microbiome patterns among samples. Sixteen microbiome species, families, domains, and orders surpassed the established expression threshold. Of the 16 categories, nine corresponded to viruses (23.07% OTU) and seven to bacteria (2.77% OTU), among which the Herperviriales order and Escherichia coli were the most abundant, respectively. HCA and PCA displayed four clusters of samples with a differentiated microbiome fingerprint. This work represents a pilot study into the viruses and bacteria that make up the human sperm microbiome. Despite the high variability observed, some patterns of similarity among individuals were identified. Further NGS studies under standardized methodological procedures are necessary to achieve a deep knowledge of the semen microbiome and its implications in male fertility.

Sperm rates of 7q11.23, 15q11q13 and 22q11.2 deletions and duplications: a FISH approach.

Genomic disorders are human diseases caused by meiotic chromosomal rearrangements of unstable regions flanked by Low Copy Repeats (LCRs). LCRs act as substrates for Non-Allelic Homologous Recombination (NAHR) leading to deletions and duplications. The aim of this study was to assess the basal frequency of deletions and duplications of the 7q11.23, 15q11-q13 and 22q11.2 regions in spermatozoa from control donors to check differences in the susceptibility to generate anomalies and to assess the contribution of intra- and inter-chromatid NAHR events. Semen samples from ten control donors were processed by FISH. A customized combination of probes was used to discriminate among normal, deleted and duplicated sperm genotypes. A minimum of 10,000 sperm were assessed per sample and region. There were no differences in the mean frequency of deletions and duplications (del + dup) among the 7q11.23, 15q11-q13 and 22q11.2 regions (frequency ± SEM, 0.37 ± 0.02; 0.46 ± 0.07 and 0.27 ± 0.07%, respectively) (P = 0.122). Nevertheless, hierarchical cluster analysis reveals interindividual differences suggesting that particular haplotypes could be the main source of variability in NAHR rates. The mean frequency of deletions was not different from the mean frequency of duplications in the 7q11.23 (P = 0.202) and 15q11-q13 (P = 0.609) regions, indicating a predominant inter-chromatid NAHR. By contrast, in the 22q11.2 region the frequency of deletions slightly exceed duplications (P = 0.032), although at the individual level any donor showed differences. Altogether, our results support the inter-chromatid NAHR as the predominant mechanism involved in the generation of sperm deletions and duplications.

A survey of small RNAs in human sperm.

BACKGROUND: There has been substantial interest in assessing whether RNAs (mRNAs and sncRNAs, i.e. small non-coding) delivered from mammalian spermatozoa play a functional role in early embryo development. While the cadre of spermatozoal mRNAs has been characterized, comparatively little is known about the distribution or function of the estimated 24,000 sncRNAs within each normal human spermatozoon. METHODS: RNAs of <200 bases in length were isolated from the ejaculates from three donors of proved fertility. RNAs of 18-30 nucleotides in length were then used to construct small RNA Digital Gene Expression libraries for Next Generation Sequencing. Known sncRNAs that uniquely mapped to a single location in the human genome were identified. RESULTS: Bioinformatic analysis revealed the presence of multiple classes of small RNAs in human spermatozoa. The primary classes resolved included microRNA (miRNAs) (≈ 7%), Piwi-interacting piRNAs (≈ 17%), repeat-associated small RNAs (≈ 65%). A minor subset of short RNAs within the transcription start site/promoter fraction (≈ 11%) frames the histone promoter-associated regions enriched in genes of early embryonic development. These have been termed quiescent RNAs. CONCLUSIONS: A complex population of male derived sncRNAs that are available for delivery upon fertilization was revealed. Sperm miRNA-targeted enrichment in the human oocyte is consistent with their role as modifiers of early post-fertilization. The relative abundance of piRNAs and repeat-associated RNAs suggests that they may assume a role in confrontation and consolidation. This may ensure the compatibility of the genomes at fertilization.

Meiotic behavior of three D;G Robertsonian translocations: segregation and interchromosomal effect.

Robertsonian translocations are one of the most frequent reorganizations in humans. Their segregational behavior and their implication in the occurrence of interchromosomal effects (ICEs) has been an important topic of research for the past 10 years. Most of the cases analyzed correspond to rearrangements with chromosomes from the D-group (chromosomes 13, 14 and 15), whereas some rare Robertsonian translocations are scarcely found in the literature, mainly those with both chromosomes from the G-group (chromosomes 21 and 22) and those involving chromosomes from both groups (D;G translocations). Results supporting/rejecting the existence of the ICE phenomenon have been reported, showing the need for more studies to characterize its distribution. In this study, sperm fluorescent in situ hybridization studies have been performed in three D;G Robertsonian translocation carriers: two men with the translocation t(14;21) and a third individual with the rare t(13;22) reorganization. Segregation and ICE results have been considered in relation to their cytogenetic features and all previously published data. The compiled information discards a particular segregation behavior related to the chromosomes involved. In contrast with this segregational homogeneity, heterogeneous ICE results were observed, indicating a significant but random distribution of such phenomenon.

Sperm microRNA pairs: new perspectives in the search for male fertility biomarkers.

OBJECTIVE: To identify candidates of fertility biomarkers among pairs of human sperm microRNAs. DESIGN: Expression data of 736 sperm microRNAs from fertile and infertile individuals characterized in previous published studies by means of TaqMan quantitative polymerase chain reaction (PCR) were reexamined. A set of microRNA pairs with the best biomarker potential were selected and validated by means of quantitative real-time (qRT) PCR in an independent cohort. SETTING: University laboratory. PATIENT(S): Semen samples were obtained from fertile (n = 10) and infertile (asthenozoospermia, n = 10; teratozoospermia, n = 10; oligozoospermia, n = 10; unexplained male infertility [UMI], n = 8) individuals. The validation cohort included 9 fertile donors and 14 infertile patients with different seminal alterations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Spearman test was used to select microRNA pairs with a correlated expression in fertile individuals and a noncorrelated expression in each infertile group. The biomarker potential of these pairs was determined with the use of receiver operating characteristic curves. The differential relative expression of each pair in fertile and infertile populations was verified (Mann-Whitney test). Those pairs with best results were validated by qRT-PCR. RESULT(S): Forty-eight pairs showed significant correlations in the fertile group. The pairs that were uncorrelated in the infertile populations and displayed the best biomarker potential were hsa-miR-942-5p/hsa-miR-1208 (asthenozoospermia), hsa-miR-296-5p/hsa-miR-328-3p (teratozoospermia), hsa-miR-139-5p/hsa-miR-1260a (oligozoospermia), and hsa-miR-34b-3p/hsa-miR-93-3p (UMI). The hsa-miR-942-5p/hsa-miR-1208 pair showed the greatest potential for detecting seminal alterations in the validation cohort (85.71% true positives). CONCLUSION(S): The pairs hsa-miR-942-5p/hsa-miR-1208 and hsa-miR-34b-3p/hsa-miR-93-3p have the potential to become new molecular biomarkers that could help to diagnose male infertility, especially in cases of UMI or when seminal parameters are close to the threshold values.

Reciprocal translocations: tracing their meiotic behavior.

PURPOSE: Segregation and interchromosomal effect studies have been performed in reciprocal translocation carriers by sperm-fluorescent in situ hybridization reporting a great heterogeneity. The divergences have been attributed to the particular cytogenetic characteristics of each rearrangement. Nevertheless, there is no consensus in the factors that are responsible for such variability. The purpose of this study was to determine which cytogenetic features influence in the segregation and interchromosomal effect outcome. METHODS: Segregation and interchromosomal effects analyses were performed in 14 reciprocal translocation carriers, selected because they presented very different cytogenetic features regarding the tetravalent pairing geometry. In each segregation study, a customized combination of probes was used to identify all the segregation products. In the interchromosomal effect study, we used a triple-color fluorescent in situ hybridization for chromosomes X, Y, and 18. RESULTS: A preferential segregation pattern with a gradually decreasing production of Alternate, Adjacent I, Adjacent II, and 3:1 segregation was observed in the segregation analysis. Some specific features have been observed to influence this distribution: size of the translocated and centric segments and the presence of centromeres from acrocentric chromosomes in the center of the cross. Aneuploidy/diploidy screening revealed increased frequencies of numerical anomalies in seven carriers. CONCLUSIONS: Our data suggest that reciprocal translocations display a more homogeneous behavior than described in the literature. The interchromosomal effects represent an additional source of imbalances in these carriers.

Effect of nut consumption on semen quality and functionality in healthy men consuming a Western-style diet: a randomized controlled trial.

Background: Human semen quality has declined in industrialized countries. Pollution, smoking, and the consumption of a Western-style diet are all hypothesized as potential causes. Objective: We evaluated the effect of chronic consumption of nuts on changes in conventional semen parameters and the potential mechanisms implicated. Design: The FERTINUTS study was a 14-wk randomized, controlled, parallel trial. A total of 119 healthy men, aged 18-35 y, were allocated to 1 of 2 intervention groups: one group was fed the usual Western-style diet enriched with 60 g of a mixture of nuts/d (nut group), and the other was fed the usual Western-style diet avoiding nuts (control group). Semen and blood samples were collected at baseline and at the end of the intervention. Dietary information was recorded throughout the trial. Changes in conventional semen parameters (pH, volume, sperm count and concentration, motility, and morphology) were determined as primary outcomes. The effect of nut consumption on sperm DNA fragmentation (SDF), reactive oxygen species (ROS) production, chromosome anomalies (X, Y, and 18), total DNA methylation, and microRNA expression were measured in sperm samples as potential causes of the changes in the seminogram. Results: Compared with the control group, improvements in total sperm count (P = 0.002) and vitality (P = 0.003), total motility (P = 0.006), progressive motility (P = 0.036), and morphology of sperm (P = 0.008) were observed in the nut group. Participants in the nut group showed an increase in the consumption of total fat, monounsaturated fatty acids, polyunsaturated fatty acids, magnesium, vitamin E, α-linolenic acid, total omega-3 (n-3) and ω-3:ω-6 ratio intake during the intervention. Participants in the nut group showed a significant reduction in SDF (P < 0.001) and in the expression of hsa-miR-34b-3p (P = 0.036). No significant changes in ROS, sperm chromosome anomalies, or DNA methylation were observed between groups. Conclusions: The inclusion of nuts in a Western-style diet significantly improves the total sperm count and the vitality, motility, and morphology of the sperm. These findings could be partly explained by a reduction in the sperm DNA fragmentation. This trial was registered at ISRCTN as ISRCTN12857940.

Cardiac papillary fibroelastoma in left ventricular apex.

Papillary fibroelastoma (PF) is a rare benign heart tumor and represents less than 10% of primary cardiac tumors. It mainly affects the cardiac valves and is often discovered during open heart surgery or autopsy. We present a case of a patient who was detected with a left ventricular apex tumor during the evaluation of the transient ischemic attack. The patient underwent surgery, and subsequently, macroscopic and microscopic examination confirmed the diagnosis of PF.

FISH studies of chromosome abnormalities in germ cells and its relevance in reproductive counseling.

Chromosome abnormalities are one of the major causes of human infertility. In infertile males, abnormal karyotypes are more frequent than in the general population. Furthermore, meiotic disorders affecting the germ cell-line have been observed in men with normal somatic karyotypes consulting for infertility. In both cases, the production of unbalanced spermatozoa has been demonstrated. Basically addressed to establish reproductive risks, fluorescence in situ hybridization (FISH) on decondensed sperm heads has become the most frequently used method to evaluate the chromosomal constitution of spermatozoa in carriers of numerical sex chromosome abnormalities, carriers of structural chromosome reorganizations and infertile males with normal karyotype. The aim of this review is to present updated figures of the information obtained through sperm FISH studies with an emphasis on its clinical significance. Furthermore, the incorporation of novel FISH-based techniques (Multiplex-FISH; Multi-FISH) in male infertility studies is also discussed.

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.

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.

Isolating mRNA and small noncoding RNAs from human sperm.

The isolation of spermatozoal RNA is a challenging procedure due to the intrinsic heterogeneous population of cells present in the ejaculate and the small quantity of RNA present in sperm. The transcriptome of these gametes includes a wide variety of messenger RNAs (mRNAs), small noncoding RNAs (sncRNAs), and highly fragmented ribosomal RNAs (rRNAs).The protocol described in this chapter to isolate both the mRNA and sncRNA fractions represents years of development towards automation. It combines a guanidinium thiocyanate-phenol-chloroform-based methodology to reduce the content of DNA and a column-based system. Both manual and semi-automated options are described, with preference given to automation for consistent results. A novel quality control procedure has been developed to assess the integrity and purity of the entire population of isolated mRNAs due to the absence of intact rRNAs.